Bio 104 Lab Manual 2010

Kingdoms of Organisms I Bio 104

Laboratory Manual

Fall 2010

LABORATORY SCHEDULE - FALL 2010 Date Exercise # Topics Sept. 7 & 9 1 Experimental Design

4 Introduction to Euglena exercise

Sept. 14 & 16 2 Introduction to Spectroscopy 3 Introduction to Microsoft Excel

4 Growing Euglena gracilis in Heterotrophic and Autotrophic Media In Light and Dark Conditions

Sept. 21 & 23 5 Kingdom Monera and Introduction to Microscopy 4 Growing Euglena gracilis – experimental design worksheet Sept. 28 & 30 LAB QUIZ 1

6 Protists and Osmotic Relationships Oct. 5 & 7 7 Fungi

8 Mitotic and Meiotic Cell Division

Oct. 12 & 14 LAB QUIZ 2 9 Evolutionary Strategies for Survival on Land: Vegetative and

Transport Structures Complete data collection for Euglena experiment 4 Growing Euglena gracilis – graphing and data analysis (in class)

Oct. 19 & 21 FALL BREAK – NO LABS

4 Euglena Outline and Graph Due in Class Friday, Oct. 23 Oct. 26 & 28 10 Plant Reproductive Structures and Early Seedling Development

11 Solar Energy Conversion Nov. 2 & 4 12 Solar Energy Conversion Data Analysis

Nov. 9 & 11 LAB QUIZ 3

5 Euglena Report Due 13 Sponges, Cnidarians Film: The Shape of Life Nov. 16 & 18 14 Worms: Platyhelminths, Nematodes and Annelids 12 Solar Energy Conversion Student Worksheet Due Nov. 23 & 25 NO LABS – THANKSGIVING BREAK Nov. 30 & Dec. 2 15 Arthropods

Dec. 7 & 9 LAB QUIZ 4 and NOTEBOOK COLLECTION 16 Molluscs

1

Exercise 1:

The Scientific Method and Experimental Design

OBJECTIVES:

This laboratory serves as an introduction to the scientific method. Specific objectives of the lab include:

• To understand what is meant by “the scientific method” and to understand both its strengths and

limitations for answering questions about the natural world,

• To understand the relationship between the scientific method and various elements of experimental

design

• To design an experiment and effectively report and interpret the results.

BACKGROUND INFORMATION:

Developing the Hypothesis

The scientific method: questions and answers

Science addresses questions relating to our curiosity about “how the world works”. Scientists attempt to

answer these questions by proposing possible explanations called hypotheses. A hypothesis is a tentative

explanation for what we observe. Hypotheses are based on observations, on information gained through

previous research, or on a combination of both.

Developing hypotheses

A hypothesis is usable only if it can be proven false (is falsifiable). The nature of science is such that we

can prove a hypothesis false by presenting evidence from an investigation that conflicts directly with a

2

prediction based on the hypothesis. We cannot, however, prove a hypothesis to be true. We can only

support the hypothesis with evidence from our investigation. A scientist NEVER concludes that the data

prove his or her hypothesis. Additional information or subsequent tests may later disprove the hypothesis

or provide alternative explanations for the phenomenon.

Scientific knowledge is therefore an accumulation of evidence in support of hypotheses; it is not to be

regarded as absolute truth. Hypotheses are accepted only on a trial basis. However, this does not mean

that scientific knowledge is flimsy and unreliable. Much of the information in your textbook, for

example, is based on many rigorous experiments carried out by numerous scientists over a period of

years. Hypotheses such as the “law of gravity" and "the theory of evolution" are well-accepted scientific

conclusions. Both hypotheses have held up because neither hypothesis has ever been falsified by a well-

designed scientific experiment. On the other hand, current scientific studies that you read about in the

newspaper or hear about on "Dateline News" -- for example, investigations on "the" gene that causes

breast cancer or "the" gene that causes dementia -- are much more preliminary and therefore more

tentative. You may even hear about studies with very contradictory results. These are based on

hypotheses still under investigation.

Predictions in hypothesis testing provide a reference point. If our predictions are confirmed, then we

have evidence to support the hypothesis. If the predictions are not supported, the hypothesis is falsified.

Either way, the scientist has gained new knowledge of the process being studied, and therefore can

generate an even MORE INFORMED hypothesis for the next investigation.

Hypothesis and the scientific method

The scientific method, then, applies only to hypotheses that are testable and have the potential to be

proved false or supported through experimentation, additional observations, or the synthesis of data from

a variety of sources. Students often think that controlled experiments are the only way to test a

hypothesis. However, many scientific advances have relied on other procedures and information to test

hypotheses. For example, James Watson and Francis Crick developed a model that was their hypothesis

for the structure of DNA. Their model could only be supported if the accumulated data from a number of

other scientists were consistent with the model. Watson and Crick won the Nobel Prize for their work,

but they did not perform a controlled experiment in the laboratory. Methods other than experimentation

are also acceptable and appropriate, depending on the question being investigated.

3

Identifying the Variables

Dependent Variable(s) (Response Variable)

The dependent variable is what the investigator measures (or counts or records). It is what the

investigator thinks will be affected by the experiment. For any experiment, there may be a number of

possible dependent variables. It is very important that the investigator specifically define which

dependent variables you will measure, how often, etc. It is up to the investigator to decide which

dependent variables are most appropriate for the hypothesis and to define them very specifically. An

investigator might also choose to measure more than one dependent variable.

Independent Variable(s) (“Treatments”)

The independent variable is what the investigator varies during the experiment. It is what the

investigator thinks will affect the dependent variable(s). Although it is possible to construct complex

experimental designs that simultaneously test the affects of more than one independent variable, most

experiments in Biology 104 should be limited to testing one independent variable at a time.

Controlled Variables (held constant by the investigator)

Since there can be only one independent variable in each experiment, all independent variables other

than the one being studied must be held constant. These are called controlled variables. The investigator

must eliminate the possibility that factors other than the "targeted" independent variable are affecting the

outcome.

Designing the Procedure

Not all of the items below are part of each investigation, but each should be considered as the

experiment is designed.

Level of Treatment

The investigator must determine appropriate values to use for the independent variable, called levels of

treatment. This judgment is usually based on knowledge of the system, or on information from the

4

scientific literature. (This is not the same as guessing.) For example, if the purpose of the experiment is

to investigate the effect of temperature on weight gain in hamsters, the scientist should acquire enough

knowledge of the physiology of hamsters to use appropriate temperatures. Subjecting the animals to

extremely high or low temperatures could kill them, and no useful data would be obtained. Likewise, the

scientist attempting to determine how much fertilizer to apply to soybean fields would need to know

something about the amounts typically used by other investigators and vary the treatments around those

levels.

Replication

Replication means that the scientist repeats the experiment numerous times using exactly the same

conditions to see if the results are consistent. (When designing experiments in your courses, work with

your lab instructor to make sure that you have a sample size that is appropriate.)

Variation is normal in biological systems. Replicating the experiment allows the investigator to see how

much variation there is. Duplicate data help the investigator determine how much variation is inherent

in the system and simply due to chance or, alternatively, if there are additional, uncontrolled variables

not accounted for. A measure of variability is essential for data interpretation. In real research the

question of adequate sample size is a complex problem, often involving statistical consultation.

Control Treatment (NOT a variable)

A control is a set of subjects that receive no treatment (or where the treatment is set at a standard value).

In all other respects they are treated exactly as those receiving experimental treatment. A control allows

the scientist to be sure that the effect on the dependent variable is in fact due to the independent variable.

For example, a control on the effect diet pills would include a group of similar subjects that receive no

drug but who do receive a placebo pill.

5

EXERCISES:

Form groups of three to four students (two pairs team up), and cooperatively design an experiment.

Together, you should:

• Formulate a hypothesis, and record it on your data sheets.

• Design and carefully define an experiment to test your hypothesis.

• Perform your experiment.

• Record results on a data sheet.

• Express your results in an appropriate form. (text, graphs, tables, etc. – materials will be provided.)

• Write a short conclusions section. This can even be an outline, as long as it’s complete. It should

address the following:

1. Review the results.

2. Did the experiment support the hypothesis? Explain.

3. Are there any follow-up experiments you could perform to improve on some aspect of your

experiment?

4. Can you think of a new investigation that would more definitively answer your hypothesis?

5. Other than your hypothesis, is there an explanation for your observations that is consistent

with your data?

This assignment will be collected at the end of class today. One paper per group is sufficient. Be sure to

include all of the group member’s names!

REFERENCES:

Morgan, J.G. and M.E.B. Carter. 1993. Annotated Instructor's Edition for Investigating Biology: A

laboratory manual for Biology. The Benjamin/Cummings Publishing Company, Inc. Redwood City, CA.

pp.2-7.

Shanholtzer, S.F. and M. E. Fanning. 1991. Termites and the scientific method. Page 195, in Tested

studies in laboratory teaching. Volume 12. (C.A. Goldman, Editor). Proceedings of the 12th

Workshop/Conference of the Association for Biology Laboratory Education

6

STUDY GUIDE:

• In an experimental situation, you should be able to identify the controlled variables, dependent

variables and independent variables.

• You should be familiar with the basic aspects of developing a hypothesis and experimental design.

• Given a set of observations, you should be able to write a well-developed hypothesis and design an

experiment to test your hypothesis. The experimental design should consider and include control,

dependent and independent variables, level of treatment, and response measurement.

7

BIOLOGY 104 LABORATORY Experimental Design Data Sheet

Investigators:______________________________________________________________________

Working Hypothesis:__________________________________________________________________

_________________________________________________________________________________

Dependent variable: ___________________________________________________________________

Independent variable: _________________________________________________________________

Control variables: ___________________________ __________________________

___________________________ __________________________

____________________________ __________________________

Procedure: Describe in one or two paragraphs, and include level of treatment, replication, control

treatment, etc. Define the measurement you are using and give a short explanation as to why you

selected this measurement. Where appropriate, you may use a diagram.

8

Results: (Show your original results here. If you wish, you may also add a graph, table, diagram or

other appropriate summarization of results.)

Conclusions: (Include any ideas for further investigation or refinements of your experiment.)

9

BIOLOGY 104 LABORATORY

Peer Review Worksheet

Reviewers:________________________________________________________________________

Thoroughly review your colleagues’ experimental design worksheet, including the procedures, results

and conclusions. Answer the following questions, referring back to their worksheet and giving specific

examples as needed.

Was the hypothesis clearly stated, and was it relevant to the study of termite behavior?

Did the experimental design include only ONE independent variable (and what was it)? Was it stated in

the hypothesis?

Did the group identify appropriate controls to account for other variables which may influence termite

behavior?

Did the experimental procedure itself effectively “control” the control variables?

10

Were the results presented clearly? Suggest possible improvements.

Did the group’s experimental data support their conclusions?

Was this experiment, as presented by the investigators, reproducible by another group? Why or why not? Did this investigation effectively contribute to your class’s body of knowledge concerning termite behavior?

Exercise 2: Spectroscopy

1

Exercise 2:

Introduction to Spectroscopy


Spectroscopy

Light absorption spectroscopy is one of the most widely used techniques in biology and chemistry.

Since the absorbancy of a solution is directly proportional to the concentration of absorbing molecules in

the solution, one can use this method to determine the concentration of a dissolved solute (or the density

of a cell culture) at any given time simply by making an absorbancy measurement. Such measurements

are performed using a spectrophotometer.

Figure 2.1 – Basic design of a spectrophotometer

How a spectrophotometer works

1. Light from a light source passes through a filter, which selects a desired wavelength (or

color) of light to pass through the sample. The wavelength is selected based on the physical


2

properties of the sample to maximize absorption. For example, if the sample is green, a

wavelength of light corresponding to non-green parts of the spectrum (i.e.: red or blue)

should be selected.

2. The selected wavelength of light travels through a small slit, then through the sample in the

cuvette or test tube.

3. The light that is not absorbed by the sample hits a detector on the far side of the sample.

Light transmittance is recorded on the meter.

4. The meter also has a scale for light absorbancy of the sample; this value is the log of the

inverse of the transmittance. Therefore, if 100% of the light is transmitted through the

sample, the absorbancy of the sample is 0.000 – no light was absorbed.

The Beer-Lambert Law and Its Applications

The absorbancy of a sample is a useful measurement; the Beer-Lambert Law demonstrates that the

absorbancy of light by a sample at a given wavelength is directly proportional to the concentration of the

absorbing molecules in the sample. Therefore, as the concentration of a sample is increased, the

sample’s absorbance increases.

A = E l c

where: A = the absorbancy of light

l = the path length (in this case, 1 cm, which is constant for the instruments we use)

c = the concentration of the sample

E = the molar absorptivity of the sample (constant for a given substance at a given wavelength)

Because this is a direct mathematical relationship, the Beer-Lambert Law can be used to determine the

concentration of a sample if its absorbance and molar absorptivity are known. Likewise, the molar

absorbtivity of a sample can be determined experimentally, by measuring the absorbance of a solution

with a known concentration and using the Beer-Lambert Law.

For example:

If a 0.01 mM ( 1 x 10-5 M) solution of bromophenol blue has an absorbancy of 0.790 at a wavelength of

590 nm, the molar absorptivity of the sample could be determined by:


3

A = E l c

0.790 = ( E ) ( 1 cm ) ( 1 x 10-5 M) and’

E = 7.90 x 104 cm-1 M-1

EXERCISES:

Measure the absorbancy of bromophenol blue solutions

Use Table 2.1 and the calculations worksheet to record your data for these exercises!

Measure the absorbancy of five samples of known bromophenol blue concentration using a

spectrophotometer. Note that the more concentrated the solution, the higher the expected absorbancy.

1. Obtain a series of BPB solutions of known concentrations, as well as one BPB solution of

unknown concentration. There are three replicate samples of each known concentration.

2. Set the wavelength to 590 nm on the Turner spectrophotometer. (Instructions for use are next to

the instrument, and in Appendix D of your lab manual.)

3. Measure the absorbance of each BPB solution, and record your results in Table 2.1.

Construct a standard curve for bromophenol blue at 590 nm

As stated earlier, the molar absorptivity (E1M) of a solution at a given wavelength can be determined

experimentally by measuring the absorbancy of a 1.0 molar solution and using the Beer-Lambert Law.

Another way to determine E1M experimentally is to measure a series of concentrations of the solution at a

given wavelength, then construct a standard curve illustrating the relationship between solution

concentration and absorbancy. At absorbancies less than 1.0, this curve should be linear, and the slope

of this curve gives the molar absorbtivity (E1M) of the solution at that wavelength.

To determine the molar absorptivity in this manner, you will graph your results by plotting the

concentration of the solutions vs. the absorbancy readings, and draw a best-fit line through the points to

create a standard curve. You will then calculate the slope of the standard curve to find the molar

absorptivity (E) of BPB at 590 nm. NOTE: To reduce tedious calculations and difficult graphing, plot


4

the solutions’ concentrations in units of µg/ml rather than molarity. We will refer to the slope of the

curve as simply the absorptivity, abbreviated E*.

1. In Table 2.1, record the A590 values for each of the three replicate samples of bromophenol blue

for each concentration. Calculate the average of each set of replicates. Use the average values

for the standard curve.

2. Using graph paper, plot the data. The concentration of BPB in µg/ml should be on the x-axis,

and the absorbancy on the y-axis. Be sure to label the graph and axes.

3. Calculate the absorptivity of the sample by using the Beer-Lambert Law to find E*. Record your

values for E* on Table 2.1.

E* = __A__ (remember, l = 1 cm)

l c

4. Use any one of your “E*” values from your table to calculate the concentration of the unknown

BPB solution using the Beer-Lambert Law. Record this on Table 2.1.

5. Now, use only your graph to estimate the concentration of the unknown BPB solution by finding

the unknown’s absorbance on the standard curve and extrapolating. Record this on Table 2.1.

6. Hand in your table and graph (figure) at the end of class today. Be sure that your graph has a

descriptive figure legend. (See Appendix B of your lab manual – Guide to Writing Lab Reports

– for an example of a descriptive figure legend).

STUDY GUIDE:

• Given a set of directions, know how to use the Turner spectrophotometer.

• Be able to determine the concentration of an unknown using both a standard curve, or E and the

Beer-Lambert Law.

• Understand the basic relationship between the concentration of a solution and its absorbancy.


5

Name: ______________________________ Section: ________________________________ Spectroscopy table and calculations sheet Table 2.1 - Absorbancy of Bromophenol Blue at 590 nm

Absorbancy at 590 nm Concentration

(µg/ml) Replicate 1

Replicate 2

Replicate 3

Average

E*

(calculated using B-L Law)

1

2

3

4

5

unknown

none

none

E*, the absorbtivity = the slope of your line (the absorbancy per unit of concentration) To find E* using the slope of the line: E* = y2 – y1 (absorbancy units) x2 – x1 (concentration)

E*, calculated by slope of the line (select any x and corresponding y values) = ____________________ Unknown concentration (using any of the calculated E* values) = _________ Unknown concentration (using the E* from slope of the standard curve) = ________ If you have absorbancy readings on five BPB samples of unknown concentration, which method would you use (standard curve or calculation using E*) to determine the concentration of each? Why? ___________________________________________________________________________________ ___________________________________________________________________________________


6

Exercise 3: Introduction to Excel

1

Exercise 3:

Introduction to Excel

BACKGROUND INFORMATION: Spreadsheets are routinely used for recording data, making calculations, and for basic graphing. The following exercise analyzes the data produced by the spectrophotometry exercise (Exercise 2). The bromophenol blue (BPB) dye concentrations and the resulting absorbance (A590) measurements are used to make a standard curve, which can then be used to determine the concentration of the unknown BPB dye sample. This introductory exercise illustrates the most basic spreadsheet and graphing features of Microsoft Excel. The tutorial, and workshops held on campus, can greatly enhance your proficiency with this spreadsheet program. Unless you are instructed otherwise, tables and graphs in biology and chemistry can be produced using Excel. Entering your data:

1. Open Excel. A clear spreadsheet should appear on your screen. A spreadsheet is arranged in rows and columns forming “cells”; each cell has its own “address” based on the row and column number. Typically, rows are numbered and columns identified by letters.

2. Set up your spreadsheet by entering labels as indicated in the figure below.

(Note: the cells in bold represent the Excel spreadsheet labels)

A B C D E F G 1 BPB conc

(µg/mL) A590

Replicate 1 A590

Replicate 2 A590

Replicate 3

2 1 3 2 4 3 5 4 6 5 7

3. Use the arrows or mouse to position the cursor and type in your raw data for the spectroscopy exercise. At this point, DO NOT enter the means OR the unknown value.


2

Make a scatter plot:

1. Now you can express your data table in figure form. To do this, first “paint” the entire

area of the spreadsheet you want to graph (in this case, highlight cells A1 thru D6). 2. Click on the “insert” tab on the top toolbar. A graphing toolbar should appear.

3. Select the “scatter” option, then choose the plot with points only (no lines!)

4. Your graph should appear on your spreadsheet. Notice that the toolbar has changed as

well. Some features of this toolbar are outlined below.

a. You can change the arrangement of your data on the figure with the “switch row/column” button. This is useful if Excel “guesses wrong” as to which values of your data table are on the x-axis and which on the y-axis. Check your graph and make sure the x-axis represents concentration, and the y-axis represents absorbancy.

b. You also have “chart layout” options on this toolbar. This places axes, titles, keys and other important things on your graph. Select “Layout 1” for your graph.

5. Take a look at your figure in Layout 1. Here, you may make any number of changes

simply by left-or-right clicking on the object in the figure.

a. Axes: Click on the x-axis and y-axis titles, and change the content to reflect your figure. Do not forget units!

b. Title: Biologists do not use titles; instead, we use descriptive legends. Click on the title and backspace so it is blank. You will add a legend later.

c. Key: There is a box or column on the right side of the figure that identifies the shapes/colors of each data point – this is the key.

d. Data labels: By clicking on individual data points, you will initiate a drop-down menu which allows you to name the point, add trendlines, etc.

e. Cosmetics: By right-clicking on the graph as a whole, a menu appears that allows you to change font, colors, etc.

6. Save your spreadsheet as it is to your G:/ drive.

Working with your figure:

1. The scatter plot you have generated is a good way to examine the scatter of the data

points, and is a simple way to assess the variability of your data. 2. Examine the individual data points on your scatter plot. Points that are significantly

different than other data are called “outliers”. Outliers can result from a number of


3

things including faulty technique or data entry errors. If you can identify erroneous data and assign a specific cause to them, they should be corrected or deleted. On the other hand, data cannot be discarded simply because they don’t fit our expectations.

3. Do you have any bad data on your plot (for example, replicate 2 for 3 ug/ml)? If you

question your lab instructor, you’ll find that he or she intentionally added twice the dye to that sample, just to irritate students. You can fix this (but not your instructor!)

4. In your DATA TABLE on the spreadsheet, click on the cell containing the offending

data point. Backspace to delete this (make it blank, not 0). Note that your figure automatically reflects this change!

Calculate the means (or averages) using the spreadsheet:

1. In your data table, label cell E1 “mean”. 2. Click on cell E2 (just below the label) to highlight it.

3. Click on the summation icon on the tool bar, then select “average” in the drop down

menu.

4. Excel paints the cells it thinks you want to average for the first concentration. Be sure to check Excel’s choice, and paint them manually if Excel is incorrect. In this case, you do not want to include the first column of data in the mean – they are not absorbancy values, but concentrations.

5. Hit the enter key. Is the mean correct?

6. If you would like to calculate means for each concentration, you may copy the formula

to other cells. To do this:

a. Highlight cell E2 (with the formula and mean). b. Select “copy” on the toolbar c. Click on cell E2, hold, and pull down to highlight the cells you want to include in

that column (E3-E6). d. Hit Enter. The means should appear in the appropriate cell.

NOTE: Alternatively, just highlight cell E2, grab the “tab” on the lower right corner of the cell and (holding down the mouse) drag it across cells E3-E6. The formula is applied to each cell.


4

Include the means in your figure:

1. Repeat the graphing procedure outlined earlier, but include the raw data and the means

(cells A1-E6). Be sure you the rows and columns translate correctly on your figure; if not, use the “switch row/column” button to fix this.

2. Label your axes.

3. While a scatter plot is a good way to look at raw data, a line connecting the means would

be useful in illustrating the data trend. To draw a line connecting the means:

a. Carefully use the mouse to touch a single data point for a mean value. b. Right-click on this point. c. From the drop down menu, select “add trendline”. d. To choose a best-fit straight line, select “linear”. You may also set the

y-intercept to “0”.

NOTE: You should have a good reason to choose a linear fit and to decide to set the y-intercept to 0. Why do we expect a linear fit for our data? Why is it acceptable to set the y-intercept to 0 in this case?

4. Sometimes it is desirable to simply connect the data points to illustrate a trend. For

example, you do not expect your Euglena growth to follow a linear course, so trying to fit your data to a straight line would be incorrect and would not accurately reflect the data trend. In this case, you should select a plot other than “scatter” when first creating your figure.

Add a figure legend: 1. In science, figures do not have titles; instead, they have descriptive “legends” beneath

the figure. A good legend contains just enough information to explain what the figure is illustrating.

2. Figures are numbered, and the figure number is the first part of the legend. For your

figure, the suggested legend is:

“Figure 1: Standard curve for serial dilutions of bromophenol blue read at 590 nm using a path length of 1 cm. The “best fit” (linear regression) line is drawn through the means of three replicate dilutions.”

3. Add this text to your figure by doing the following:

a. First, be sure “title” is deleted from your figure. b. Click on the figure itself to select that item on your spreadsheet.


5

c. Click on the “insert” tab in the toolbar, and choose “header/footer”. d. Select “custom footer”, then type in the legend on the left or center box. e. To see what your finished figure looks like, choose “print preview”.

Print the graph Make the desired chart active. If it looks OK, just click on the printer icon. Use the options in the 'Page Setup" menu under "file" to change the orientation [landscape or portrait] or to change your figure legend. Print a section of a worksheet Suppose you want to print your table as well, but you don’t want to print the entire Excel worksheet. To do this, there are a number of selections under the “File” menu that allow you to set the print area, change the page setup, or preview a printed area. Experiment with this menu on Excel. Save your files and exit Remember to save your file to a USB drive or to your Goucher account.


6

Additional Tips to Help You “Excel” Graphing Data From Non-adjacent Columns in Your Spreadsheet Cells containing the data that you want to graph may not always be adjacent. For example, you might want to plot only the x values and the mean values several columns away in your spreadsheet. 1. Select specific columns by painting them individually. First paint the x-axis column by itself.

2. Move the cursor to the top of next desired column. Hold down the control key and paint this column.

3. Repeat step 2 for any additional columns. 4. Graph the selected data, as previously.

Inserting Excel Graphs Into Word Documents 1. With the graph active, click on the COPY icon.

2. Open a WORD document 3. Go to the location in the text where you want the figure to appear.

4. Click on the PASTE icon [or find paste in the edit menu]. 5. The chart should appear in a box that can be moved or resized as needed.

NOTES: A simple copy/paste function can also be used to place a fully-active Excel table into the Word document. Data can be entered, edited, formatted within this table. If a simple, non-editable image of the table is to be inserted into the Word document, use the “Paste Special” command and insert it as a picture or PDF image. Graph or table picture inserts can be formatted to select for various types of text “wrapping” around the picture as follows: 1. Double-click on the inserted graphic to make it active. 2. Select “Layout” from the menu, then choose the appropriate text wrapping style for your document.

Exercise 4: Growing Euglena gracilis

1

Exercise 4:

Growing Euglena grac i l i s in Heterotrophic and Autotrophic Media in Light and Dark

PART 1 – Background Information and Experiment Setup BACKGROUND: The organism used for this study, Euglena gracilis, is a flagellated protist that will be studied during your laboratory on the Protists. In brief, E. gracilis is an organism with chloroplasts that can function as either a heterotroph, obtaining nutrients from the environment; or an autotroph, using light to create its own food. In this experiment, you will be growing Euglena in heterotrophic and autotrophic media in both light and dark conditions. Both types of media include water and small amounts of minerals and other micronutrients the organism requires. In addition, the heterotrophic medium contains sucrose, a fixed form of carbon the organism can use for energy. During the course of the project you should come to understand:

1. The difference between autotrophic and heterotrophic growth 2. The value of replication in scientific experiments 3. The use of the spectrophotometer in indirect measurements of cell density and

growth. In addition, you will (hopefully) master a number of skills, notably:

1. Good aseptic (sterile) technique 2. Macroscopic and microscopic observation of cultures 3. Use of the Sequoia-Turner spectrophotometer 4. Careful labeling and record keeping 5. Communication (oral and written) of your data and interpretation of results.

EXPERIMENT SETUP: This laboratory will be set up during laboratory hours, and its progress will be followed for four weeks outside of regular laboratory hours. It is imperative that you and your lab partner plan a schedule for accumulating data from this experiment!


2

Inoculate Cultures 1. Each student will be provided with a culture of Euglena, three tubes of sterile heterotrophic

medium (blue caps) and three tubes of sterile autotrophic medium (green caps). 2. Students should form pairs and label their tubes with tube number, experimental conditions

and student initials. The four different experimental conditions are listed below, and each pair will have three duplicate tubes for each condition. One member of each student pair should incubate their tubes in the light; the other member, the dark. See Figure 4.1 for experiment overview.

a) Autotrophic medium, light conditions (AL) - label AL-1, AL-2, AL-3 b) Heterotrophic medium, light conditions (HL) - label HL-1, HL-2, HL-3 c) Autotrophic medium, dark conditions (AD) - label AD-1, AD-2, AD-3 d) Heterotrophic medium, dark conditions (HD) - label HD-1, HD-2, HD-3

3. After observing the demonstration of aseptic transfer technique, inoculate each of your six

tubes with ONLY two drops of pure Euglena culture. Be careful to use sterile technique! Data Collection 1. The Sequoia-Turner spectrophotometer will be used to indirectly monitor the growth of the

cultures via increases in cell densities. Follow the hints below for the best, consistent readings throughout the experiment.

a) The use of this instrument was demonstrated in an earlier lab. User instructions are placed next to the instrument and will remain there for the duration of the experiment. They may also be found in Appendix C of the lab manual.

b) All readings for this experiment should be taken at a wavelength of 525 nm. (NOTE: Check the wavelength setting before each set of measurements)

c) For consistency, use the same spectrophotometer for all readings. d) To “blank” the spectrophotometer, use an un-inoculated tube of growth medium -

autotrophic medium, if measuring autotrophic cultures; heterotrophic medium, if measuring heterotrophic cultures. Keep your blanks with your cultures and use them throughout the experiment.

e) Agitate the cultures before reading by rolling tubes quickly between your hands. DO NOT mix by inversion or the cultures will become contaminated!

f) Always wipe the outside of the tubes before reading. g) Any “negative” readings should be recorded as zero.


3

Figure 4.1 – Flow chart of the procedure for the Euglena experiment


4

2. Take an initial reading (Day 0) of each inoculated Euglena culture. Record these readings in

Table 4.1 (light grown cultures) and Table 4.2 (dark grown cultures). 3. Plan to take an optical density reading of all cultures every two to three days for four weeks.

You and your lab partner MUST have data for the same days for all tubes, so coordinate a schedule and stick to it. A good time to take readings is every Monday, Wednesday and Friday after 104 lecture.

4. It is possible that after a number of days you notice some changes in the appearance of the

cultures. These changes should be recorded, as well as the absorbency, since they will be critical to your data interpretation in your lab write-up. You should also record the reading from the light-meter to monitor the light intensity that the light-grown cultures are exposed to.

5. At the end of four weeks make sure that both you and your partner have all of the data for all

12 tubes. Complete Tables 4.1 and 4.2 by calculating average absorbency values for each triplicate set of OD525 readings.

Culture Storage 1. Each table will be assigned two test tube racks - one for dark-incubated tubes, one for light-

incubated tubes. Your instructor will label these racks. 2. Each rack will also contain two “blanks”, one of heterotrophic medium and one of

autotrophic medium. These are to be used by the students at that table throughout the experiment to zero (or “blank”) the spectrophotometer.

3. Each student pair should have one student who places their tubes in the dark; the other

student places their tubes in the light. REFERENCES:

Bio. Sci. 104 Laboratory Manual Appendix B, The Research Paper McMillan (2006) Writing Papers in the Biological Sciences Starr et al. (2009), p. 355


5

PART 2 – Euglena Experiment Design Worksheet

Name: _________________________________ Section: Tues PM Thurs AM Thurs PM In the next several weeks you will finish collecting the data for the Euglena experiment. In preparation for the data analysis and laboratory write-up, answer the following questions. Refer back to the Scientific Method and Experimental Design exercise performed on the first day of Bio 104 lab. You may discuss the questions with a lab partner or classmate, but each individual should complete the worksheet. During the experiment, a single strain of Euglena was grown in two different types of media. The autotrophic medium contains water and small amounts of minerals and other micronutrients, and the heterotrophic medium contains the same components, plus sucrose. Cells inoculated into both types of media were grown in light or dark conditions for four weeks. What is the purpose of the Euglena experiment? In some respects, the Euglena experiment is really a comparison of growth under four different conditions. What are the four conditions? Be sure to specifically state the independent variable(s) for each. What is the dependent variable? 1) 2) 3) 4)


6

In addition to the OD525 data in Tables 4.1 and 4.2, you will summarize your culture growth data in a clear. easy-to-read manner by making one graph for your lab report. Think about how you would present these data. What variable should be on the x-axis? Y-axis? Should you manipulate your raw data (triplicate OD525 readings for each time point) in any way before plotting it on the graph? Why or why not? _____________________________________________________________________________ Turn the completed exercise in to your instructor.


7

PART 3 – Graphing and Data Analysis

In this exercise, you will use Microsoft Excel to generate a preliminary figure (graph) for your Euglena culture growth data. Before you begin: 1. In Tables 4.1 and 4.2, record all OD525 readings taken by your lab group for your Euglena cultures to date. 2. Calculate the average optical density (OD525) for each set of triplicate readings taken for each time point (day), for each culture set. Record the averages in Tables 4.1 and 4.2 (if not done earlier). 3. If necessary, convert “date of reading” (Sept 15, Sept. 18, etc.) to “days of growth” (day 0, day 3, etc.). Entering your data: 1. Open Excel.

2. Set up your spreadsheet, labeling as is illustrated below: day # goes in column A; average optical density values for each day are entered in rows under corresponding treatments (columns B-E). (Note: the cells in bold represent the Excel spreadsheet labels)

A B C D E 1 Day

Number Heterotrophic Light

Autotrophic Light

Heterotrophic Dark

Autotrophic Dark

2 0 3 3 4 etc. 5 6 7

NOTES: Be sure to enter the days of growth in column A. DO NOT enter dates. Enter average optical density values for each condition and day. DO NOT enter raw optical density data.


8

Generate your figure:

Now you can express your culture growth data in figure form. To do this, first “paint” the entire area of the spreadsheet that you want to graph.

1. Click on the “Insert” tab on the top toolbar. A graphing toolbar should appear.

2. Select the “Scatter” option and choose the plot with points connected with straight

lines.

3. Your graph should appear on your spreadsheet. Notice that the toolbar has changed as well. In the Layout tab, select “Layout 1”. At this point, you may make any number of changes simply by left-or-right clicking on the object in the figure.

a. Axes: Click on the x-axis and y-axis titles, and change the content to reflect your

figure. Do not forget units!

b. Title: Do not use figure titles; instead, use descriptive figure legends. Click on the title and backspace to delete it.

c. Cosmetics: By right-clicking on the graph as a whole, a menu appears that allows

you to change font, colors, etc.

4. Add a figure legend to your graph. To do this, click on the “Insert” tab and select “Header/footer.” Insert your legend as a custom footer.

a. Begin your legend with “Figure 1:” b. THINK CAREFULLY when composing your legend. In one or two short sentences, try to describe what the figure represents. We will give you feedback when we look over your outline. c. Be aware that you have a limited number of characters in a footer, so be concise!

5. Once your figure is complete, go back to your original spreadsheet and label it as “Table 4.3: “ followed by an appropriate table heading.

Print your figure and your spreadsheet. Save your file! IMPORTANT: Save this file. When you are finished collecting data, add them to your data table and revise your figure. Your revised figure and data table should be included in your outline.


9

Part 4 – Outline of Euglena Paper Think of the outline as a “rough draft” of your lab report. In general, any issues that you feel should be addressed in your report should be included in your outline. For each major section of the report, outline brief statements that summarize your ideas. Use the guidelines below and consult “The Research Paper” (Appendix B of the lab manual) to help you put information into the correct sections of your outline. Below is an incomplete “skeleton” of an outline to guide you – feel free to add to this, as is necessary, for your paper. Point values for each section are indicated in parentheses (35 points total).

I. Cover Page (1)

• Title of paper • Name, lab section, lab partner’s name, date

II. Introduction (3)

• State the question being asked and your basic experimental approach. • Information about Euglena that is relevant to your investigation. • Types of media, how it meets Euglena’s nutritional requirements, etc. • Anything else pertinent…

III. Materials and Methods (1) • Cite the lab manual for procedures, and note only significant changes that you’ve

made. Do not get bogged down in detail in this section – it should be short and to the point.

IV. Results (10)

• Focus on your group’s data to identify trends in growth of cultures over time, under different experimental conditions

• Describe growth trends quantitatively • Summarize macroscopic observations (appearance of cultures - “cloudy’, color

changes, etc.); if microscopic observations of cultures made, describe appearance of cells in each culture; DO NOT interpret your data in this section.

• You MUST turn in Figure 1 (the Excel graph that you made in class, using your own data; update this figure with final OD525 data, if necessary)

The graph should have Time (Days of Growth) on the x-axis and Optical Density (525 nm) on the y-axis.


10

Graph the average OD525 values for each triplicate data set. You should have one graph with four lines on it. The key should read: “Heterotrophic Light”, “Heterotrophic Dark”, “Autotrophic Light” and “Autotrophic Dark”.

Include a complete figure legend.

• “Raw” data Tables 4.1 and 4.2 should be appended to the outline. Photocopies of the original tables are fine for the outline (but complete data from these tables at the end of the experiment will be included as Table 1 and Table 2, respectively, in the final paper). The spreadsheet table of average OD525 used to make your graph (Figure 1) should also be included as Table 3. DO NOT forget to give these tables appropriate headings!

V. Discussion (20)

• This is the section in which you analyze/interpret your results. • Discuss results for each of the four individual treatments, one at a time

Relate growth trend under each condition to what you know about nutrition in Euglena; compare with growth under other conditions

Relate macroscopic appearance of cultures, microscopic appearance of cells, to individual growth conditions

Do the results make sense? • Briefly address any possible sources of error.

VI. Abstract, References and Appendices

These sections are not necessary for this outline (but are required for the paper!)

REMEMBER: This outline is intended to help you in writing your final paper. If you do a good job, we can return the outline with a lot of feedback. Also, writing a complete outline now will make writing your final paper a much simpler task.


11

Part 5 – Final Euglena Paper Completion of this exercise requires a written Research Report that will comprise 20% of your laboratory grade. For this assignment, you will need to follow the format below. Additional information about writing biology research reports may be found in Appendix B of this lab manual. Because most Bio 104 students are inexperienced at preparing such reports, this assignment was completed in two steps:

(i) A preliminary draft outline of the Research Report was collected. This draft included a figure of Euglena culture growth data, collected up to that point. The draft was graded and returned with comments, to help you prepare the final version of the report.

(ii) The final version of the report will now be prepared, using suggestions from the

draft outline, and turned in for a final grade.

Below is a brief outline of the format requirements and additional comments that are specifically relevant to the Euglena experiment.

1. Title page 2. Abstract (optional)

a. An abstract is a brief summary of your results and conclusions. b. A well-written abstract is worth up to 5 extra points.

3. Introduction

a. State the nature and purpose of the Euglena experiment. b. Give background information on the Euglena organism, heterotrophic vs.

autotrophic growth methods, etc. and cite references for this information. For this report, your textbook and this lab manual are the minimal required references.

4. Materials and Methods

a. This section should be very brief , and include any changes or additions to the written procedure in the lab manual. (For example, if you chose to observe your cultures under a microscope at the end of the experiment, this should be mentioned here.)

b. You may cite the lab manual for specific procedures, rather than rewriting everything.


12

5. Results

a. This section also a short overview of the results of the experiment. This overview, however, should contain no data interpretation.

b. A single summary graph exhibiting the average growth trends for Euglena cultures in under all four conditions must be included. The horizontal axis should be: Time (days of growth), and the vertical axis should be, Optical Density (525 nm). Label this graph Figure 1 and include a good descriptive legend.

c. This section must also include Table 1 and Table 2, showing the raw OD525 data taken from Tables 4.1 and 4.2, respectively. (NOTE: Place Tables 4.1 and 4.2 at the end of your report, as appendices.) In addition, Table 3 gives the means of each triplicate set of OD525 readings. (This is the Excel spreadsheet that you made to produce Figure 1.)

d. Any additional observations or data (e.g., macroscopic appearance of cultures, microscopic appearance of cells in each culture, etc.) should be presented here in clear, easy-to-follow paragraphs.

6. Discussion

a. This section is the most heavily-weighted in your lab report grade. b. Analyze your results in this section. c. If you have done any additional work, discuss your findings here and include

whether or not they support other results. d. If any questions arise regarding your work or results, they may be discussed

here. e. Any ideas for future work may be included. f. Any possible sources of error, or inconsistencies in your results should be addressed.

7. References

a. Include any reference you have used in doing the experiment or preparing your report.

b. Use the correct format from your style guide.

8. Appendix

a. You must include your raw data Tables 4.1 and 4.2 from your notebook. b. You must include your previously-graded draft outline and figure The template used by your instructors to grade the final paper is provided below.


13

Bio 104 Euglena Lab Report Name _______________________________ Grading Template Lab Section (circle): TU PM, TH AM, TH PM Introduction (10 total) Question clearly stated (5) ___ Relevant background, Euglena biology (5) ___ Materials and Methods (3 total) (3) ___ Results (35 total) Tables Heading (s) (2) ___ Data entered (Table 1 - data from Table 4.1,

Table 2 – data from Table 4.2) (2) ___ Means calculated (Table 3) (2) ___ Figure 1 Axes labeled, scaled properly (2) ___ Lines drawn (2) ___ Data all on one graph (2) ___ Figure legend complete, accurate (3) ___ Written description Description of individual growth trends (5) ___ Quantitative (not qualitative) description of

trends; references to figures, tables in text (6) ___ Macroscopic observations (5) ___ Doesn’t include "discussion" (4) ___ Discussion (40 total) Possible reasons for observed growth responses under different

experimental conditions; interpretation of data (30) ___ Expected vs. unexpected results (5) ___

Possible sources of error, how error might alter results (5) ___ References (3 total) Correct citations in text and at end of paper (3) ___ Format (9 total) General organization (2) ___ Writing style (7) ___ Extra Points (up to 10 total possible) Abstract (3) ___ Describe any changes to protocols in Methods section (1) ___ Microscopic observations in Results, relevance of

observations in Discussion (5) ___ Possible future experiments (1) ___ Points Subtracted: Graded outline not appended to final report (-5) ___ Total Points: _______


14

Table 4.1 - Data for light grown cultures of Euglena gracilis Name: ____________________________ Date of Day 0: __________________ Light intensity: ________________ Lab Section: _______________________ Lab partner: __________________

Optical Density at 525 nm after days of growth indicated

Day / Date

Heterotrophic Tube #1

Tube #2

Tube #3

AVERAGES

Autotrophic Tube #1

Tube #2

Tube #3

AVERAGES


15

Table 4.2 - Data for dark grown cultures of Euglena gracilis Name: ____________________________ Date of Day 0: __________________ Lab Section: _______________________ Lab partner: __________________

Optical Density at 525 nm after days of growth indicated

Day / Date Heterotrophic Tube #1

Tube #2

Tube #3

AVERAGES

Autotrophic Tube #1

Tube #2

Tube #3

AVERAGES


16

Exercise 5: The Monara

1

Exercise 5:

The Kingdom Monera and Introduction to Microscopy


The Monerans

The Monerans represent the simplest forms of life on earth and some are thought to resemble the earliest

living cells. Their cells exhibit a prokaryotic type organization; they lack membrane-bound nuclei,

mitochondria and other organelles typical of the eukaryotes. These simple forms of life have been and

remain enormously successful, certainly outnumbering and probably outweighing (in total biomass) all of

the “higher” forms of life combined.

Within this ancient group considerable evolutionary diversity is evident in both the structure and the

mode of nutrition of the different subgroups; not surprising, as the Monerans have had about 3.5 billion

years to evolve. Some bacteria in this kingdom are pathogenic, causing disease in other organisms.

Others, however, play quiet but essential roles in the recycling of nutrients of dead plants and animals.

All prokaryotes are classified as Monerans according to the five kingdom system of classification, since

all other organisms have a eukaryotic cell structure. There are two groups of Monerans: the Eubacteria

and the Archebacteria (Table 5.1). Although both are prokaryotic in cell organization, recent studies of

nucleic acid sequences (the DNA of the organisms) indicate that they are barely (if at all) related to one

another. Certain Archebacteria are thought to have given rise to modern eukaryotes.

Exercise 5: The Monera

2

Figure 5.1 - A longitudinal section of the AO One-Fifty Series Microscope


3

Table 5.1 - The two groups of Monerans

Eubacteria Archaebateria Cell wall peptidoglycan non-peptidoglycan Examples and features

saprobes (decay/recycle organic matter) pathogenic (disease-causing) photosynthetic bacteria chemoautotrophic bacteria

Methanogens (live in anaerobic conditions / fix nitrogen) Halophiles (require extreme salinity) Thermophilic (require extreme heat)

The Microscope

To examine members of the Kingdom Monera, you will need to use a light microscope. The light

microscope is the most basic and widely used tool of the biologist. You will practice using the compound

microscope, become familiar with all aspects of this instrument and learn how to use an ocular

micrometer to measure sizes of objects examined. Skilled use of the microscope enables you to learn as

much as possible about the organisms you are studying.

EXERCISES:

Basic Microscope Skills

Obtain a microscope from the cabinet, supporting it with both hands. Take a few minutes to review the

names and functions of the principle parts of the microscope; these are illustrated in Figure 5.1.

Base - with built-in illuminator and transformer control knob, the base holds the entire

microscope upright. The illuminator provides light from the bottom, and the transformer control

knob controls the intensity of the light.

Stage - is the place a specimen rests for examination. It features a slide holder that secures the

slide. The slide can be moved across the stage by turning the two attached knobs.


4

Arm - is the piece that holds the eyepieces and objective lenses above the stage. NEVER carry a

microscope by the arm only!

Revolving nosepiece - contains three objective lenses, which can be used to increase the

magnification of a specimen. The objective lenses magnify an object by 4x (scanning power),

10x (low power) and 45x (high power). Most of the magnifying power of the microscope resides

here.

Iris diaphragm - and its control lever adjust the amount of light allowed into the viewing field

from the illuminator. It also reduces scattered light, which can greatly reduce resolution at higher

magnifications. If a specimen seems “washed out” or lacking in color, there is too much light in

the field, so the iris diaphragm should be closed down until appropriate lighting is achieved.

Substage condenser- and its control knob adjust the intensity of light allowed into the viewing

field by focusing transmitted light into a coherent beam. Proper control of light quality with the

condenser and iris diaphragm can greatly increase resolution and overall image quality.

Coarse and fine focus adjustment knobs - are used to bring the specimen into focus. (The coarse

is the “outside” knob; the fine is “inside”)

Reversible body tube - has the eyepieces on one end; the objective lenses on the other.

Binocular eyepieces - are the parts you look through. One eyepiece contains a pointer, the other

an ocular micrometer. The eyepieces are 10x in magnification.

Use of the microscope: Viewing a slide of colored threads

Obtain a prepared slide of three colored threads from your instructor. Practice your microscope skills

with this slide until you are comfortable with the instrument.

1. Raise the nosepiece using the coarse adjustment knob. This provides greater access to the stage

when a slide is positioned.


5

2. Rotate the nosepiece so that the 4x scanning objective is in operating position. Note that the

lenses “click” into position. Always begin microscope work at scanning power!

3. Open the iris diaphragm approximately half way.

4. Turn the in-base illuminator to the 5V setting.

5. Place your slide of colored threads on the stage and position the specimen directly above the

center of light from the condenser (over the center of the hole in the stage).

6. Raise the microscope condenser (by means of the substage adjustment knob) until the top of the

condenser is approximately the thickness of piece of paper beneath the slide.

7. Rotate the coarse adjustment knob to lower the nosepiece until the positive stop is reached. Look

through the eyepiece and, without disturbing the coarse adjustment setting, slowly rotate the fine

adjustment knob to move the objective lens up or down until specimen detail is in the sharpest

possible focus.

8. Use the thumb wheel located between the eyepieces to adjust the interpupillary distance. The

left eyepiece tube can be focused to compensate for differences between the eyes.

a) The correct procedure is to bring the specimen into the sharpest possible focus with the

fine adjustment knob using the right eyepiece only (covering the left eyepiece).

b) To focus for the left eye, first turn the knurled ring on the left eyetube fully

counterclockwise. Now view the specimen with the left eye only and turn the knurled

collar clockwise until the specimen is again in sharp focus. DO NOT adjust the fine

adjustment knob during this procedure.

9. Adjust the light intensity for maximum contrast by changing the aperture of the iris diaphragm.

Lower light intensity is often better. You should have noticed by now that the slide consists of

three different colored threads intersecting one another. Move the stage control knobs until you

are viewing an intersection of two thread colors. As you focus with the fine adjustment, notice


6

that only one thread is in sharp focus at a time, and the other blurred. By focusing up and down

you can perceive depth that cannot be visualized when the focus stays at one level. This

technique is referred to as optical sectioning of the three-dimensional specimen. In all of your

microscopic work you will find it valuable to “keep one hand on the fine adjustment”, moving it

slightly up and down to make yourself constantly aware of the depth dimension.

a) What colored thread is uppermost in your slide? ____________________________________

b) Which thread is in the middle? _________________________________________________

10. Now rotate the nosepiece to other objectives without changing the position of the coarse

adjustment knob. Only minor fine adjustment should be required because the AO Series One-

Fifty microscope objectives are parfocal. Change to the high power objective (40x).

a) Notice that at a higher power, you perceive less depth. The depth of field (the vertical

distance in focus at one time) decreases as magnification increases. For this reason, the

optical sectioning effect becomes much more pronounced at higher magnification. Avoid the

tendency to go directly to the highest magnification, because careful use of the lower powers

frequently provides a better understanding of spatial relationships between the structures you

see.

b) You should also note that the iris diaphragm setting must be changed whenever an objective

is changed. As you increase magnification more light is required to view the specimen.

11. Now you are ready to observe some representatives of the Kingdom Monera in your microscope.

Refer to the above instructions whenever necessary.

Heterotrophic Eubacteria

Most common of the Monerans, including those most often found in humans, fall into the large group

generally referred to as the eubacteria (or true bacteria). Included in this mostly heterotrophic group are

many beneficial species as well as several notorious pathogens.


7

Prepared slides of the three most common shapes of bacteria have been set up for you at the demo scopes

using an oil immersion lens (under oil immersion, the magnification is 1000x). Table 5.2 illustrates these

three bacterial shapes. View these slides, but please DO NOT adjust the focus!

Table 5.2 - Three common bacterial shapes:

Do a cheek smear to find bacteria in your mouth:

Now that you are familiar with the appearance of bacteria under the microscope you are ready to look at

some bacteria of your own.

a. Use the wide end of a toothpick to scrape the bases of your teeth where they meet the gums.

b. Smear the scrapings on a slide to make a thin film about the size of a dime, and let it dry.

c. Now, pass the slide gently, smear side up, over a low flame. This kills the bacteria and

adheres them to the slides.


8

d. Add several drops of crystal violet stain to the slide and allow it to stand for one minute. You

may want place the slide on a paper towel before adding the crystal violet, as it stains

clothing as well as bacteria!

e. Wash the slide gently in water to remove the excess stain and blot with a paper towel to dry.

Be careful to avoid the smear.

f. Add a drop of water (if necessary) and a cover slip, and examine the slide under the high

power of your compound microscope.

1) Most bacteria are small and have few visible features at this magnification, so be

patient - it may be difficult to find them. However, with careful observation you

should be able to find some of your own oral bacteria. You can usually find some

adhering to the large purple epithelial cells from your mouth.

2) Classify the bacteria you see according to the three general shapes, if possible.

____________________________________________________________________

____________________________________________________________________

3) Compare the relative sizes of the bacteria and epithelial cells of the mucosal lining of

your mouth. Which are larger? _________________________________________

4) Draw an epithelial cell and bacteria at 450x magnification on the sheet provided in

your manual. Check the Appendix of your manual for tips on doing a scientific

drawing.


9

BIOLOGY 104 LABORATORY: OBSERVATIONS Name of Specimen: ________________________________ Date Observed: ______________

Preparation: ______________________________________ Magnification: ______________

Natural Environment: ______________________________

Comments and Observations:

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________


10

Using the ocular micrometer

The ocular micrometer is mounted in one of the eyepieces of your microscope; the pointer is mounted in

the other eyepiece. By rotating each ocular, you can determine which eyepiece contains the ocular

micrometer and pointer respectively. Move your slide on the mechanical stage so that the ocular

micrometer is easily visible in the field. Concentrate on the dimensions of the ocular micrometer as

scanning, low power and high power objectives are clicked into place in turn. Does the apparent size of

the ocular “ruler” change as objectives are changed? _________________________________________

Now repeat the examination of the appearance of the ocular micrometer with a check epithelial cell in the

field and brought into sharp focus. Measure the length and width of the same cell in ocular micrometer

units at each magnification (scanning, low and high power) and record your measurements in Table

5.2. Use the instructions below to do this more easily.

1) You may find that it is difficult to measure the same cell with each objective. Select a cell at

scanning power in the center of your viewing field, and note its shape and those of

surrounding cells. When you click over to the 10x objective, the cell will remain in the center

of the field. Remember to focus using only the fine adjustment after switching objectives!

2) Also, remember that the ocular micrometer can be positioned by rotating the eyepiece - you

can measure length and width of a cell simply by adjusting the micrometer, and allowing the

slide to remain in place.

Table 5.2 - Dimensions of a cheek epithelial cell

OBJECTIVE

DIMENSION Scanning Low Power

High Power

units µm units

µm Units µm

Length

Width


11

Work / Calculation space:

3) Answer the following questions concerning the use of the ocular micrometer.

a) Does the apparent size of the epithelial cell change when viewed under each of the

objectives? ___________________________________________________________

b) Do the real dimensions of the cell change? __________________________________

c) If the structure in the field changes in size as the objectives are changed, but the size

of the ocular “ruler” remains constant, what do you conclude about the length

represented by an ocular micrometer unit under these different magnifications?

_____________________________________________________________________

4) As you know by now, ocular micrometer units are arbitrary units. Before a biologist uses a

microscope regularly, an ocular micrometer is often calibrated using a special slide called a

stage micrometer. The stage micrometer provides a second “ruler” that is used to determine

the size of an ocular unit for each combination of ocular and objective lenses: i.e., each

magnification of your microscope. Because calibrating an ocular micrometer can be

somewhat tedious, Table 5.3 is provided for you. (Appendix D has instructions for ocular

micrometer calibration.) Table 5.3 provides approximate calibration values for each

magnification of your microscope.

a) Using Table 5.3, you can determine the real size of the cells you have been observing.

Simply multiply the observed size (the number of ocular units) by the calibration factor


12

(µm per ocular micrometer unit). Record your data in Table 5.2, and record the actual

dimensions of both epithelial cells and bacterial cells in your drawing.

b) Now you know the approximate sizes of the epithelial cells and bacterial cells in your

cheek smear. Are these sizes consistent with the expected cell sizes of prokaryotic and

eukaryotic cells? ______________________________________________________

Table 5.3 – Approximate calibration data for the ocular micrometer of the AO 150

Objective

Ocular Micrometer Units

Stage Micrometer Distance, µm

µm Per Ocular Micrometer Unit

4X

92

2000

21.7

10X

100

1000

10

45X

100

220

2.2

Bacterial Diversity

You have seen that eubacteria may be classified by three general shapes: coccus, bacillus and spirilla.

However, the incredible diversity of bacteria requires that many other methods of classification be

available to identify species. A few of these methods are described in the following exercise.

1. Gram staining – View the diagram of the gram staining process

The gram stain is used to identify bacteria based on one exterior feature. All eubacteria have

peptidoglycan-containing cell walls, but some have an extra external layer made up of

lipopolysacccharide (LPS). Gram staining differentiates between these two types.


13

a. “Gram positive” bacteria do NOT have LPS layers, so the hydrophilic crystal violet stain

used in the first step of the process adheres to the peptidoglycan cell wall, but washes off of

the LPS layer of gram negative bacteria when they are treated with ethanol or acetone.

b. The pink lipid stain Safranin O is then able to stain the LPS layer in the gram negative

bacteria. This is called a counterstain, and is necessary because many bacteria are transparent

and cannot be viewed without staining.

c. View the slide of mixed gram positive (purple) and gram negative (pink) bacteria.

d. Gram staining is often the first test done in identifying a species of bacteria, especially for

medical reasons. Why would a doctor be interested knowing whether a bacterial infection is

gram positive or negative? (Recall that most antibiotics are introduced in a water-based

medium.)

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

2. Cellular morphology – The patterns of cell growth in bacteria can be diverse as well. View the

demonstration materials showing various cellular growth patterns. These are useful in that they

give you a clue as to how some of these species are named! Try to match the names of each

species to the growth patterns shown.

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________


14

3. Colony morphology – When plated on growth medium, bacterial species may have a diverse

array of colony types. Pigmentation, colony shape, colony thickness and growth patterns can

help identify the bacteria.

View the demonstration plates and photographs of different bacterial species. Try to identify the

different morphological features of each.

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

4. Spore formation – Under the right conditions, some bacteria have the ability to form endospores.

View the example photographs or slides. What is the selective advantage for species with the

ability to form spores?

_____________________________________________________________________________

_____________________________________________________________________________

5. Utilization of oxygen – The availability of oxygen is critical for the survival of some bacterial

species (obligate aerobes) while others thrive without oxygen (anaerobes). Still other bacteria can

live without oxygen, but will happily utilize it if it is present (facultative anaerobes). One way in

which we can classify aerobic capacity is with an “agar deep stab.” An inoculating needle is

coated with the bacteria of interest and stabbed into deep agar. If growth appears at the top of the

stab it indicates that the bacteria can grow aerobically; growth deep in the agar indicates the

bacteria prefer low-oxygen environments.


15

If available, view the agar stabs and determine the aerobic capacity of the bacterial species.

___________________________________________________________________________

___________________________________________________________________________

6. Biochemical features – Overall, bacteria use a huge array of biochemical pathways, utilizing

energy from a wide variety of sources. Culture media with different nutrients may be used to

help characterize species. Other biochemical tests look for the bacteria’s ability to produce a

certain metabolite (for example, gas or alcohol in fermentation).

If available, view demonstrations of some of the biochemical assays used to identify bacteria.

7. Utilization of light – Some eubacteria are autotrophic and can perform photosynthesis. These

species contain membrane stacks, much like chloroplasts (but they are NOT separate organelles

like chloroplasts!) Do not let the color of some of these bacteria fool you – not all

photoautotrophic species are green (or greenish).

View the plate or culture of Rhodospirilium rubrum. Its purple color is from the photosynthetic

“accessory pigments” phycoerythrin. This species is heterotrophic and colorless in high oxygen

environments, yet becomes photoautotrophic (and red-purple in color) under low oxygen

conditions.


16

Cyanobacteria (“blue-green algae”): Photoautotrophic Eubacteria This large and distinct group of photosynthetic Monerans is common in the soil as well as in freshwater

and marine environments. Their characteristic blue-green color results from the presence of another type

of photosynthetic accessory pigment called phycocyanin. Cyanobacteria may occur as single cells,

frequently are grouped as colonies within a gelatinous matrix, or they may form thin filaments or chains

of cells with a mucilaginous or gelatinous sheath. Some filamentous types regularly form heterocysts,

cells in which nitrogen fixation occurs. Some cyanobacteria may also form achinetes, which are special

cells that can survive high stress environmental conditions.

Several kinds of cyanobacteria are available for you to work with. Examine a sample of each type with

your microscope. (Remember, low light is better when viewing biological specimens such as these!) For

those types with a gelatinous sheath, a small amount of dilute India ink may help make the sheath more

easily visible (don’t forget to note this stain on your drawing!) As you examine the cyanobacteria, do the

following exercises:

1. Make a careful drawing in your lab notebook of a cell, colony or sheath of each type. Note the

name of the specimen, the magnification, and other pertinent information.

2. Summarize the characteristics of the living cultures by filling out Table 5.4.

Table 5.4 - A comparison of living cyanobacteria

Name

Oscillatoria Anabaena Gleocapsa

Cell size and shape

Heterocysts present?

Gelatinous matrix or

sheath present?

Motility (describe)


17

BIOLOGY 104 LABORATORY: OBSERVATIONS Name of Specimen: ________________________________ Date Observed: ______________




_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________


18

BIOLOGY 104 LABORATORY: OBSERVATIONS

Name of Specimen: ________________________________ Date Observed: ______________




_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________


19

BIOLOGY 104 LABORATORY: OBSERVATIONS

Name of Specimen: _________________________________ Date Observed: ___________

Preparation: ______________________________________ Magnification: ____________

Natural Environment: ___________________________________


_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________


20

Monerans Used in the Manufacture of Foods

Many kinds of bacteria are unwelcome intruders in our homes because they can cause food spoilage.

However, a number of species are used in the manufacture of foods. The products of bacterial

metabolism produce characteristic textures, smells and flavors. Table 5.5 lists examples of kinds of

foods prepared by fermentations involving pure or mixed cultures of Moneran species. In many cases a

single chemical produced by bacteria is responsible for the unique flavor and/or odor. Examples of these

fermentation products are available in the hallway outside the laboratory for you to taste and smell.

The use of bacteria in food production is not limited to this sampling; in fact, bacteria are becoming

increasingly important as a source of protein supplements for human or animal diets. Such single-cell

proteins, or SCP’s, can be a valuable addition to diets in areas where there are shortages of meats.

Bacteria multiply and grow very rapidly (some cells double in every 20 minutes in some species!) to

produce a high-yield, high-protein “crop”. Bacterial SCP’s have been produced from cultures of

Methlophilus, Methylotrophus, and the photosynthetic Scenedesmus and Spirulina species.

STUDY GUIDE: You should be able to:

• Identify different cyanobacteria and their distinguishing features.

• Identify the three shapes of heterotrophic eubacteria.

• Explain differences between the cyanobacteria and heterotrophic eubacteria.

• Describe the relevance to humans of some Monerans

• Compare prokaryotic and eukaryotic cells, as in the cheek smear exercise.

• Name some foods Monerans are used in the manufacture of.

• Understand the use of an ocular micrometer – be able to convert the measurement of a cell from

ocular units to actual size, given a calibration chart.

• Be able to name crucial parts of the microscope.

• Know the components of a good scientific drawing.

REFERENCES:

Starr et al. (2009), pp. 342-343


21

Name: _____________________________

Section: _____________________________

ASSIGNMENT:

Take a moment to think about and compare the heterotrophic eubacteria and photoautotrophic

cyanobacteria. On the sheet provided at the end of this lab, write a short paragraph to summarize the

major differences you observed. Be sure to compare the cell sizes of the eubacteria and cyanobacteria.

This paragraph will be collected at the end of class today.

____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________


22

Table 5.5 - Examples of foods manufactured with the aid of pure or mixed cultures of Moneran species Food or flavoring

Bacteria responsible for fermentation Comments on role of bacteria and nature of metabolic product

A. Fermented milk

Sour cream

Streptococcus sp. or Leuconostoc sp. Produce desired acidity in inoculated cream

Buttermilk Streptococcus sp. or Leuconostoc sp.

Produce desired acidity in skim milk

Acidophilus milk Lactobacillus acidophilus

Allow lactose-deficient individuals to consume milk

Yogurt Lactobacillus bulgaricus and Streptococcus thermophilus

Provide texture and tart flavor

Cheeses (a variety of soft and hard cheeses)

Streptococcus sp. , Leuconostoc sp., Lactobacillus sp.

Give the desired taste of cheese (although some cheeses are flavored by fermentation products of fungi)

Swiss cheese Propinionobacter sp. Propionic acid produced by this bacteria gives Swiss cheese its characteristic flavor

B. Fermented meats

Various sausages Pediococcus cerevisiae and various lactic acid bacteria

Imparts a preservative effect and adds tangy flavor to meat

C. Alcoholic bevarages Eastern wines Leuconostoc sp. and Lactobacillus sp. Yeast fermentation produces the ethanol in wine; however,

wines produced from Eastern U.S. grapes are too acidic and must be made palatable by bacterial metabolism.

D. Vinegar Acetobacter sp. and Gluconobacter Ethanol produced by yeast fermentation is converted to acetic acid by bacterial metabolism.

E. Fermented vegatables (sauerkraut, poi) Leuconostoc sp., Lactobacillus sp., Pediococcus sp., Streptococcus sp., Pseudomonas sp., some coliforms

Organic acids produced by bacterial metabolism contribute to the texture, flavor and aroma of fermented vegatables.

1

Exercise 6:

The Protists and Water Relations in Living Cells

THE PROTISTS:


Protists are eukaryotic organisms with membrane-bound nuclei and other cellular organelles. Many

members of this group are unicellular and as such they offer an excellent opportunity to examine the great

diversity in form and function that exists within the basic limits of a single eukaryotic cell. However, it is

deceiving to think of these as simple organisms. Often, protistan cells are far more complex than many of

the individual cells of multicellular organisms.

In this laboratory we will investigate how some basic biological functions are performed by representative

protists. As you examine them, try to gain an appreciation for the variety within this group AND

concentrate on the relationship between form and function represented by different protists with different

life styles.

EXERCISES: (Use the Bioreview sheets to assist you!)

CILIATES - Paramecium multimicronucleatum and Blepharisma

Movement and behavior:

Spread a drop of Protoslo on a microscope slide, spread a few cotton fibers in it, then add a drop of

Paramecium culture (this portion of the exercise may also be done using Blepharisma, a rose-colored ciliate

that moves more slowly than Paramecium). Examine the slide under the lowest power of your compound

microscope. Adjust your microscope diaphragm and light intensity for the best contrast. Carefully observe

the swimming motion of these single cells for a few minutes, and answer the following questions:

Exercise 6: The Protists

2

Figure 6.1 – Paramecium anatomy

© Carolina Biological Supply Company – reprinted by permission


3

1. Do these single-celled organisms have anterior and posterior ends? ______________________

2. Is one side always uppermost?___________________________________________________

3. How do these protists react to barriers?

a. How do they avoid them? ___________________________________________________

b. How do they get through tight places?__________________________________________

Feeding your Paramecium:

Coat the tip of a dissecting needle or toothpick with yeast stained in Congo Red dye. Add just enough yeast

so that the drop of Paramecium suspension is pink - not red. Put a coverslip on the slide and begin careful

observations under fairly low-light conditions.

1. How do Paramecia react to the food? _________________________________________

2. Describe the formation of food vacuoles. __________________________________________

____________________________________________________________________________

____________________________________________________________________________

3. Trace and diagram the path of food vacuoles on an observation sheet. This may not be as

simple as it sounds. Patience! Digestion takes place within the vacuoles. Congo Red is a pH

sensitive dye that is red above pH 5.0 and turns to blue at pH 3.0 (more acidic).

4. Paramecia normally feed on bacteria rather than on Congo Red stained yeast. You may find

that some Paramecia become more selective once they are filled with food vacuoles. If so,

describe any apparent selectivity you observe.

___________________________________________________________________________


4

Prominent structures and locomotion:

Now that your specimens have slowed down, switch your microscope to 100x and use the Bioreview Sheet

to identify prominent structures of Paramecium anatomy.

1. Locate the two (sometimes more) contractile vacuoles. These collect water from the cell and

discharge it to the exterior. Why are they important? (Contractile vacuoles will be examined

further during the Water Relations portion of the lab.) Locating the vacuoles seems difficult to

do, but patience will be rewarded. Look for “bubbles” in the Paramecium that seem to expand,

then suddenly disappear.

____________________________________________________________________________

____________________________________________________________________________

2. Select a quiet specimen and study the action of the cilia (use subdued light and high power).

a. Do all cilia beat in unison? _______________________________________________

b. Describe how the cilia beat. _______________________________________________

c. How might this rhythm be coordinated? _____________________________________

3. To identify some of the structures in the protists, you may wish to work with a partner and try a

staining technique. Methylene blue stains the nucleus and cytoplasmic granules of a cell. The

stain was prepared for you in a diluted form (10 mg stain / 100 ml 95% ethanol). Apply the

stain to an empty slide a drop at a time, and allow the stain to dry on the slide. Then add one to

two drops of Paramecium and view under the microscope.

Can you see the macro- and micro- nuclei of Paramecium? ____________________________


5

BIOLOGY 104 LABORATORY: OBSERVATION SHEET

Name of Specimen: ____________________________ Date Observed: ______________

Preparation: __________________________________ Magnification: ______________

Natural Environment: _________________________


____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________


6

Figure 6.2 – Euglena anatomy



7

FLAGELLATES: Euglena gracilis

Many different groups of protists utilize one or several flagella for locomotion. Some are photosynthetic,

some heterotrophic and some such as Euglena gracilis, may have both capabilities.

Movement and behavior:

Examine Euglena first under low power, then utilize high power (use a cover slip!). Be sure to adjust the

light for maximum clarity. Take your time and get the best possible image from your microscope, and make

notes on the Bioreview sheet as necessary.

1. Observe and describe the swimming motion. Does the body change shape? You may wish to

slow the little beasts down using Protoslo or let the slide dry a little. Look for the large

locomotive flagellum (tricks: use dilute India ink, and lower the light on the microscope.

Another trick is to use flagella stain – put some on the slide, allow it to dry, and add Euglena).

_______________________________________________________________________________

_______________________________________________________________________________

_______________________________________________________________________________

2. What other organelles can you identify in Euglena? What are their functions? If you wish, you

and your lab partner may try the staining technique you used on the ciliates.

_______________________________________________________________________________

_______________________________________________________________________________

_______________________________________________________________________________


8

Figure 6.3 – Amoeba anatomy



9

Euglena’s reaction to light:

In the dark room observe the bottle of Euglena that is illuminated from one side and describe the distribution

of the cells.

1. Is the orientation of Euglena photopositive or photonegative? __________________________

2. What organelle is responsible for this response to light? _______________________________

3. What is the advantage to Euglena of this response? __________________________________

___________________________________________________________________________

AMOEBOID PROTISTS: Amoeba

Observe living amoebae using a raised coverslip (clay under each corner) over a wet mount prepared by

your instructor, making notes where necessary on your Bioreview sheet. The amoeba may be difficult to

find! Compare the movement of amoebae to that of other protists you have seen. To facilitate and

encourage movement, Chilomonas, a dinoflagellate, may be provided as Amoeba food. Watch as Amoeba

consumes food with its pseudopod.

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________


10

Figure 6.4 – Physarum life cycle



11

SLIME MOLDS: Physarum

These organisms have both animal-like and plant-like characteristics and share developmental similarities

with some fungi. While some slime molds may produce a multinucleate mass of undifferentiated cytoplasm

or aggregations of cells, these are not true multicellular organisms. Amoeboid stages are present at some

point in the life cycle, and many have a flagellated stage as well. This bizarre mixture of characteristics has

fascinated and perplexed biologists for years. Some species are endoparasites of plants, but most live on

decomposing plant material. Recently slime molds have been used as model systems for basic research on

cell motility and development.

Observe cultures of Physarum on display. The plasmodium you see results from fusion of swarm cells.

Describe cytoplasmic streaming:

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

AUTOTROPHIC PROTISTS: The Algae

The algae are a heterogeneous group of primitive photosynthetic organisms. The various algal divisions are

unrelated evolutionary remnants that, except for the green algae, have not given rise to any of our present

day plants.

Examine the representative living or preserved specimens of algae available in the laboratory.

Diatoms and desmids:

These green algae species are mostly unicellular. They have a protective covering made of silica, which

gives them a glassy appearance under the microscope.


12

Volvox:

These particular green alga forms colonies. The flagellated cells are held together by a gelatinous material

and arranged to form what resembles a hollow ball. The colony spins due to the beating flagella of

individual cells.

You may see a colony with smaller daughter colonies inside. The daughter colonies are released when the

parent colony breaks apart.

Multicellular algae (various representatives):

Different types of multicellular algae are represented in this collection: types of phaeophyta (brown algae),

rhodophyta (red algae) and chlorophyta (green algae).

In addition to representing the ancestral stock that probably gave rise to the plants, the Chlorophyta (green

algae) provide examples of all the major evolutionary trends exhibited by the autotrophic protists

(multicellularity, structural specialization and complex life cycles).

You may wish to remove a piece of the multicellular algae Ulva and examine it under the microscope.


13

SUMMARY QUESTIONS ON PROTISTS: Name: ___________________________

Section: _________________________

Based on your observations of the protists, answer the following questions:

1. Is the classification of protists based on unicellularity? Name organisms observed in lab to support your

answer.

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

2. You have observed three broad and informal categories of protists in the laboratory. Identify these

categories, and a representative organism from each of these.

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

3. Can you use cell size as a criterion to distinguish between prokaryotic and eukaryotic cells? Support

your answer by using examples of cells observed in this lab and the moneran lab.

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________


14

CELL WALLS, MEMBRANES AND OSMOTIC RELATIONSHIPS:


A basic property of living cells is that they can control, or at least modify, their internal environment.

Since all cells are bounded by membranes, the selective permeability of membranes plays a vital role in

the movement of substances into and out of cells. Most cell walls, in contrast, are freely permeable and

do not affect the movement of solutes. The physical and chemical properties of cell membranes make

them permeable to water and to a few other small molecules but not to most large molecules.

Passive transport of substances across membranes is usually via diffusion. Diffusion is simply the

movement of molecules from a region where they are relatively concentrated to a region where they are

more dilute. The dissolved gases, oxygen and carbon dioxide, move into and out of cells via simple

diffusion across the cell membrane. Therefore, the directions and rates of their movements are

determined by differences in concentration between the inside and outside of the membrane

(concentration gradients).

Cells are aqueous systems, and the movement of water itself is vitally important. It usually moves across

cell membranes via osmosis. Osmosis is a special case of diffusion and refers to movement of water

across a selectively permeable membrane in response to differences in solute concentration. Water moves

in the direction of higher solute (or osmotic) concentration as determined by the total number of solute

particles in solution, not by their size or their weight. Other properties of solutions such as boiling and

freezing points also depend on the solute concentration.

The terms hyperosmotic and hypoosmotic refer to a solution that is more concentrated or less

concentrated than a second solution. These somewhat confusing terms are meaningless unless the two

solutions involved are specified. For example, a cell with a high internal salt concentration would

probably be hyperosmotic to a freshwater environment, and the water would be hypoosmotic to the cell.

Isoosmotic solutions have the same osmotic concentration. It will be helpful to become accustomed to

these terms and to the consequences for water movement by osmosis, both beneficial and detrimental, as

illustrated by the following experiments.


15


Name of Specimen: ____________________________ Date Observed: ______________

Preparation: __________________________________ Magnification: ______________

Natural Environment: _________________________


____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________


16

EXERCISES:

Plasmolysis and turgor pressure in plant cells

The vacuole and cytoplasm of a plant cell contain a variety of dissolved substances (sugars, amino acids,

inorganic salts) and thus have an osmotic potential greater than water (they are hyperosmotic to water).

When placed in pure water, the cells will take up water by osmosis and increase in volume. This

swelling will eventually exert pressure on the stiff cell wall. Such water pressure on the cell wall from the

inside is called tugor pressure and the cells are said to be turgid (as crisp lettuce is after being soaked in

water.) Conversely, a cell exposed to a hyperosmotic (more concentrated) environment will lose water.

As cell volume decreases, the cytoplasm shrinks away from the cell wall, a phenomenon known as

plasmolysis.

1) Observe Elodea cells in a turgid condition (Elodea is a fresh-water plant, only two cells thick!).

Make a wet mount of an Elodea leaf in water. Add a coverslip and observe, adjusting the microscope

for optimum focus and contrast.

a) Locate the cell wall, vacuole and chloroplasts. Recall that the cell membrane is just inside

the cell wall. Although Elodea cells are eukaryotic, it will be difficult to see the nucleus

without stain. It may be visible as a vaguely purple/grey structure.

b) Make a careful drawing of a typical Elodea cell in water.

c) The cell is in a hypoosmotic environment. There is a higher solute concentration in the leaf

than in the water, so water enters the leaf cell’s cytoplasm. What prevents the leaf cell from

bursting?

____________________________________________________________________

2) Now, make a wet mount of an Elodea leaf in 1 M glucose. Add a coverslip and observe, again

adjusting the microscope for optimum focus and contrast.

a) Are the cells turgid or plasmolyzed? _____________________________________________


17

b) Do all cells react in the same way? ______________________________________________

c) How long does it take for Elodea cells to react to the 1 M glucose? _____________________

d) Draw a typical cell under high magnification on the same sheet that you drew the leaf in

water. Label your drawing showing the positions of the cell wall and cell membrane.

e) Is the cell cytoplasm hyper- or hypoosmotic to the glucose solution? __________________

3) Describe and explain any differences between the appearance of the cells in water and 1 M glucose.

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

Osmotic regulation in animal cells

Plant have cell walls to prevent them from bursting when they are in hypoosmotic solutions (the solution

outside of the cell is less concentrated than the cell itself). Animal cells do not have cell walls, and must

cope with changes in osmotic conditions in a more active manner. Examine osmotic regulation in a fresh

Paramecium suspension as outlined below. As you perform this exercise, think about the differences in

structure of plant and animal cells, and the adaptations of each cell type that allow them to respond to

osmotic changes.

1. Use the Paramecium slide from the feeding exercise earlier, or make a fresh slide of Paramecium

suspension. Add a few cotton fibers in an attempt to slow down and trap a specimen.

2. Locate the contractile vacuoles. These will appear as “bubbles” inside the Paramecium that enlarge,

then suddenly disappear. This part of the exercise requires careful observation and patience!

3. Time the contraction rates under low power. How many times do these vacuoles contract per minute?

__________________________________________________________________________________


18

4. Now add 1-2 drops of 0.25% sodium chloride. Has the rate of contraction increased or decreased? Did

you expect the contraction rate to increase or decrease, based on what you know about osmosis?

__________________________________________________________________________________

__________________________________________________________________________________

a. P. multimicronucleatum is a common fresh water species. Seawater contains about

3.5% salt. Would the rate of vacuole contraction be the same for a marine ciliate in the

same (0.25% sodium chloride) medium? ____________________________________

b. Why?_________________________________________________________________

STUDY GUIDE:

You should be able to:

Identify the protists observed in lab.

Identify and describe an example of each of the broad catagories of protists observed in lab.

Describe any specific adaptations of the protists observed.

Describe the differences between the Moneran and Protista kingdoms, using specific laboratory

examples.

Understand the following key words and concepts, and their application:

Diffusion, Osmosis, Hyper and hypo osmotic, Isoosmotic, Turgor pressure, Plasmolysis

REFERENCES:

Starr et al. (2009), Chapter 22

Hickman et al. (2008), Chapter 11

1

Exercise 7:

The Fungi


In this laboratory you will observe representatives of each of the major groups of fungi. The fungi are

eukaryotic organisms, differing from plants and animals by one or more important features. All true fungi

have heterotrophic nutrition and may be either saprobic or parasitic. They have cell walls with chitin as a

major polysaccharide component. They reproduce, both sexually and asexually, with non-motile spores

and multicellular fungi have their cells organized as filaments called hyphae.

Both saprobic (decomposition of dead materials) and parasitic (degradation of organic matter in living

organisms) fungi secrete digestive enzymes which break down organic matter, then absorb the products of

this extracellular digestion. Such activities are ecologically and economically important. Fungal

decomposition is critical in the recycling of carbon, nitrogen and other nutrients. Without the degradative

activities of fungi, bacteria, and microbe-assisted invertebrates, the surface of the earth would be littered

with solid waste and nutrient availability for new growth would be limited. From an economic standpoint,

the degradative activity of fungi can be very costly. For example, mildew and rotting of leather, paint,

furniture, etc. are common problems in areas in which flooding or high humidity promote fungal growth.

The fungi are also important in food production. Fermentations produce solvents (including ethanol that is

also imbibed) and are sources of meat-tenderizing enzymes, pharmaceuticals (e.g., antibiotics, steroid

precursors of birth control pills), etc. Some fungi produce hallucinogens that play a significant role in some

religious practices.

From a purely scientific perspective, fungi such as Neurospora crassa and the yeast Saccahromyces

cerevisiae have contributed greatly to our understanding of genetic regulation of biological processes and

continue to be important eukaryotic “model organisms”.

Exercise 7: The Fungi

2

ZYGOMYCETES

Examining spoiled bread:

“Bread molds” belong to either the Zygomycetes or Ascomycetes. The color you see in bread mold is

usually imparted by the presence of asexual spores, which are formed by mitosis. The vegetative hyphae

are usually colorless. Sometimes, you can smell moldy bread without seeing the mold, which usually

indicates the presence of a mycelium, which is a mass of hyphae. In addition to the smell test, the fungal

mycelium in bread can be detected by examination under a dissecting microscope.

If available, examine some “spoiled” bread and note the color of each bread mold.

Sexual reproduction in zygomycetes:

One common zygomycete bread mold is Rhizopus stolonifer, known commonly as black bread mold. When

haploid (1N) “+”and “-“ strains of the fungus grow together and come into contact, fusion of cells and

nuclei from each strain results in formation of a diploid (2N) nucleus. The 2N nucleus undergoes meiosis

and new 1N spores are produced within an elaborate structure called the zygospore.

This heavily-pigmented, ornately-decorated wall of the zygospore can easily be distinguished from the

pigmented, smooth walled sporangia containing asexual spores (products of mitotic cell division). The

latter, terminal sporangia look like “lollipops” on a stick. The zygospore differs in having the original “+”

and “-“ mating cells (gametangia) still attached to either side.

Examine a demonstration slide showing a zygospore. What is the significance of sexual reproduction in this

fungus, which also is capable of reproducing asexually?

______________________________________________________________________________________

______________________________________________________________________________________


3

EUMYCETES

The classification of the two major groups of multicellular eumycetes, the Ascomycetes and the

Basidiomycetes, is based upon the type of spore that they produce during sexual reproduction. Each spore

type represents a “variation on a theme” during development. Spores produced by meiotic cell division

either are retained within the cell in which meiosis occurred (the “sac”-like ascus), or spores are extruded

from this cell, becoming attached to the outside of the cell in which meiosis occurred (the “club”-shaped

basidum). A schematic representation of these processes is shown in Figure 7.1 below.

DEUTEROMYCETES

The Deuteromycetes, or “imperfect” fungi, have no known method of sexual reproduction, and thus cannot

be classified as Ascomycetes or Basidiomycetes. As more is known about the organisms in this group, they

are reclassified, as is appropriate. One well-known former deuteromycete is Penicillium, whose sexual

stages identify it as an ascomycete (Talaromyces).

Carnivorous fungus:

The Deuteromycete to be studied today is a carnivorous fungus species, Arthrobotrys conoides, which is

found in soil and in fresh and salt water. This fungus traps and digests roundworms, or nematodes, by

forming “lassos” with its hyphae. The adhesive lasso traps the nematode while additional hyphae are

produced which penetrate the worm’s body, digest then absorb the contents. Today you will inoculate a

plate of A. conoides with Rhabditis (nematodes) and watch as the nematodes are trapped.

1. Obtain a plate of A. conoides and a culture of Rhabditis from your instructor. Examine the fungus under

a dissecting microscope. Are there any structures resembling lassos present? _____________

2. Using sterile technique, inoculate the plate with the nematode culture.

a. Check the plate under the microscope to be certain that nematodes were transferred. Continue

to watch for a while - perhaps you will see a nematode captured.


4

b. Check the plate periodically for the capture of nematodes by the fungus, if you did not observe

a capture in the first several minutes.

c. Describe what you see: ________________________________________________________

_______________________________________________________________________________

3. Check the plate periodically throughout class, and also observe the demonstration plates, which were

inoculated 24 hours and 72-96 hours previously.

a. Describe any differences in the appearance of the nematodes or fungus in the demo plates as

compared to your plates.

____________________________________________________________________________

___________________________________________________________________________

b. Why do you think “lassos” appear after inoculation of the plates with nematodes?

____________________________________________________________________________

c. Does the digestion of nematodes have any effect on fungal growth? _____________________

____________________________________________________________________________


5

Figure 7.1 Asexual and sexual reproduction in ascomycetes and basidiomycetes. 1) Asexual reproduction (mitotic) via conidia in each haploid (1N) mating type. 2) Mating to form first N+N dikaryotic cell, then hyphae 3) Fusion of N+N nuclei to form diploid (2N) nucleus. 4) Meiosis to re-establish 1N spores of each mating type (ascospores within ascus; basidiospores on basidium).


6

BASIDIOMYCETES

While much of the mycelium of a basidiomycete is generally hidden within the substrate on which the

fungus is growing, you will be familiar with the reproductive structures (or basidioscarps) of the

mushrooms, toadstools, puffballs and shelf fungi. Some economically important diseases of crop plants (the

“rusts” of wheat and the “smuts” that infect corn and other grains) are also caused by basidiomycetes. A

major identifying characteristic of this group is the basidium, a club-shaped cell formed during sexual

reproduction, and the four (usually) basidiospores which develop on its surface.

The basidiomycete with which you are probably most familiar is the common edible mushroom, Agaricus

bisporus (bisporus, because this species has only two basidiospores per basidium).

1. Look at the demonstration slides of a thin section wet mount of the gill area to see the basidia

and attached basidiospores. How are the basidiospores attached to the basidium?

_______________________________________________________________________________

2. Look at cross-sections and longitudinal sections (prepared slides) of the basidiocarp of

Coprinus sp. Look for the hyphae that make up the gill area. They are much intertwined, but

can be seen fairly clearly in the mycelial connection at the base of the basidium.

3. Observe the demonstration of spore prints set up in the lab. This is something you may enjoy

doing for yourself with wild species of basidiomycetes that you find.


7

ASCOMYCETES

In addition to bread molds, the Ascomycetes include a wide variety of forms such as truffles, yeasts and

powdery mildews. A major unifying characteristic of the group is that during sexual reproduction spores

form within a sac-like structure, the ascus. Most of these fungi also reproduce asexually by means of

conidia, spores formed singly or in chains at the tips of special hyphae (Figure 7.1)

Penicillium and Aspergillus:

You have probably seen some of such blue- or green-colored molds on overripe oranges and on preserves or

jelly. These likely were Penicillium and Aspergillus species. The individual “+” and “-“ mating strains of

these ascomycetes were formerly classified as Deuteromycetes and the original names (Penicillium and

Aspergillus) are still commonly used to describe these strains. Some species are important commercially,

including those used in the production of antibiotics and in cheese making.

Observe the demonstration Penicillium culture. Note the white mycelium and green conidia. Examine the

chains of conidia produced by a Penicillium mycelium growing on orange skin (demo slide).

NOTE: This exercise requires the use of Penicillium cultures and penicillin. If you are allergic to

penicillin, please let the lab instructor know and please leave the lab while the remainder of the class

performs this experiment!

Set up of Fleming’s experiment (This week):

Alexander Fleming's fortuitous discovery that bacterial growth is inhibited by Penicillium eventually led to

the burgeoning antibiotic pharmaceutical industries and contributed to extended life expectancy and

decreased infant mortality. Today you will repeat Fleming's experiment.

1. Each pair of students will be given 2 nutrient agar plates and 2 tubes of soft agar medium. Your

instructor will review aseptic techniques.


8

2. Using a sterile transfer pipette, inoculate each soft agar tube with 2 drops of Micrococcus luteus culture.

Swirl rapidly but avoid bubbles. Pour each tube onto a nutrient agar plate. Tilt gently to spread the

liquid top agar. Cover and let solidify right side up (about 5 minutes).

3. When the top agar has solidified, inoculate one edge of the first plate with a loopful of Penicillium

notatum. Cover, label the underside of the plate with your name, date, and microbial species, and

incubate inverted at room temperature in your drawer.

4. Transfer a penicillin impregnated disc from a sterile cartridge to the center of the second plate. Label

and incubate with the first plate.

Observations (Next week):

Examine your plates in the next laboratory period and record your observations. The presence of bacteria

is indicated by cloudiness of the top agar. You will be provided with a sterile plate for comparison.

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

______________________________________________________________________________________

Sexual reproduction in ascomycetes:

One common ascomycete used in teaching laboratories is Sordaria fimicola. Recall that in sexual

reproduction in ascomycetes spores form in a sac-like structure called an ascus. An agar plate with

Sordaria is provided on your lab bench. This plate was innoculated with two strains of Sordaria; one strain

has a brown spore color and the other strain a tan spore color. Notice the groups of hybrid perithecia on the

plate, near the edges where the two Sordaria strains meet. The perithecia are structures (a type of ascocarp)

that contain many asci.


9

1. Locate the perithecia on the plate. Under the dissecting scope, the perithecia look like black

bulbs. The best place to find hybrid perithecia is along the edge of the plate, where the two

strains meet and grow together.

2. Crush perithecia to release spore-containing asci.

a. Using a dissecting needle, carefully remove a few of the perithecia and place them in a drop

of water on a slide.

b. Put a cover slip over the perithecia, and place a kimwipe over the cover slip.

c. Using your thumb, gently crush the perithecia without breaking the cover slip

d. View your slide under a compound microscope. The perithecia, if crushed correctly,

should release many asci with arrangements of eight spores within each. If you found

hybrid perithecia, you will see both tan and brown sprores arranged in the same ascus!

3. Remember, the spores are the product of meiosis, with one mitotic division following meiosis,

to produce eight spores. Therefore, it makes sense to see two different spore colors in one

ascus, if the perithecia resulted from a mating of brown and tan spore-producing Sordaria. The

segregation of spore color during meiosis is dependent upon both 1) independent assortment

of chromosomes with the black and tan spore color alleles and 2) crossing-over between the

allele-containing regions of these chromosomes, as is shown in Figure 7.3.

Powdery mildew on leaves: “Powdery mildews” are common fungal diseases on plant leaves. The white powdery material on lilac

(Syringia sp.) leaves consists of the mycelium and abundant conidia produced by the fungus Microsphaeria

alni.

Look at the demonstration showing cross-sections of infected lilac leaves and you will see how the powdery

mildews got their name. Note also the presence of another type of ascocarp called a cleistothecium, which

contains asci and ascospores, identifying this fungus as an ascomycete.


10

LICHENS

Lichens, which appear to be single organisms, are actually two organisms living in very close association.

They consist of a fungus plus a green alga or a cyanobacterium. They are included here because the fungus

portion is often an ascomycete, sometimes a basidiomycete.

The lichen association has traditionally been viewed as a symbiosis, with the fungus providing physical

protection and increased water and mineral nutrient availability to the alga (phycobiont). The heterotrophic

fungus (mycobiont) benefits from the absorption of sugars and other organic molecules from the

photoautotrophic alga. Some scientists now view the lichen association more as a form of “controlled

parisitism”, where the fungal partner actually harvests (kills) many of the algal cells, which reproduce just

fast enough to maintain the algal cell population. The association between the fungus and alga is highly

specialized. Although each partner can be grown separately under defined laboratory conditions, neither is

found as a free-living organism in nature.

Observe the examples of crustose, foliose and fruticose forms on display.

1. How could you prove that the lichens really consist of two organisms?

______________________________________________________________________________

2. How could you determine whether the relationship is symbiotic or parasitic?

______________________________________________________________________________

Lichens exist in ecological niches in which most higher plants could not survive (e.g., on the surfaces of

rocks, places subjected to intermittent drying, and with extreme daily temperature cycles).

Observe the demonstration slides of squashed lichen (fresh wet mounts and prepared slides). Be able to

identify the fungal and algal components.


11

FUNGI IN THE FOOD INDUSTRY

On a table outside the laboratory are examples of foods or food flavorings that were produced with the aide

of fungal plus bacterial (i.e., double) fermentation. You may taste each sample if you wish (subject to

availability).

1. Soy sauce (produced by fermentation with Aspergillus)

2. Tempeh (boiled skinless soybeans fermented by a Zygomycete)

3. Beer or wine (contain ethanol produced by yeasts)

4. Bread (CO2 produced by yeasts is the leavening agent)

5. Nata de pina (a cooked and sugared by-product of fungal fermentation). This is a delicacy in the

Philippines and is often used as candy or served as a dessert

6. Sauted mushrooms (basidiomycete fruiting bodies sauteed in garlic and butter)

STUDY GUIDE:


• Understand the similarities and differences between the major groups of fungi, and

other eukaryotic organisms

• Understand the ecological and economic importance of fungi

• Identify reproductive structures characteristic of Zygomycetes, Ascomycetes,

Basidiomycetes, Deuteromycetes (asexual, sexual)

• Explain the nature of lichen associations and the benefits for each fungal and algal

symbiont

• Identify uses of fungi in the production foods and beverages

REFERENCES:

Ahmadjian, Vernon. (1982) The nature of lichens. Natural History 91(3): 31-37.

Starr et al. (2009), Chapter 24


12

ASSIGNMENT:

Complete Table 7.1 comparing the sexual reproduction of ascomycetes, basidiomycetes and zygomycetes.

Focus on the reproductive structures of each, and how the spores are released from these structures. You

have seen an example of each of these structures in lab today!

Table 7.1 – Sexual reproduction in fungi

Reproductive structure

Spore arrangement and release

Ascomycetes

Zygomycetes

Basidiomycetes

Exercise 8: Meiotic Cell Division

1

Exercise 8:

Mitotic and Meiotic Cell Division


Mitosis

You are already familiar with the important aspects of mitosis and cytoplasmic division

(cytokinesis) that occur in eukaryotic organisms. Replication of cells by these processes is

responsible for the growth of a plant or animal embryo into an adult, as well as for asexual

reproduction of a wide variety of organisms. Uncontrolled cell division can produce malignant

tumors and other forms of cancer. Remember that nuclei produced by mitosis have the same

amount and kind of genetic material (chromosomes) as the parent nucleus. Figure 8.1

illustrates the basic process of mitotic cell division.

Meiosis

Meiosis is another kind of nuclear division. It also occurs only in sexually-reproducing

eukaryotes, but results in daughter nuclei with half the amount of genetic material as the parent

nucleus. This process is a prerequisite for sexual reproduction in higher organisms. In animals,

meiosis occurs in special tissues or glands (gonads) and results in the formation of eggs or

sperm; in plants and fungi meiosis results in the formation of spores. Eggs, sperm and spores

all have only one half the amount of genetic material as the parent nucleus, or a single set of

chromosomes.


2

An example of meiosis may help you understand the reduction in genetic material without a loss

of chromosome number (known as “n”). Humans have 23 pairs of chromosomes (n=23), one

maternal set and one paternal set. An egg carries only the maternal set (23 chromosomes, all

different) until fertilized by a sperm, which carries a paternal set (23 chromosomes). An egg,

sperm or spore that contains only one set of chromosomes is known as a haploid (1n) cell.

Upon fertilization, the zygote has two complete sets of chromosomes, which are homologous,

and is a diploid cell (2n). Repeated cell division is responsible for the growth of the human

body from the original zygote. To sexually reproduce, meiosis takes place in the gonads to

produce eggs or sperm. In this way, a diploid organism produces haploid gametes which can

recombine to form a new, genetically distinct, diploid organism. Figure 8.2 illustrates a basic

life cycle for a diploid organism. Note the alteration between haploid and diploid states.


3

Figure 8.1. Stages of mitotic cell division in animal cells (top row) and in plant cells (bottom

row)


4

Figure 8.2. Life cycle of a diploid organism

Figure 8.3. A comparison of mitosis and meiosis


5

EXERCISES:

CHROMOSOME BEHAVIOR IN A MODEL OF MITOSIS

Review the process of mitosis, as is shown in Figures 8.1 and 8.3, then use three sets of pipe cleaner

“chromosomes” in a hands-on exercise demonstrating the segregation of chromosomes in this process.

You should first “replicate” the genetic material in each of the three different chromosomes (forming an

“X” using two same-colored and sized pipe cleaners). Be sure that the products of mitosis are two

identical daughter cells. Once you have completed “mitosis”, have an instructor check your lab group

before you continue on to meiosis.

CHROMOSOME BEHAVIOR IN A MODEL OF MEIOSIS

You will now use pipe cleaners of different colors as models to study what happens to individual

chromosomes during meiosis. Independent assortment, which occurs in meiosis, is one way of

introducing genetic variability in the products of meiosis. Crossing over in meiosis also contributes to

genetic variability (this subject is covered in detail in Genetics courses). Use Figure 8.3 to help guide

you through the basic stages of meiosis. Figures 8.4 and 8.5 (below) show detailed stages of meiotic cell

division leading to the production of gametes in animals (sperm and egg) and in higher plants (pollen,

egg).

1. Again, use three sets of pipe cleaners to model chromosome segregation during meiosis in an

individual with three different chromosomes (n=3). Follow the movement of chromosomes through

the two divisions of meiosis. It is important to remember that, although the description of meiosis is

divided into “phases”, the process of meiosis is a continuous one.

a. Pair the homologous chromosomes for Prophase I.

1) In our imaginary individual, there is one long chromosome from each parent. These

are homologous, and should be paired together.

2) Same situation for the short and intermediate-length chromosomes.


6

3) Notice that these chromosomes have already replicated, so each chromosome consists

of two chromatids joined at the centromere.

4) When the homologous chromosomes pair up, the arms of the chromatids may cross

over one another, exchanging pieces of chromosomal DNA. Crossing over can result

in the exchange of genetic information between the homologous chromosomes; this

results in genetic recombination. (In this case, do not cross over the chromatids.)

b. Align the paired chromosomes for Metaphase I.

1) Notice there are a number of different ways the chromosome pairs can align. How

many different alignments are possible?

____________________________________________________________________

2) Describe or diagram the possible alignments (use colored pencils to help you!).

c. For one of the possible alignments, manipulate the “chromosomes” through the remaining

stages of Meiosis I (Anaphase I and Telophase I).

1) You should have two “daughter cells” at the end of Meiosis I.

2) Each daughter cell should have a complete long chromosome (with both chromatids)

and a complete intermediate-length chromosome, and a short chromosome.


7

3) Draw or describe these daughter cells.

d. Now manipulate the “chromosomes” through Meiosis II, in which the sister chromatids

separate. Do this for both the daughter cells from Meiosis I.

1) How many daughter cells result from both Meiosis I and II? _______________

2) Draw or describe these cells. Keep these daughter cells to use in part three of this

exercise.


8

2. Answer the following questions about the process of meiosis. You may wish to review meiosis with

the pipe cleaner chromosomes as you answer.

a. What events in meiosis result in genetic variability?

___________________________________________________________________________

b. What aspects of meiosis are similar to mitosis? ___________________________________________________________________________

c. What aspects of meiosis are different from mitosis?

___________________________________________________________________________

d. If an organism has four pairs of chromosomes (n = 4), how many different gametes are

possible? When answering this question, assume no crossing over and that the maternal and

paternal sets of chromosomes are different. Hint: you may use extra chromosome pairs if you

want. Diagram your results on scrap paper as you work through the combinations.

___________________________________________________________________________ ___________________________________________________________________________

3. To understand the genetic diversity that results from the process of meiosis, you will “fertilize” one of

your daughter cells from part one with one daughter cell from the lab group across the table. You

will then repeat the process of meiosis, and examine the resulting “second generation” daughter cells.

a. Combine one of your daughter cells with a daughter cell from the lab group across the table

to simulate “fertilization.” The resulting cell is a diploid cell, made from the fusion of two

haploid cells. This diploid cell has two copies of each chromosome (2n) - one from mom

(you) and one from dad (across the table).

b. Now, pretend that the diploid cell has grown into a fine young diploid organism is now ready

to produce haploid gametes. “Replicate” the chromosomal material by adding one pipe

cleaner to each chromosome (the same color as the original chromosome). Each


9

chromosome should look like an “X” , with two sister chromatids. You now have a cell that

is ready to enter the meiosis stage of the cell cycle.

c. Repeat the cycle of meiosis as in part one of this exercise.

1) Prophase I - pair the homologous chromosomes

2) Metaphase I - align the paired chromosomes

3) Anaphase I and Telophase I - pull apart the homologous pairs and form two daughter

cells.

4) Meiosis II - the sister chromatids separate. (Do for both daughter cells)

d. You should now have four daughter cells.

1) Draw or describe these cells.

2) How do these cells differ from the daughter cells you made in part one?

_______________________________________________________________

4. Answer the following questions about meiosis, fertilization and the resulting genetic diversity.

a. How are the events of meiosis and fertilization responsible for creating progeny with genetic

information different from that of their parents?

__________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________


10

b. Briefly explain how meiosis is important in keeping the chromosome number constant from

generation to generation.

__________________________________________________________________________ __________________________________________________________________________

c. Which stage in meiosis reduces the amount of chromosomal material carried by the nucleus?

__________________________________________________________________________

STUDY GUIDE:

You should be able to: Answer the questions on the previous pages concerning meiosis.

REFERENCES:

Hickman et. al. (2008) pp. 52-55, 77 – 80


11

Figure 8.4. Animal meiosis (Copyright Carolina Biological Supply Co., BioReview Sheet #1610)


12

Figure 8.5. Plant meiosis (Copyright Carolina Biological Supply Co., BioReview Sheet #1630)

1

Exercise 9:

Evolutionary Strategies for Survival on Land: Vegetative Structures


A variety of evidence indicates that modern seed plants ultimately derived from photosynthetic green

algae (Chlorophyta). In order to successfully invade terrestrial environments a variety of adaptations

evolved to address the problems of acquiring and maintaining water and nutrients, physical support,

protection from ultraviolet light, and the ability to withstand climates in various ecological niches. The

purpose of this lab is to examine in detail some of the adaptations plants developed to address these

problems.

The Bryophytes are plants that have invaded the land but still require a moist environment They illustrate

not only adaptations useful to plants in making the transition from aquatic to terrestrial life, but also show

increased complexity and division of labor within the plant body. Although they have parts that resemble

stems, roots and leaves, they do not have xylem and phloem and are thus not “vascular” plants.

The primitive vascular plants (whisk ferns, club mosses, horsetails and ferns) and the seed plants

(gymnosperms, angiosperms) represent a major advance in the plant kingdom. They have specialized

conducting tissues that facilitate the bulk transport of water, dissolved minerals and the products of

photosynthesis throughout the plant. Such transport is rapid and occurs over considerable distances (e.g., to

the tops of the tallest trees). Consequently, vascular plants can grow much larger than nonvascular plants.

Collectively, vascular plants are referred to as “tracheophytes” because the cell wall architecture of

individual xylem cells (tracheids) superficially resembles cartilaginous rings of the human trachea.

Primitive vascular plants are generally confined to wet habitats and their leaves and roots do not exhibit the

types of adaptations that higher plants have to avoid desiccation in drier environments. Photosynthetic

structures such as leaves, however, are poorly developed in most primitive vascular plants. In contrast, the

more advanced seed plants have evolved a variety of adaptations in their leaf structures, stems and root

systems to facilitate photosynthesis while avoiding desiccation. They are found in almost every ecological

Exercise 9: Vegetative Structures

2

niche on Earth.

In this exercise, you will examine living representatives of the nonvascular bryophytes, some primitive

vascular plants, gymnosperms and angiosperms. As you examine the plants, try to determine the features of

each specimen that allow the plant to obtain and transport water and nutrients, physically support itself, and

permit gas exchange. You will find it helpful to fill out Table 9.1 as you proceed through the laboratory.

EXERCISES:

DIVISION: BRYOPHYTA (Mosses, Liverworts and Hornworts)

The Bryophytes are photosynthetic, i.e., they have an autotrophic mode of nutrition. They have root-like

rhizoids to aid in absorption of water and minerals from the soil. They do not have specialized water or

food conducting (“vascular”) tissues and are thus termed non-vascular plants. The radially symmetrical

mosses have leaf-like appendages attached to the vertical stem-like axis. Most authorities believe that

bryophytes represent an evolutionary dead end; nevertheless these organisms do exhibit a primitive level of

tissue organization and adaptation to terrestrial habitats.

Liverworts:

Examine the living gametophyte (haploid) plants of Marchantia.

1. Note the flattened, Y-shaped, dichotomously branching thallus. Remove a piece of the thallus,

and examine the lower surface using the 4x objective of your microscope. Locate the slender,

hairlike unicellular rhizoids and the long, flattened multicellular scales.

2. Examine the upper surface of the thallus and observe the pores at the centers of hexagonal

patterns of cells. These pores facilitate the rapid exchange of gases between the atmosphere

and the internal photosynthetic chambers; however they cannot be opened and closed like

stomates in higher plants. How does this affect the water balance of the plant?

____________________________________________________________________________


3

3. Think for a moment about the gas exchange in this plant, and answer the following questions:

a. What gases are moved through these pores? __________________________________

b. In what processes are these gases involved? __________________________________

c. How do these gases move through the pore? _________________________________

4. Asexual reproduction in Marchantia can occur very simply. While the thallus continues to

grow and form new branches anteriorly (at the tip), the posterior end disintegrates. New and

separate plants are formed when the posterior disintegration reaches a branch point.

Another, more elaborate means of asexual reproduction involves multicellular structures called

gemmae that develop within cup-shaped outgrowths of the upper surface of the thallus. Locate

any gemmae (if present) on the thallus of the Marchantia.

Mosses:

Moss spores develop into new gametophyte plants. The gametophyte begins as a branching filament

(protonema) that later develops into an erect, radially symmetrical haploid plant.

1. Examine the green "leafy" base of Polytrichum, a common moss. Are the leaf-like appendages

true leaves?

___________________________________________________________________________

2. Pull off a one of the “leafy” structures and examine it under a microscope. Where and how are

gases exchanged in the plant?

____________________________________________________________________________

3. Examine the base of the plant and find the root-like rhizoids. What two functions do you think

these structures perform?

____________________________________________________________________________


4

PRIMITIVE VASCULAR PLANTS (Tracheophytes)

Vascular tissues are an adaptive advantage for photosynthetic plants living on land. Xylem cells transport

water and minerals absorbed from the soil and provide support while phloem cells carry the products of

photosynthesis to growing and non-photosynthetic parts of plants. These remarkable transport systems

enable some tracheophytes to grow very large, compared to the non-vascular Bryophytes. There are

representatives of three tracheophyte divisions available to examine in lab.

DIVISION: Arthrophyta or Sphenophyta (horsetails or scouring rushes)

The only living representatives of the horsetails are about 25 species of the genus Equisetum. Their stems

are hollow and jointed. Leaves (microphylls) are much reduced and borne in whorls at the nodes. Observe

the representative species of horsetails available in the lab.

DIVISION: Psilophyta (whisk ferns)

Psilotum nudum is a living representative of plants that are abundant in the fossil record. These simple

plants interest botanists because they illustrate a basic structural pattern of branches from which leaves may

have evolved. Psilotum consists of a rhizome (an underground stem) with rhizoids (rootlike filaments)

plus leafless aerial stems that branch dichotomously (into two equal parts).

If available, observe the small whisk fern Psilotum.


5

DIVISION: Pterophyta (also called Pteridophyta, ferns)

During the Carboniferous period of the Paleozoic era (about 300 million years ago) the ancestors of these

plants were tree sized and formed great forests. The remains of these carboniferous forests contributed to

the coal deposits being mined for fuel today. Ferns are more closely related to the seed plants than to the

more primitive whisk ferns, club mosses, and horsetails.

Examine each of the variety of fern species available in the laboratory. Note the horizontal stem (rhizome),

and the large, pinnately compound leaves (also called fronds).

Break off a piece of fern leaf to examine under the microscope. Are stomata in the upper or lower leaf

epidermis? Why do you think this is?

_____________________________________________________________________________________

_____________________________________________________________________________________

Describe the shape(s) of fern leaf epidermal cells. ____________________________________________

______________________________________________________________________________________

What is the structural advantage in the shape(s) you observe?

______________________________________________________________________________________

THE GYMNOSPERMS

Conifers are the most numerous and widely distributed of the gymnosperms. The tallest plants (the coastal

redwoods of California and Oregon) and plants with the longest life spans (Bristlecone pines of the Sierra

Nevada, known to survive for several thousand years) are conifers. Familiar representatives in the eastern

U.S. include pines, spruces, cedars, firs, hemlocks, junipers and cypresses. All are perennial trees with

woody stems. With few exceptions, they retain their leaves for two or more years and are therefore

evergreen. In this exercise we will study the anatomy and morphology of conifers from the Goucher

campus.


6

1. Leaf arrangement

a. Look at specimens of pine branches that have been brought into the laboratory. Note

the needle-like, photosynthetic leaves borne on short shoots. How many needles are

clustered on each short shoot?

_____________________________________________________________________

b. If several species of pine are available, examine them and record the number of leaves

in a cluster (fascicle). Does this number vary between species? Also, note the length

and other obvious features of these leaves. Such characteristics are used to distinguish

one species from others and thus are taxonomically important.

_____________________________________________________________________

2. Leaf structure

3.

a. With your microscope, examine the cross section of a pine needle under low power and

then at higher magnification.

1) Note that the outer surfaces of the epidermal cells are covered with a heavy

water-proof layer of cutin and that there is a layer of thick-walled

sclerenchyma cells just beneath the epidermis.

2) Find the depressions in the epidermis. Stomata are located in these protected,

sunken chambers beneath the leaf surface. Note that rows of stomata often

appear as visible white lines on the leaves of conifers such as white pine and

hemlock.

b. How would the location of stomates in these leaves be a useful adaptation for surviving

such periods of drought? (Remember that pines are evergreen, retaining their leaves

through the winter months when ground water may be frozen and therefore not

available for absorption by the roots.) _______________________________________

______________________________________________________________________


7

c. Locate the central cylinder of vascular tissue surrounded by the endodermis (a layer of

water-proof cells also found around the conducting cells of roots). Photosynthetic cells

(mesophyll) are located between the surface layers and the central core of vascular

tissue.

Other Gymnosperms:

Observe the living cycad and ginkgo specimens. Neither of these plants look like a “typical” gymnosperm

(for example, a pine). Why do you think these plants are classified as gymnosperms? (Hint: think about the

primary criterion for gymnosperm classification!)

______________________________________________________________________________________

______________________________________________________________________________________

VEGETATIVE STRUCTURES OF FLOWERING PLANTS

The two groups of angiosperms (flowering plants), monocots and dicots, differ in the anatomy of their

leaves, stems and roots, in the structural patterns of their flowers and seeds, and in some aspects of their

growth.

In this exercise you will look at the structure of leaves, stems and roots of representative flowering plants.

1. Leaf arrangement and morphology - Examine the leaves of young bean plants, geranium, zebrina

and other species available in the laboratory, and locate the structures and examples outlined below.

Leaves are appendages of the shoot, the above ground part of a plant. Appropriate to their primary

role in photosynthesis, leaves generally have:

a. an expanded and flattened portion (the lamina or blade) for capturing sunlight.


8

b. The blade usually is attached to the stem by a narrow, stalk-like petiole.

c. The place on a stem where a leaf (or leaves) is (are) attached is called a node and portions of

stem between leaves are called internodes.

d. Leaves are arranged on stems in several possible ways (note that these are important

characteristics in plant identification keys):

1. spiral or alternate pattern: one leaf per node

2. opposite pattern: two leaves per node

3. whorled: three or more leaves per node

e. Tiny strands of vascular tissue (veins) connect all parts of the blade to conducting cells (xylem

and phloem) in the stem. The arrangement of veins in the leaf (venation) is parallel in the

monocots (grasses and their relatives) and netted in dicots (broad-leaved flowering plants).

f. Leaves may be either simple (single blade) or compound (several to many leaflets per leaf).

This is the only way to determine whether you are looking at a branch or a compound leaf:

there is only one bud per leaf, and therefore no buds are associated with the leaflets of a

compound leaf.

1. Pinnately compound leaves have leaflets arranged on opposite sides of a common axis.

2. In palmately compound leaves, all leaflets are attached at the same point at the end of

the petiole. In the axil (angle between the base of the petiole and the stem) there is a

bud.

2. Leaf epidermis and internal anatomy

Obtain a leaf from a bean or geranium plant. Make a ragged tear in the leaf, remove a small piece from the

torn lower edge that appears colorless (lower epidermis) and mount it in a drop of water on a slide. Observe

the isolated leaf epidermis under low and high power.


9

Note that pairs of bean shaped guard cells are scattered throughout the lower epidermis. They constitute the

stomatal apparatus and changes in turgor of the guard cells result in changes in the size of the opening

(stoma) between their inner surfaces. What role do the stomata play in controlling:

1. The rate of photosynthesis? ______________________________________________

2. The amount of water lost from leaves? _____________________________________

3. The amount of water moving up a stem? ____________________________________

4. What cellular organelle present in the guard cells is not found in other cells of the

epidermis? Postulate as to why this is.

_____________________________________________________________________

Examine the cross section of a Syringa (lilac) leaf.

1. With the aid of Figure 9.1, locate the following: upper and lower epidermis, stomata, cuticle,

palisade and spongy parenchyma of the leaf mesophyll, intercellular spaces and veins. Can you

recognize xylem and phloem cells in the veins? _____________________________________

2. Compare the internal structure of the lilac (Syringa) leaf (a mesophyte) to that of leaves from a

variety of plants adapted to specific environments. Record features of the adaptations below:

a. Leaves specially adapted to dry environments (e.g.; Nerium, a xerophyte)

_____________________________________________________________________

b. Leaves from wet habitats (e.g. the water lily Castalia, a hydrophyte; and Elodea, a

submerged hydrophyte) (prepared slides, cross sections, demonstration microscopes).

_____________________________________________________________________


10

c. What structural differences do you see and how do these modifications relate to the

specific habitats of each species?

_____________________________________________________________________

_____________________________________________________________________

Figure 9.1: A stereoscopic view of a portion of a typical leaf.

Stems: organs of transport, support and storage:

Examine a prepared slide of Coleus stem tip (longitudinal section, demonstration microscope). Use

Figure 9.2 to help you locate the apical meristem, a region of dividing cells which gives rise to stem

tissues and, at periodic intervals, to leaves (which develop from leaf primordia). At the base of each

leaf, dormant apical meristems are formed. These are called lateral (or axillary) buds and are the

feature that defines true leaves (megaphylls) in the ferns and all higher plant groups. Eventually, further

down the stem, lateral buds are released from dormancy and become lateral branches. The regions of

the stem at which leaves develop are called nodes. Regions of the stem between the nodes are called

internodes.


11

Figure 9.2. A longitudinal section through the tip of a Coleus stem.

3. Examine the prepared slides (cross sections, demonstration microscopes) of stems of the

following plants:

a. Medicago sativa (alfalfa)

b. Helianthus annicus (sunflower)

c. Tilia (linden or bass wood), all dicots, and

d. Zea mays (corn), a monocot.

How do patterns of distribution of vascular bundles differ in these plants? ______________

___________________________________________________________________________


12

Roots: organs of absorption, transport, anchorage and storage:

Roots are the part of a plant axis below the soil surface. They may be classified by form as either tap roots

or fibrous roots.

1. Examine the tap root of a carrot. Note that the main root is many times larger in diameter than

the tiny branch (or lateral) roots. A tap root may be greater in diameter than the stem. Also

examine the fibrous root system of a bean plant.

a. Would fibrous roots or tap roots be more efficient in absorbing water and minerals

from the soil? Why?

_____________________________________________________________________

_____________________________________________________________________

_____________________________________________________________________

b. Which type of root is more obviously adapted for storing nutrients?

_____________________________________________________________________

2. Obtain a young radish or grass seedling. Place the root in water (to prevent drying out) and

examine it under a dissecting microscope. Locate the following structures: root cap, root

hairs. In what way do root hairs aid in the absorption of water and minerals by the root?

___________________________________________________________________________

3. Examine the demonstration slides of developing lateral roots in young willow (Salix) roots.

Branch roots originate from the pericycle, a layer of cells that retain the capacity to become

meristematic. When a lateral root is formed, a new meristem complete with root cap develops

from dividing pericycle cells. Eventually the young branch root pushes outward, puncturing the

cortex and epidermis. Be sure you understand the differences in origin of lateral roots and

the lateral branches of the shoot.


13

What is the most striking difference between the internal structures of roots and shoots? Hint:

what cell (tissue) type is found at the center?

___________________________________________________________________________

___________________________________________________________________________

Complete Table 9.1, which summarizes general information about vegetative (non-reproductive)

structures, their functions in various plant groups, and how they have enabled these organisms to adapt to

terrestrial environments.

STUDY GUIDE:


Recognize monocot and dicot stem types.

Recognize and understand various adaptations of leaves for the environment.

Recognize and understand how stomata work.

Recognize spiral, opposite and whorled leaf patterns.

Identify xylem, phloem, mesophyll and epidermal layers in a leaf cross-section.

Know the role of the pericycle in roots, and the role of the apical meristem in shoots.

Identify representative vascular and non-vascular plants.

REFERENCES:

Starr et al. (2009), Ch. 23.2-23.4; 23.6-23.8; 29.


14

TABLE 9.1 – SUMMARY TABLE FOR BRYOPHYTES, PRIMITIVE VASCULAR PLANTS, GYMNOSPERMS AND ANGIOSPERMS

Water/nutrient uptake and transport (include structures)

Gas exchange (include structures & locations)

Specific adaptations to environment (include leaf, stem, root adaptations)

Bryophytes (example: moss)

Primitive Vascular Plant (example: fern)

Gymnosperm (example: pine)

Angiosperm (example:

cherry tree)


15

USE OF A KEY FOR IDENTIFICATION OF GYMNOSPERMS OF THE GOUCHER CAMPUS Correct identification of organisms is an essential first step in any type of investigation. The most frequently employed identification aid is the key, a standard tool for identifying organisms - from Monera to Vertebrates. The experience and skill that you acquire in identifying gymnosperms today will prove useful when you identify (or "key out") virtually any organism. The more familiar you are with any given key and the terminology it employs, the easier it will be to arrive at an identification. A key consists of a series of paired statements. You decide which of the pair applies to your specimen and the key will direct you to another pair of statements. With each choice you progressively eliminate a number of possible species until only one possibility remains. A variety of gymnosperm specimens are on the Goucher campus. You will use the map of the campus given to you in class to locate some of these specimens (your instructor or TA will serve as a guide). Use the key provided to identify four or more unknown specimens including at least one with "scale-like" leaves, one with long (>3 cm) leaves, one with short (<3cm) leaves, and one with broad flat leaves. As you go through the identification process, try to get a feeling for the major types of gymnosperms and for the diversity of form that they exhibit. (In the event of inclement weather, samples of the gymnosperms will be available in lab.) It will be more efficient if you work in pairs with one person reading the choices while the other makes the decisions. As you proceed through the key, fill out Table 9.2 recording the number and characteristic of each choice selected. This will allow you to retrace your path through the key should you go astray.

Helpful Hints for the Use of Keys

1. Examine the specimen in a general way, noting such characteristics as arrangement, shape,

venation, margins and surface of leaves, cone size and shape, etc. Read the labels - in some cases they provide useful information such as whether the sample came from a tree or a bush.

2. Always begin to use a key at the first pair of statements. Avoid the temptation of jumping to the

middle of a familiar key - this often leads to an incorrect answer. 3. Read BOTH alternatives before selecting one. 4. If you encounter any unfamiliar terms, look them up in the glossary of your textbook, in a

dictionary, or in the references available in the laboratory. 5. If the size of a structure is needed, measure carefully and measure several samples (remember, there

are variations in any species). 6. If it is not clear which of a pair of alternatives is correct, select the one that seems to best apply to

the unknown specimen. If it is impossible to decide, arbitrarily select one and follow it through several additional steps in the key. If this is done for both alternative choices, it usually will become obvious which of the two is the correct choice.

7. Once a tentative identification is made with a key, check the identification with a detailed

description, illustration or photograph in one of the reference books available in the laboratory.


16

KEY TO GYMNOSPERMS OF THE GOUCHER CAMPUS Use of this key. During the summer it may be difficult to distinguish between evergreens and deciduous trees and shrubs. This is usually evident from the darker year-old leaves below the new growth. During the winter, and through April the evergreens stand out more clearly. A key consists of sets of alternatives, and when the proper choice is made, one choice leads to the next alternative, until a name is arrived at. Thus, for example, "White pine" (Pinus strobus) is found by choosing: 1a, 2a, 3a, and 25a. One number guides the reader to the next. Much depends upon careful reading of the alternatives, and careful examination of the plant! 1. a. Leaves needle shaped, narrow with parallel sides or minute and scale- like, pressed against the branchlets and seemingly part of them. Leaves never hairy. CONIFERS, see ................................................... 2 1. b. Leaves broader, with a flat or rolled-up leaf blade, either hairy or glabrous (smooth) ................................................................................................... 38 2. a. Leaves needle-like ................................................................................................................... 3 2. b. Leaves minute, scale-like....................................................................................................... 34 3. a. Needles in fascicles (clusters) of 2 to 5 along the branches. Genus Pinus, see ................................................ 25 3. b. Needles attached individually to the branches ........................................................................ 4 4. a. Needles flat, often appearing in 2 rows along horizontal branches ................................................................................................................................... 5 4. b. Needles not flat, but round, or square or triangular in cross section, pointing in all directions........................................................................................... 15 5. a. Needles longer than 3 cm., gradually tapering to a long point Cunninghamia lanceolata "CHINA FIR", (S.W. China) 5. b. Needles shorter, coming to abrupt point or blunt end............................................................. 6 6. a. Underside of the needles with a yellowish green cast. Midrib elevated. Needles tapering to a sharp point................................................................ 7 6. b. Underside of the needles with a whitish cast. Midrib grooved on top. Tip of needle blunt ....................................................................................... 8 6. c. Leaves pointed at tips, arranged on slender, deciduous branchlets. Cones globular (spherical) Taxodium distichum "BALD CYPRESS", (S.E.U.S.)


17

7. a. Underside of needle greenish yellow. Needles pointing sideways and upwards. Shrubs Taxus cuspidata "JAPANESE YEW" (with many horticultural varieties, and closely allied species) 7. b. Underside of needle yellowish green. Needles more clearly in two rows. Shrubs or trees Taxus baccata "ENGLISH YEW" (with many horticultural varieties, and closely allied species) 8. a. Leaves not elevated on stalks. Leaf scars on branchlets circular (use hand lens). Comes pointing straight up ............................................................. 9 8. b. Leaves with narrow stalks. Leaf bases slightly elevated. Cones pendulous (hanging down) or various....................................................................... 11 9. a. Needles whitish on both sides. Abies concolor "WHITE FIR" (W. U.S.A.) 9. b. Needles glossy green on top, white underneath .................................................................... 10 10. a. Buds resinous Abies balsamea "BALSAM FIR" (Canada, E. U.S.) 10. b. Buds not resinous Abies alba "SILVER FIR" (C. Europe) NOTE: There are many other firs, some of which are in the Baltimore area. 11. a. Needles pointing in all directions, whitish green. Cones with long three-clefted bracts between scales. Pseudotsuga taxifolia "DOUGLAS FIR" (W. U.S.A.) 11. b. Leaves mostly pointing sideways, glossy green above......................................................... 12 12. a. Needles with finely toothed margin (use hand lens!) Tsuga canadensis "EASTERN HEMLOCK" (E. N.Am.) 12. b. Needles with smooth margin ................................................................................................. 13 13. a. Needles more than 2 mm. wide, with indistinct white bands on underside. Cone scales round. Tsuga chinensis "CHINESE HEMLOCK" (W. China) 13. b. Needles 2 mm. wide or less, with distinct bands of white stomata underneath ................................................................................................................ 14


18

14. a. Branchlets slightly hairy (use hand lens!) Tsuga caroliniana "CAROLINA HEMLOCK" (Smoky Mts.) 14. b. Branchlets glabrous (smooth) Tsuga sieboldii "SIEBOLD HEMLOCK" (Japan) 15. a. Some of the needles in clusters on short spur-like branches. Cedrus atlantica "ATLAS CEDAR" (N. Africa) 15. b. Needles all along regular shoots............................................................................................ 16 16. a. Needles decurrent, i.e., merging with the branch................................................................ 17 16. b Needles on little stalks, raised above the branch (Use microscope or hand lens!) genus Picea, see ................................................. 21 17. a. Needles spirally arranged, blunt. Cone with toothed scales. Cryptomeria japonica "CRYPTOMERIA" (Jap., China) 17. b. Needles opposite or in whorls of 3. Cones berry-like.......................................................... 18 18. a. Trees of conical shape, usually with both needles and scale-like leaves ..................................................................................................................... 19 18. b. Shrubs or subshrubs ............................................................................................................... 20 19. a. Needles with whitish marks underneath, curved................................................................... 35 19. b. Needles greenish, or with whitish marks above only. Needles straight, with transitions to the scale type usually present on the younger branches. Common tree. Juniperus virginiana "RED CEDAR" (E. U.S.A.) 20. a. Needles about 1 cm. long. Bush 1-2 m. tall Juniperus communis "JUNIPER" (E. U.S.A. Eur.) 20. b. Needles at most 5 mm. long. Flat creeping plant Juniperus horizontalis "CREEPING JUNIPER" (E. U.S.A.) 21. a. Needles less than 1 cm. long. Young branches with short hairs between needles (lens!) Picea mariana "BLACK SPRUCE" (N. U.S., Can.) 21. b. Needles longer than 1 cm ...................................................................................................... 22


19

22. a. Needles blue, very sharp, arising almost at right angles to the branches. Picea pungens "BLUE SPRUCE" (Rocky Mts.) 22. b. Needles green or bluish green, pointing forward .................................................................. 23 23. a. Main branches curving upward, lateral branches drooping. Cones longer than 10 cm. Picea abies "NORWAY SPRUCE" (Europe) 23. b. Lateral branches stiff, not drooping....................................................................................... 24 24. a. Branches yellowish to orange, needles green. Buds at the base with several sharply pointed scales. Picea rubens "RED SPRUCE" (E. U.S., Can.) 24. b. Branches dull brownish. Needles glaucous (waxy). Buds without pointed scales. Picea glauca "WHITE SPRUCE" (N. U.S., Can.) 25. a. (Key to the Pines) Needles in clusters of 5 Pinus strobus "WHITE PINE" (E. N. Am.) 25. b. Needles in clusters of 2 - 3..................................................................................................... 26 26. a. Needles in clusters of 3 ......................................................................................................... 27 26. b. Needles in clusters of 2 .......................................................................................................... 28 27. a. Needles more than 15 cm. long Pinus taeda "LOBLOLLY PINE" (SE U.S.A.) 27. b. Needles shorter, stiff Pinus rigida "PITCH PINE" (E. U.S.) 28. a. Young branches purplish brown often with bluish cast…………………………………..29 28. b. Young branches light brown to gray without bluish or purplish cast ....................................................................................................................... 30 29. a. Needles occasionally in clusters of 3, less than 1 mm. thick, but needles can be 7.5cm or longer. Pinus echinata "SHORT-LEAF PINE" (E. U.S.) 29. b. Needles always in 2's (rarely 3's), 1 mm. thick or more. Most common pine on campus. Pinus virginiana "SCRUB PINE" (E. U.S.) 30. a. Needles more than 7 cm. long ............................................................................................... 31 30. b. Needles less than 7 cm. long.................................................................................................. 32


20

31. a. Needles snapping when bent. Pinus resinosa "RED PINE" (E. N. Am.) 31. b. Needles folding when bent. Pinus nigra "AUSTRIAN PINE" (C. Europe) 32. a. Shrubby plant, with many stems from the ground up. Needles dull green. Pinus mugo "MOUNTAIN PINE" (C. Europe) 32. b. Trees with single trunk .......................................................................................................... 33 33. a. Needles bluish green. Bark on older trees orange in Upper part of tree. Cones knobby. Pinus sylvestris "SCOTS PINE" (N. Europe) 33. b. Needles green. Bark brown. Cones with thick spines on cone scales. Pinus pungens "TABLE MOUNTAIN PINE" (E. U.S.) 34. a. Branchlets flattened, arranged in one plane .......................................................................... 35 34. b. Branchlets square in cross section, branched in all directions.............................................. 37 35. a. Underside of leaves green...................................................................................................... 36 35. b. Underside with white marks. Cone round with angular scales. Chamaecyparis obtusa "HINOKI CYPRESS" (Japan) 36. a. Cones with elongate woody scales. Thuja occidentalis "ARBOR VITAE: (E. N. Am.) 36. b. Cones rounded, with curved point on each scale. Chamaesyparis thyoides "WHITE CEDAR" (coastal U.S.) 37. a. Conical tree. Cones berry-like. Juniperus virginiana "EASTERN RED CEDAR" see 19b 37. b. Flat bush Juniperus horizontalis CREEPING JUNIPER" see 20b 38. a. Leaves distinctly fan shaped Ginkgo biloba "MAIDEN HAIR TREE" (China) 38. b. Leaves otherwise ANGIOSPERMS


21

Name: ___________________________ Section: __________________________ TABLE 9.2 – SUMMARY OF GYMNOSPERM IDENTIFICATION For each specimen identified, record the number of each choice in the key and the characteristic you selected. Place final identification at the bottom of the box. Example: 1. needlelike leaves 2. needles in fascicle, etc.

Specimen # Genus and Species: Common Name:





22

1

Exercise 10:

Plant Reproduction BACKGROUND:

Plants can reproduce either asexually (by vegetative means) or sexually. Most of us have participated in

the vegetative propagation of some of our favorite plants by making cuttings of houseplants or dividing

clumps of garden perennials. Many plants reproduce in nature exclusively by vegetative means.

Horticulturalists utilize asexual modes of replication (cloning) to produce new individuals identical to the

original plants so desirable strains can be maintained without variation. Some seedless fruit varieties

such as the navel orange must be propagated asexually because they are sterile.

The more prevalent mode of creating new plants in nature is by sexual reproduction. This process

insures variability by the recombination of parental traits and generally results in the production of large

numbers of offspring. Both of these characteristics of sexual reproduction favor evolutionary change.

Even the most primitive plants have methods of sexual reproduction. An examination of the sexual

reproduction of primitive to more advanced plants illustrates some of the evolutionary steps necessary to

progress from single-celled algae to modern seed plants. For all of the plant groups we will examine

today, note that the complete sexual cycle involves the following:

1. The formation of special cells that divide by meiosis and produce either spores (primitive

plant groups) or gametes (advanced plant groups).

2. In primitive plant groups, there is development of haploid, multicellular, male and female

gametophyte structures from spores. Gamete formation in primitive plants is via

differentiation of gametophyte tissues. In higher plants, there is a reduction of the

gametophyte phase to a single cell (an egg, or sperm carried by pollen).

3. Fertilization (which restores the diploid number of chromosomes).

4. Development of the zygote to form a multicellular sporophyte plant (or structure). At sexual

maturity, meiosis occurs in the sporophyte, beginning the cycle anew.

Exercise 10: Plant Reproduction

2

The plants you will examine today exhibit a phenomenon called alternation of generations, which is the

occurrence of separate haploid and diploid stages in their life cycles. A general evolutionary pattern in

the plant kingdom is for the diploid (spore-producing or sporophyte) stage of the life cycle to become

increasingly larger and more dominant relative to the haploid (gamete-producing or gametophyte) stage.

Figure 10.1 Polytrichum life cycle.


3

EXERCISES:

DIVISION: BRYOPHYTA (Mosses, liverworts and hornworts)

Mosses will be used today as an example of a more primitive plant (specifically, the moss Polytrichum).

Recall that mosses grow in damp, moist areas. They must, because they rely on water for their sexual

reproduction. Unlike the seed plants you are familiar with, mosses have male gametes that must SWIM

to the female gametes for fertilization to take place. Mosses are also among those plants with a dominant

gametophyte stage – that is, the green plant you commonly think of as “moss” is a haploid plant. (Before

you examine the reproductive structures of the moss you may find it helpful to review the general

structure of the plant.)

Polytrichum reproductive structures:

Polytrichum has reproductive structures located at the tops of separate male and female haploid,

gametophyte plants. The male reproductive structure is the antheridium, and the female the

archegonium.

1. Look at the representative male and female plants.

2. Male gametes (sperm) released from the antheridia of male plants swim to the egg in the

archegonium of a female plant, where fertilization takes place.

3. Think about the environment in which you find mosses. How is this environment essential to

sexual reproduction?

______________________________________________________________________

4. How does the DENSITY of moss growth affect sexual reproduction?

______________________________________________________________________


4

5. The sperm and egg fuse together to form the diploid zygote. This zygote develops into the

brown sporophyte (a stalk with a capsule) attached to the top of the green gametophyte. Look

at the demonstration sporophyte growing from the female gametophyte in the dissection scope.

6. Examine the capsule of the sporophyte more closely. The remains of the archegonium may be

retained as the calyptra (cap) that encloses the spore capsule at the tip of the sporophyte.

Observe the exposed top surface of the capsule with a hand lens or dissecting microscope.

7. Recall that this sporophyte is diploid (2N). Examine an opened sporophyte capsule and locate

the spores inside.

Are these spores haploid (1N) or diploid (2N)?

_____________________________________________________________________

What process is involved in spore formation?

______________________________________________________________________

8. Think about the brown sporophyte structure and the release of the spores inside. Answer the

following questions:

Why do you think the sporophyte grows taller than the rest of the plant?

______________________________________________________________________

Why do you think the capsule has the calyptra (cap)?

______________________________________________________________________


5

Figure 10.2 Fern life cycle.


6

PRIMITIVE VASCULAR PLANTS (Tracheophytes)

DIVISION: Pterophyta (also called Pteridophyta, ferns)

Living plants of one representative division of primitive vascular plants are available in the laboratory: the

ferns. Remember that a general evolutionary trend in the plant kingdom is for increasing dominance of the

sporophyte phase in the life cycle. Unlike bryophytes, the plant you typically think of as a fern is a

sporophyte (diploid) plant. However, like bryophytes, ferns rely on motile sperm for sexual reproduction

and also rely on spores to spread their gametes. There are several stages of the fern life cycle to examine,

each representing an important stage in the eventual development of a sporophyte (diploid) fern. For each,

note whether the plant you are observing is a gametophyte or sporophyte. The sporophyte stage of the life

cycle dominates in the ferns, but the small gametophyte usually is a nutritionally independent

(photosynthetic) and separate plant.

Fern sporophyte:

Look at the under surfaces of the leaves and locate small yellow to brown spots on some of them. These

sori contain many sporangia and the leaves on which they are borne are called sporophylls (spore-bearing

leaves). The spores are the products of meiosis in the fern, and are haploid. The spores fall from the

underside of the leaves and onto the ground, where they germinate.

Fern gametophyte:

On germination, fern spores develop into a haploid filament of cells that differentiates to form a heart-

shaped gametophyte. The mature gametophyte has both male and female reproductive structures.

Examine a mature gametophyte under a dissecting microscope.

1. On the lower surface locate rhizoids. Among the rhizoids look for male antheridia. This is

where sperm develop.

2. Locate female archegonia, also on the lower surface near the notch in the heart-shaped plants.

Only the necks of the archegonia are visible. Sperm from the antheridia swim to the egg in the

archegonium.


7

Young sporophyte:

Following fertilization, the embryonic sporophyte (diploid) develops within the archegonium, just like in

mosses. It forms four lobes. One develops into the "foot", a second grows into the soil and becomes a

“root” (actually, a root-like shoot, or rhizome), a third develops into a leaf and the fourth becomes a stem.

When the first roots and leaves become functional, the young sporophyte is nutritionally independent of the

gametophyte, which then dies.

Examine the young sporophyte stage of the fern, and identify the gametophyte and sporophyte parts of the

plant. This will grow into what you commonly recognize as a fern.

GYMNOSPERMS

As you learned earlier in this exercise, primitive plants such as mosses and ferns have motile sperm. As a

result, they require water to be present for sexual reproduction to take place. Most gymnosperms represent

a real advance in plant reproduction; they have pollen instead of motile sperm, and water is not required for

the male gametophyte (pollen) to reach the female gametophyte (egg). Instead, most gymnosperms rely on

wind to disperse the pollen.

As in the ferns, the “pine tree” you are familiar with is a diploid plant, and the cones are the sexual

reproductive structures. Many gymnosperms produce both male and female cones on the same tree, with

the male cones located on lower branches and the female cones on higher ones. Meiosis occurs in both the

male and female cones, producing a haploid egg (in the case of the female cone) or pollen grains (in the

case of a male cone). Fusion of the egg and sperm (from the pollen) produce a diploid zygote, which

develops within a seed inside of the female cone. The seed is relatively exposed, developing on the surface

of the scale in female cones, thus gymnosperms are designated as “naked” seed plants. Protection of the

developing embryo within the seed represents another evolutionary advance within this group of plants.

Pollination and fertilization:

Overview of pollination process:

1. Pollen grains are released from the male cones in late spring (usually toward the end of May).

Massive amounts of pollen are carried by air currents. The pollen is somewhat sticky and


8

attaches to the female cones. At about the same time, the young female (seed) cones open and

pollen shifts down into the pollen chamber. Examine male and female pinecones and

gymnosperm pollen grains available in the laboratory. Look at the demonstration slide of pine

pollen, and notice the airy “wings” that aid in dispersal.

2. Pollen grains germinate on the female cones and each produces a pollen tube (containing

sperm nuclei) that digests its way toward the egg nucleus. These events in the pine life cycle

unfold very slowly and fertilization may not occur until over a year after pollination. Seeds are

not shed from the female cones until two years after these cones begin to form.

a. Look at a branch from a scrub pine (found on campus!) See if you can locate three

years of pinecone development on the branch. What differences do you see in the

scales of the immature and mature pinecones?

_______________________________________________________________

b. Examine the slide of a cross-section of a seed-bearing cone, and locate an ovule.

These ovules may mature into a seed if fertilized. Note that the eventual seed will be

exposed and will NOT be surrounded by sporophyte tissue (i.e., “naked” seed).

3. The scales of seed cones open for a second time to shed seeds. In some species such as jack

pines, scales open to release seeds only after being subjected to extreme heat as in forest fires.

After a fire, seeds that have accumulated over many years are released so that the species is re-

established in the burned area. In most seed cones, scales open in response to changes in

moisture content.

Observe the demonstration of soaked pinecones. (These cones have already shed their seeds).

What happened to the cone when soaked? ____________________________________

In the adjoining bin are cones that were soaked several hours ago then removed from the

water. How did they respond to drying?__________________________________


9

Figure 10.3 Pine life cycle.


10

Figure 10.4 Angiosperm life cycle.


11

ANGIOSPERMS

Angiosperms are the flowering seed plants. The plant embryos in angiosperm seeds are protected with a

seed coat and provided with nutritive tissues (endosperm) that feed the embryo through the early stages

of germination. Like gymnosperms, male gametes are spread using pollen; subsequent fertilization then

leads to seed formation. Unlike gymnosperms, the seeds are not exposed (“naked”), but are enclosed

within sporophyte tissues which develop into a fruit.

To understand sexual reproduction in the angiosperms it is necessary to be aware of the structure of

flowers (where the essential steps take place) and to understand the role of each part of a flower in the

total process.

The complete sexual cycle in angiosperms involves:

1. The formation of special cells that divide by meiosis.

2. Development of haploid male and female gametophyte structures within the

flower (each reduced to a single cell)

3. Pollination

4. Fertilization (which restores the diploid number of chromosomes in the

zygote).

5. Development of the seed (which contains the embryo that develops from the

zygote) and fruit (which develops from ovary and “accessory” tissues).

6. Seed and fruit dissemination from the parent plant.

7. Seed germination and growth of a new sporophyte plant.

Parts of a “complete” flower:

Flowers exhibit great variation in form, size, color of petals, number and arrangement of parts, symmetry

and the ways that they are borne on the plant (whether singly or in clusters). A flower is considered to be

a compressed shoot (i.e., an axis with leaf-like appendages). Identify the following parts of the

complete flower illustrated in Fig 10.5:


12

1. Sepals: The most leaf-like part and the outermost appendages of the flower, typically green;

collectively referred to as the calyx.

2. Petals: Internal to the sepals, may be white or colored; collectively call the corolla.

3. Stamens: Internal to the petals; the male parts of the flower; consist of a stalk (filament)

that supports the terminal anther in which pollen grains are formed.

4. Pistils (carpels): Female parts found at the center of the flower. They consist of three parts:

an enlarged basal ovary (containing one or more ovules, a slender stalk (the style) and a

somewhat enlarged tip (the stigma).

Figure 10.5 Flower structure


13

Flower dissection:

Several kinds of flowers representing both monocot and dicot species, will be available in the laboratory.

Be sure to note the name of each plant you examine and whether it is a monocot or a dicot (take a careful

look at and dissect at least one of each). Floral parts in monocot flowers are present in 3's or

multiples of 3 while dicot flower parts occur in 4's or 5's.

Examine the representative flowers with your lab partner. For each kind of flower you examine, note in

Table 10.1:

1. whether all floral parts are present (whether it is complete or incomplete) and the number of

each,

2. whether floral parts are attached below, form a tube around, or are attached above the ovary,

and

3. the symmetry of the flower (bilateral or radial).

Table 10.1 Representative monocot and dicot flowers

Flower name: Monocot /

Dicot

Complete /

Incomplete

Multiples of petals,

stamens and pistils

Symmetry of

flower


14

DEVELOPMENT OF MALE GAMETOPHYTES IN ANGIOSPERMS:

POLLEN TUBE GROWTH

Anthers contain cells that undergo meiosis, producing haploid cells that eventually develop into gamete-

(sperm-) producing gametophytes called pollen grains.

1. Remove an anther from one of the flowers you have examined, crush it in a drop of water on

a microscope slide, add a cover slip and examine it under high power. Depending on the

stage, you may find cells undergoing meiosis, tetrads (groups of four) of haploid

microspores or developing pollen grains. If a yellow, powdery substance is present on the

anthers, this is mature pollen.

2. Look at the demonstration slide of pollen tube growth.

FRUITS

A fruit is a ripened ovary plus any other associated flower parts. There are three kinds of fruits based on

the number of ovaries and flowers involved in their development:

1. Simple fruits are derived from a single ovary, which may have one or a number of

carpels.

2. Aggregate fruits are derived from a number of ovaries belonging to a single flower.

3. Multiple fruits are formed from a number of ovaries from different flowers that

grow together.

Some simple fruits have a fleshy pericarp, (derived from the ovary wall), others have a dry pericarp. Dry

walls may split at maturity allowing the seeds to escape (dehiscent) or they may not split (indehiscent).


15

A variety of fruits are available in the laboratory. Representatives of most of the types listed on Table

10.2 are on display, and those that are edible may be tasted as well as examined as botanical specimens.

1. Locate and note (on Table 10.2) the common name of an example of as many of the

listed types of fruit as you can find. The Bioreview sheet on fruits, photographs and

descriptions of fruits in your text, and display cards next to sample fruits will be

helpful in identifying each fruit type.

2. A number of simple fruits (derived from a single ovary) have multiple seeds. How

is this related to the structure of the ovary in the pistil of the flower from which the

fruit developed? (HINT: Examine an orange)

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

3. Examine the fruits of a tomato and an orange that have been cut in half. Both are

simple fruits with multiple seeds. How many carpels were united to form the ovary

of each of these fruits?

_________________________________________________________________

4. Feel free to munch as you observe, dissect and classify the fruits on display. But

please don't consume the last example of each kind on display until all students in

the lab have had an opportunity to complete this exercise. (Yum!) You may not be

able to fill in ALL the blanks in the chart below – due to the availability of ripe fruit,

we may not have on display an example of each variety listed


16

Table 10.2 Fruits on display

accessory fruit

achene

berry

capsule

compound: aggregate

compound: multiple

drupe

hespiridium

legume (pod)

nut

pome

samara

silique

pepo

Seed structure of monocots and dicots:

Use a dissecting microscope to examine the following kinds of seeds. Figures 10.6 and 10.7 should

help you locate the external and the internal structures of the seeds.

1. Garden bean (Phaseolus vulgaris), a dicot:

a. Examine the external structure of an intact, soaked seed. Find the prominent hilum

and micropyle. The hilum is a scar left from the stalk attaching the ovule to the


17

ovary wall. The micropyle is an opening in the integuments of the ovule through

which the pollen tube entered.

b. Remove the seed coat and gently split the seed open. Find the following parts:

Cotyledon – an embryo or “seed leaf” that stores or absorbs food

Embryo – the immature sporophyte

Radicle – this part is destined to develop into the root

Figure 10.6 Garden bean seed and seedling structure (dicot plant)

2. Corn (Zea mays), a monocot: The corn kernel is really a fruit which developed from

an ovary.

a. Examine a soaked corn grain. The thin, transparent wall (pericarp or seed coat) is

made up of the fused seed coat and fruit coat or ovary wall. The cotyledon is the

large shield shaped structure visible through the wall.

b. Cut the kernel in half lengthwise to reveal its internal structures. In corn there is

only one cotyledon that functions in absorbing stored food from the endosperm.

Both the embryonic shoot is covered with a sheath called the coleoptile.


18

Figure 10.7 Corn seed and seedling structure (monocot plant)

STUDY GUIDE:

Complete Table 10.3, reviewing the evolutionary trends in plant reproduction.

You should also be able to:

1. Describe reproduction in bryophytes and identify sporophyte and gametophyte stages,

2. Describe reproduction in ferns and identify sporophyte and gametophyte stages,

3. Describe reproduction in gymnosperms,

4. Determine whether a germinating seed, plant or flower is a monocot or dicot,

5. Recognize the parts of a flower,

6. Identify a simple, aggregate and multiple fruit,

7. Explain spore formation, fertilization and seed development in angiosperms,

8. Recognize the parts of a seed (in bold type) and their functions.

REFERENCES: Bio Review Sheets on Fruit Types, and Seed and Seedling

Starr et al. (2009) Ch. 23; 31.1-31.5


19

Table 10.3 A comparison of plant reproduction methods BRYOPHYTES

(MOSS) PRIMITIVE VASCULAR

(FERNS)

GYMNOSPERMS (PINE)

ANGIOSPERMS (BEAN)

Is the plant sporophyte or gametophyte dominant?

Where does meiosis occur? What are the products of meiosis?

Location and form of the female gametophyte structure?

Location and form of the male gametophyte structure?

What is the method of getting the male gamete to the female gamete (fertilization method)?

Describe the sporophyte produced as the result of fertilization.


20

1

Exercise 11: Solar Energy Conversion


Photosynthesis, the solar energy conversion process of plants, provides the basis for almost all food chains

on this planet. Respiration is the process by which cells release energy from food stuffs. Respiration

occurs in both plants and animals, while photosynthesis occurs only in those plant, moneran and protistan

cells that contain the green pigment chlorophyll. In this exercise you will use an oxygen electrode to

measure the rates of both respiration and photosynthesis in a suspension of Euglena cells.

The changes that occur in photosynthesis are summarized in the following equation:

light

CO2 + H2O (CH2O) + O2

(substrates) (energy source) (products)

The rate of the overall process can be estimated as rate of carbon dioxide consumption, the rate of

carbohydrate synthesis or the rate of oxygen evolution. The photosynthetic rate is influenced by the

availability of substrates, temperature and light intensity. Note that the process requires an input of energy

in the form of light. The above equation correctly summarizes the overall chemical balance of the process

but does not reveal its complexity. The large number of individual reactions that make up photosynthesis

can be divided into those that require light (the primary, photochemical or “light” reactions) and the

biosynthetic or “dark” reactions.

The overall changes of respiration are exactly opposite the changes in photosynthesis. Cellular food

reserves (e.g., starch in many plant cells) are hydrolyzed to simple sugars that are broken down to CO2 and

H2O in a many-staged process that releases energy in small, usable amounts that can be utilized to do

cellular work. The rate of respiration can be estimated by measuring the rate of consumption of oxygen, or

the rate of evolution of carbon dioxide.

Photosynthetic organisms release energy from foodstuffs by respiration at all times; thus, they are

constantly consuming oxygen. Their cells need ATP for work and synthesis reactions just as do those of

Exercise 11 – Solar Energy Conversion

2

animals or microorganisms. Photosynthesis occurs only in the light; therefore, oxygen is produced by the

organism only in light conditions. During the daytime, photosynthesis and respiration occur simultaneously

and the net amounts of oxygen and carbon dioxide produced or consumed depend on the relative rates of the

two processes.

In this laboratory you will explore a few of the many variables that affect the rate and efficiency of

photosynthesis. There will be several exercises conducted at once; be sure to go to each exercise (even if

you are not responsible for the primary data collection) and understand the purpose of the lab. The results of

all of these exercises, as well as a data analysis, will be done next week in lab. By the end of next week, you

should have a good understanding of the process of photosynthesis and a few of the many variables

involved in the process.

In this exercise, you will measure the changes in oxygen concentration of Euglena cultures in both light and

dark conditions. In the dark, only respiration will take place in the culture, and oxygen concentration should

drop. In the light, both photosynthesis and respiration will take place. Because the rate of photosynthesis in

daylight is many times the rate of respiration, the oxygen levels are expected to rise in a light-grown

Euglena culture. (Remember, light grown Euglena cultures have chloroplasts, which are required for

photosynthesis.) The groups will then repeat the experiment using dark-grown Euglena, which should be

lacking in chloroplasts. Will dark grown cultures produce oxygen in a lighted environment?

EXERCISE:

Measurement of gas exchange in dark grown and light grown cultures of Euglena gracilis.

Dissolved oxygen meter:

1. The YSI Model 58 Dissolved Oxygen Meter is used to measure dissolved oxygen in the field or

laboratory. The instrument consists of a probe and a meter that converts the signal from the

probe into concentration of dissolved oxygen displayed on a digital meter. Instructions for the

oxygen meter are in Appendix F.

2. The dissolved oxygen meter will be used in this experiment to monitor the rates of respiration

(in the dark) and of simultaneous respiration and photosynthesis (in the light) of a Euglena

gracilis culture. Information about the metabolic activity of this Protist can help explain your


3

growth data from Exercise 4.

Materials needed:

Dissolved oxygen meter and probe

Magnetic stirrer

Cultures of Euglena gracilis: dark grown and light grown cultures (both on heterotrophic media)

will be available

BOD bottles (sample chamber)

Black cloth or aluminum foil to exclude light

Colorimeter to measure cell density of cultures

Light meter / PAR sensor (Licor)

Calibration of the dissolved oxygen meter:

Calibration consists of exposing the oxygen probe to air at 100% humidity and adjusting the O2 CALIB

control so the meter reads exactly the calibration value for water vapor saturated air at the altitude at which

the measurements are made. The probe automatically compensates for temperature fluctuations. We will

assume that with Goucher's elevation of less than 50 meters above sea level, the calibration value should be

set at 99.5.

1. Set the function switch to % mode.

2. Place a one inch layer of water in the bottom of a BOD bottle to provide 100% relative

humidity. Insert the oxygen probe into the neck of the bottle.

3. Set the function switch to ZERO and adjust the meter to read 0.0. Switch back to % air

saturation mode.

4. When the meter has stabilized, unlock the O2CALIB control locking ring and adjust the display

to 99.5. Relock the ring to prevent accidental changes in the calibration setting.

Dissolved Oxygen Measurement:

For accurate determination of dissolved oxygen concentration, the liquid in the sample chamber must be

stirred so that the oxygen depleted layer at the membrane surface of the probe is flushed away. This is

accomplished with a magnetic stirrer. Set up this experiment in a dark room with a light source and a water

insulator (the insulator captures the heat generated by the light.)


4

1. With the instrument calibrated, transfer a stirred culture of Euglena to the sample chamber. At

the same time transfer a second sample of the stirred Euglena culture to a test tube for

determination of cell density. If the culture was grown in the dark, bubble compressed air

through the suspension to raise the concentration of dissolved oxygen.

2. Place the probe in the sample chamber and turn on the magnetic stirrer. Cover the sample

chamber with black cloth or foil so that all light is excluded. Turn on the light source (this is to

maintain constant temperature conditions for both the light and dark readings.) Use a light

meter to check the light intensity, and make any necessary adjustments so that the intensity is

approximately the same for each lab group.

3. Turn the function switch to O2ZERO and readjust if necessary.

4. Turn the function switch to read dissolved oxygen in mg/L (0.01 mg/L scale). Wait five

minutes for the instrument to stabilize. After five minutes begin to record your readings for

oxygen in Table 11.1 (time 0). Continue readings every five minutes for 25 minutes.

5. Remove the black cloth or foil so that your Euglena cells can receive daylight. Watch the

response of the meter. Is there a change in the direction of movement of the meter readings?

Note that the 25 minute reading is the last reading in the dark and the beginning of the light

period. Continue 5 minute readings for 25 minutes in the light.

6. When you have finished taking readings, remove the probe from the sample chamber, rinse in

water and leave the probe in a bottle with a layer of water on the bottom. Return the Euglena

suspension in its BOD bottle to the instructor.

7. Measure the absorbance of your Euglena suspension at 525 nm in the Turner colorimeter. This

will provide a relative measure of cell density so that you can compare the metabolic rates of

your cell suspension with rates of other suspensions measured at different times.

8. Be sure your instructor has a copy of your group’s data from Table 11.1 before you leave

today!


5

9. ASSIGNMENT: Make one graph illustrating the changes in oxygen concentration (y-

axis) over time (x-axis) for both the light and dark-grown cultures. Bring this graph for

next week’s data analysis lab!

Table 11.1: Oxygen concentrations for dark and light grown Euglena suspensions.

Dark Grown Culture

Light Grown Culture

Dark Condition

Light Condition

Dark Condition

Light Condition

Time (minutes)

Reading

(mg O2/L)

Time

(minutes)

Reading

(mg O2/L)

Time

(minutes)

Reading

(mg O2/L)

Time

(minutes)

Reading

(mg O2/L)

0

0

0

0

5

5

5

5

10

10

10

10

15

15

15

15

20

20

20

20

25

25

25

25

O.D. (525 nm): ________ O.D. (525 nm): ________ Temperature (°C): ________ Temperature (°C): ________ Light Intensity: ________ Light Intensity: ________


6

Exercise 12:

Solar Energy Conversion Data Analysis

OBJECTIVES:

You have already collected data in Exercise 11 for the Oxygen concentrations in light and dark grown

Euglena gracilis experiment. Today, you will use these data to compare the rate of oxygen consumption

or production in the Euglena cultures. Specifically, you will use two different computer programs to do

the following:

In Microsoft Excel, you will

• Prepare a graph to summarize your group’s raw data for both light and dark grown Euglena

by plotting oxygen concentration (mg O2/liter) against elapsed time (minutes).

• Calculate the RATES of respiration (oxygen consumption) and net photosynthesis (oxygen

synthesis minus respiration) for each culture and condition.

In PRISM, you will

• Use data sets from the entire class to compare the oxygen evolution of dark and light-grown

Euglena cultures in light or dark conditions;

• Learn and understand the concepts of normal distribution, probability, standard deviation,

variance and standard error; and

• Understand the data from a statistical point of view, and determine whether or not your data

are likely to be statistically significant.

In lab today, you will complete four figures: one in Microsoft Excel and three in GraphPad Prism. You

will also do three statistical analyses in Prism. Using these data, you will answer the questions on the

“Photosynthesis and Respiration” worksheet in this exercise. Your typed, prepared answers, figures and

statistical analyses will be collected next week in lab, and will be graded as your second “lab report” of

the semester. Although you may discuss this assignment with your lab partner, please DO YOUR OWN

WORK on the worksheet questions.

Exercise 12: Solar Energy Conversion Data Analysis

2

Replication: Replication means that the scientist repeats the experiment numerous times using exactly the

same conditions to see if the results are consistent. Variation is normal in biological systems.

Replicating the experiment allows the investigator to see how much variation there is. Duplicate data

helps the investigator determine how much variation is inherent in the system and simply due to chance

OR, alternatively, if there are additional, uncontrolled variables not accounted for. A measure of

variability is essential for data interpretation. In real research, the question of adequate sample size is a

complex problem, often involving statistical consultation.

For the purposes of our experiment, the instructors have pooled class data (when possible) and will

provide you with a set of the BEST CLASS DATA to use. Some class data will not be included because

of equipment malfunctions, problems with culture densities, problems with stirring and a variety of other

reasons.

EXERCISES:

Calculate the rates of respiration and net photosynthesis

For this part of the exercise, use YOUR OWN GROUP’S DATA SET and Microsoft Excel.

We want to compare the rate of oxygen production/consumption in both the light-grown and dark-grown

cultures in each condition:

Dark grown in the dark

Dark grown in the light

Light-grown in the dark

Light-grown in the light

The information your original data gives is simply the amount of oxygen (mg/L) in each culture over

time. You will prepare a figure illustrating this data, with the concentration of oxygen (mg/L) on the y-

axis and time in minutes is on the x-axis.


3

1. From START, open Microsoft Excel.

2. Set up your spreadsheet similarly to the example below:

A B C D E F G

1 0 5 10 15 20 25

2 Dark in dark

3 Dark in light

4 Light in dark

5 Light in light

Note that we are listing each condition separately, and that time (minutes) is along row 1. Data

listed is mg oxygen/L in the culture.

3. Enter YOUR GROUP’S DATA into your spreadsheet.

4. Highlight the entire area of the table. 5. Click on the Insert tab to bring up the Charts toolbar.

6. Select the chart for Scatter, and choose Scatter with only markers.

7. Your chart should appear on your spreadsheet. Make sure your chart displays each condition

in the key; if it does not, try the “Switch Row/Column” tab (Menu:Data/Switch

Row/Column) to correct it.

8. Select the Layout tab. This tab brings up a toolbar that allows you to add axis labels, titles,

and change the positioning of the key (what Excel calls a legend). Use this toolbar to:

a. Label the x-axis “Time (minutes).

b. Label the y-axis as “Oxygen Concentration (mg/L).

c. Change the location of the key (if you wish).

9. Select the Design tab to reveal a different toolbar. At the far right, choose the “move chart

location” tab to save your figure as a new sheet. Your figure will now fill the screen.


4

10. EXAMINE YOUR DATA. Do they look fairly linear?

11. Use EXCEL to draw the best fit straight line, the “trendline” for each data set. The SLOPE

of that line is the rate of NET oxygen production (in mg O2/minute). To draw your

trendlines:

a. Select one data point from the first data set (dark grown in dark) and right-click on it.

b. From the drop-down menu that appears, select Add Trendline.

c. The trendline menu will appear. Select the linear fit and select Display equation on chart.

These selections fit a best fit line to your data, and give you the equation of that line on

your graph. The SLOPE of that line is the rate of oxygen increase/decrease for that

condition.

12. Now make a similar trendline for each of the other data sets, following the above

instructions.

13.

14. Note that your trendlines are LINEAR, which means that you are assuming that net

photosynthesis (or respiration rates) are CONSTANT in your experiment.

15. Adjust the cosmetics to best display the data. You may notice that the equations are difficult

to read on the chart, because they are close together. You may MOVE the equations around

the graph by clicking and dragging. Just be sure to move the equations near the correct lines.

16. You may notice that “linear” appears in your key four times. If you wish to eliminate this,

you can click on the key (remember, Excel calls this a “legend!) and a surrounding box will

appear. Move the lower border of the box upward by clicking and dragging – you are

essentially blocking the “linear” labels from appearing.

17. Be sure you have an appropriate figure legend! Select the Layout tab to change the toolbar.

One of the options is to add a text box to your figure. Add the text box, and type in your

legend. This figure is Figure 1 for your lab assignment.

18. If possible, print out a copy of Figure 1. If printing is not possible, save your figure to disk

or your Magellan folder. Your figure should look something like this (depending on your

data – and WITH a legend!):


5

y=‐0.097x+9.8829

y=‐0.0854x+6.5738

y=‐0.0253x+12.191

y=0.0643x+11.581

0

2

4

6

8

10

12

14

0 5 10 15 20 25 30

OxygenCo

ncen

tra.

on(m

gO2/L)

Time(minutes)

darkindark

darkinlight

lightindark

Figure 1. Changes in oxygen concentration over time in light- and dark-grown Euglena gacilis cultures in light and dark conditions at 23°C. The light intensity was held at 150 Einsteins/min and oxygen levels were measured using a YSL dissolved oxygen meter.

Standardize the data so the rates may be compared directly

(This part of the exercise has been done for you by the instructor.)

We now have the RATES of net photosynthesis or respiration by each culture in each condition. These

rates are in mg oxygen/minute/L. We would like to be able to directly compare these rates; however, the

light-grown and dark-grown cultures had different numbers of Euglena cells in them, and this affects the

relative rates of oxygen production or consumption. We can use the optical density measurements for

these cultures to determine the amount of oxygen gained/lost in mg of oxygen/minute/Euglena cell. If we

know the rate of net photosynthesis or respiration for each Euglena CELL, we can easily compare

the rates between both culture types, and among data sets generated by other student groups.

The slopes obtained from your graph (Figure 1) represent the rates of oxygen loss or gain for each

culture in mg oxygen/L/minute. Examine your Figure 1. For the light-grown Euglena in light, notice

that the slope is positive, indicating a net gain in oxygen production. The other conditions have negative

slopes, and thus negative rates of net oxygen production (they represent oxygen loss).


6

The rates of oxygen production for each condition FOR EACH LAB GROUP were put into an Excel

spreadsheet by your instructor, and the following manipulations were performed. These calculations

simply took each group’s data and converted them from mg oxygen/L/minute to

mg oxygen/minute/Euglena cell. These conversions allow us to directly compare all class data, and the

light and dark cultures.

1. Multiply each rate (slope) by 0.125 L. This is done because the bottles of Euglena were 125

mL in volume. The resulting rate values represent changes in actual amounts of O2 produced

(net) per minute in the 125 mL culture.

2. Now, to compare the rates of the different cultures, the rates should be expressed to

represent comparable numbers of cells. The optical density (OD525) of your cultures was

determined after you took your oxygen readings (one reading for the light-grown culture, and

one for the dark-grown culture). A conversion factor for Euglena was used to determine the

number of cells per mL in your culture. Conversion factor = 1.0 OD525 unit = 8.85 x 104

cells/mL. Example:

3.

Light-grown 0.457 x (8.85 x 104) = 40,445 cells/mL x 125 mL = 5.056 x 106 cells

Dark-grown 0.177 x (8.85 x 104) = 15,665 cells/mL x 125 mL = 1.958 x 106 cells

NOTE: The total number of cells in each experiment was calculated by multiplying cell density values

x 125 mL volume for the culture.

4. The rates of net O2 production were then “normalized” on a per cell basis by dividing each

rate value (mg O2/min) by the total number of cells (mg O2/min/cell)

5. The rates for each condition, and for each lab group in the class, are listed in Table 1

(provided by your instructor). You will use these rates to complete the Prism part of the

exercise.


7

Using bar graphs and error bars to compare treatments Use the data in Table 2, provided by your instructor, for this exercise. You will also use GraphPad Prism for your data analysis. For this experiment, we are interested in comparing treatments in both the light and dark grown cultures.

To do this, we will use the pooled class data and a statistical software package called PRISM.

We think you will find Prism easy and fun to use. Try it out. Most things are intuitive, but there are a few

places where some trial and error is required. The guidelines below are prototypes for students, using the

tricks we have learned so far. If you find errors, or run into problems, please let us know. Please pass on

any new tricks you discover.

Comparing the dark grown cultures in the dark and light conditions

The first comparison of our data will be to see if the light or dark condition has any substantial effect on

the respiration of the dark-grown Euglena. If there IS an effect, the average respiration and error bars for

our data will be considerably different for each condition. Use the CLASS DATA for the dark grown

cultures only.

1. Launch Prism by double-clicking on the “Prism” icon on the desktop. To begin, select the

following options:

a. Create a “New Project

b. Select the “Type of Graph” tab and choose “One Grouping Variable”, “Scatter

Plot – Vertical”, and hit OK.

2. Enter your data, using one column for all the replicates for each treatment or category. You

should title the columns with each treatment (“Dark in dark” and the other “Dark in light”, etc.).

NOTE: Do not forget to enter negative values as negative!

3. Select the “graph” tab and examine your scatter plot. It looks cluttered with too many data points

and titles, and you want to look at only two conditions at a time anyway. To do this, you will

select which data sets you wish to examine two at a time.


8

4. Choose “Change” to reveal a drop down menu, and select “Remove/Replace Data Sets.”

5. To view only the dark-grown data, remove the “Light in Dark” and “Light and Light” data and

click OK.

6. Now your scatter plot is much easier to examine. Each data point is represented, and the

horizontal lines represent the mean. Do these data sets look very different from one another? Is

there a lot of variability in the data? You can begin to analyze this with error bars to express the

standard error of the mean.

7. Go back to the “Change” drop down menu and select “Graph type.” This time choose “Column

Bar, Vertical”and hit OK.

8. You now have a column bar graph illustrating the same data.

9. Add error bars by going back into the “Change” menu and choosing “Column Appearance.”

Select (for all data sets): “Appearance: Bar (one bar per column)”, “Plot: Mean & SEM”, and

“Error Bars – Both (above AND below)”. Click OK.

10. Now your graph has error bars bracketing the mean (which is the height of the column).

11. To label your axis and add your figure legend, simply click in that area of the graph. You may

also change font size, colors and other cosmetic factors.

a. BE SURE TO WRITE A PROPER FIGURE LEGEND (this is Figure 2) that

describes your graph and experiment. Think about what this graph represents.

Does the condition of light or dark have an effect on oxygen consumption in the

dark grown culture?

b. BE SURE YOUR AXES ARE LABELED ACCURATELY, especially in terms

of units.

12. You may change the colors of your columns and other cosmetic things by using the “change”

drop down and “column appearance.” Once you are satisfied with your graph, print out a copy

for your lab report.


9

Comparing the light grown cultures in dark and light conditions

Our second comparison is between the two conditions for the light-grown cultures. Keep in mind that

you must now enter positive and negative values. Remember, the Euglena culture in the dark condition

demonstrates net oxygen consumption (respiration) and the same culture in the light condition

demonstrates net oxygen production, which is the result of oxygen being produced at a higher rate in

photosynthesis than it is consumed in respiration.

Repeat the above steps to make a PRISM bar graph comparing these two data sets. Use the CLASS

DATA from the data sheet for the light grown cultures for each condition. Label this figure Figure 3 and

give it a proper legend. Print out a final copy for your lab report.

Comparing the light and dark grown cultures in the light conditions

A third comparison is between the two different cultures – light and dark grown – in the light condition.

Again, you will enter positive and negative values, and these rates will represent respiration and net

photosynthesis.

Make a PRISM bar graph comparing the two data sets, and label it Figure 4 (give it a good legend!) Print

this graph for your report.


10

t-tests

When reporting t-test results, you do not need a graphical determination. Instead, report the probability

statistic “P”, which should be less than 0.05 for statistical significance (95% confidence level). It is

important to note here that for complete credit you must INTERPRET and UNDERSTAND your t-test as

well as simply reporting it. Your instructor explained the use of a t-test in prelab (and will do so again,

upon request!).

1. From the data table you are interested in analyzing, select “Analysis: Built-in Analysis”; “Type:

Statistical Analysis” and “t-tests (and non parametric tests)” in window; Data to Analyze:

Selected data sets”. Click OK.

2. In the t-test window, select “Unpaired t-test”; under “Options: two tailed,” and a confidence

interval of “95%”. Click “OK”.

3. Your statistical output will appear automatically on the screen and will include a “P value” and a

statement as to whether or not your means are statistically different.

4. You may want to print this page, or at least record the P value and whether or not there is a

significant difference between the two data sets.

5. If you like, you may look in the Prism help menu for a brief statistic description. It is located at

the lower left hand corner of the screen.


11

Solar Energy Conversion Data Analysis – Student worksheet (25 points total) Each student should turn in:

Figures 1-4 (one in Excel, the others in Prism) T-test results, either as printouts of your analysis or a table with P values and

interpretations A copy of your group’s original data (Table 1; made in Excel) A copy of the class data, provided by your instructor (Table 2) Typed answers to the questions below – do your OWN work!

PART ONE: BACKGROUND (3 points total)

1. Give a concise and explicit statement of the purpose of our current experiment (Measuring Oxygen Levels in Euglena): (1 pt)

2. Variables: In the first lab of the semester (seems so long ago!), your lab group designed experiments to determine why termites displayed an unusual behavior. Part of designing your experiments involved the selection of appropriate variables. Below, identify the variables in our current experiments: (2 pts)

a. Dependent variable – what the investigator measures (or counts or records). What is

measured here?

b. Treatment variables – Independent variables which are varied by the investigator during the experiment. Specify the treatments in this experiment:

c. Controlled variables – Independent variable which are held constant throughout the

experiment to remove factors that could affect our interpretations. What did we control in this experiment?

d. Uncontrolled variables – Independent variables that were not held constant throughout the

experiment. In this experiment, we could not keep the number of cells in each culture (light and dark grown) constant. How would this affect our results if we did not normalize the data on a per cell basis?


12

PART TWO: DATA ANALYSIS (18 points total)

1. Use Figure 1 to answer the following questions: (6 pts) a. What is this figure illustrating, and what do the slopes of the best-fit lines represent?

b. In this figure, the best-fit lines have negative slopes for three conditions and a positive slope for one condition. What is the significance of this?

c. Using this figure, how would you estimate the true rate of photosynthesis? (The true rate of

photosynthesis is the rate of photosynthesis without taking into account the oxygen consumed due to respiration.)

2. Your instructor converted the rates determined in Figure 1 to mg oxygen production/min/cell. What was the purpose of this conversion? (1 pt)

3. Use Figures 2-4 and what you know about photosynthesis, respiration and Euglena growth to answer the following questions: (9 pts)

a. Explain the difference (or lack of difference) in rates of net oxygen production seen between the light and dark conditions for the dark grown Euglena suspension (Figure 2).

b. Explain the difference (or lack of difference) in rates of net oxygen production seen between the light and dark conditions for the light grown Euglena suspension (Figure 3).

c. Explain the difference (or lack of difference) in rates of net oxygen production seen between the light and dark grown Euglena suspensions in light conditions (Figure 4).


13

PART TWO (cont.) 4. Use your pre-lab notes and t-test results to answer the following: (2 pts)

What is the purpose of t-test? Explain how the p-value generated in these tests can be used to determine whether two data sets are significantly different from each other, at a given level of confidence. What confidence level was selected in analyses of your data in Figures 2-4? Which means (Figs. 2-4) appeared to be significantly different from each other, based upon calculated p-values?

PART THREE - THE IMPORTANT QUESTION: (4 pts) Your data has shown that the rate of photosynthesis is much greater than that of respiration. Why is that significant?


14

1

Exercise 13:

Porifera and the Cnidarians


The sponges exhibit a simple cellular level of organization and consist of a loose aggregation of cells

embedded in a gelatinous matrix. A skeleton of spicules made of calcium carbonate or silica (or both)

provides structural support. Sponges are sessile filter feeders, relying on water currents for food. The

currents are supplied by the animal’s choanocytes, flagellated cells that move water through the sponge

and trap nutrients.

Two animal phyla, Ctenophores and Cnidarians, exhibit tissue level organization. Cnidarians have

epithelial cell layers for lining, secretion and protection; muscle tissue for movement; and nervous tissue

for sensing and reacting to stimuli. There are two basic body forms, and both exhibit radial symmetry.

The polyp (hydroid) form is usually sessile while the medusa (jellyfish) is free swimming (or “errant”).

All Cnidarians have stinging cells, or cnidocytes, that discharge nematocysts to aid in protection and/or

prey capture. Some Cnidarians produce toxins as well, which are delivered into prey via the nematocysts.

EXERCISES:

MOVIE:

Watch the selected sections from the DVD series “The Shape of Life.” These video clips for the Porifera

illustrate sponge feeding and the different cell types. The Cnidarian sections focus on polyp and medusa

anatomy, moon jelly reproduction and the nerve net of a sea anemone. You will have the opportunity to

interact with live examples of some of these animals in class today.

PHYLUM PORIFERA:

Examine the various sponges in the laboratory.

Exercise 13: The Cnidarians

2

1. Notice the canals throughout the sponges; water carrying nutrients enters these canals, flows

through the sponge (where the nutrients are trapped by cells called choanocytes) and then

flows out of the sponge via excurrent canals.

2. Feel a representative sponge and notice the rough texture due to the sponge’s skeleton, made

up of spicules or spongin. View the demonstration slides for examples of spicules. There are

many different forms of these between sponge species.

3. One sponge of note has a glass fiber “tail.” These particular specimens are quite rare as they

live in very deep water where the ocean floor is murky and muddy. What advantage would

such a sponge have over a similar sponge without a glass “tail”?

__________________________________________________________________________

PHYLUM CNIDARIA:

CLASS HYDROZOA

Both polyp and medusa stages are represented in this class, although certain species may have only one

stage or the other (e.g., Hydra). The polyp stage may produce more polyps asexually via budding to form

a colony, or it may give rise to medusae - again via budding. It is the medusa stage that reproduces

sexually. This may seem similar to the alternation of generations in plants but, here, only the gametes are

haploid. Both polyp and medusa are diploid organisms.

Hydroids:

Hydroids are hydrozoans in which both the polyp and medusa stages occur. The polyps are usually small,

delicate and rather plant-like in appearance and are typically found attached to pilings, rock or seaweeds.

Many hydroids grow in colonies with continuous connections. View the slides and drawings of hydroids

such as Oblelia or Pennaria to identify the various zooids (polyp types) in the colony.


3

1. Hydroid Structure:

The main stem (stolon) of the colony usually gives rise to two kinds of individuals or zooids:

the gastrozooids with tentacles for feeding and the gonozooids that produce medusae.

2. Hydroid Reproduction:

Reproduction in hydroids is sexual and asexual. The tiny planktonic medusae release their

gametes into the sea where fertilization takes place. The zygotes develop into free-swimming

planula larvae that eventually settle, attach and produce polyps.

Other Hydrozoa:

Some more complex colonial hydrozoans have several highly specialized types of zooids (i.e., they exhibit

polymorphism).

Examine the examples on display.

1. In Physalia (Portuguese Man-of-War), special zooids produce the gas filled float. Several

distinct (and potent) types of dactylozooids bear nematocysts in cnidocytes but cannot feed.

2. Millipora (fire coral), although not a true coral, is prominent on coral reefs. Like corals, the

colony is supported by a calcium carbonate exoskeleton that is secreted by the animal itself.

Five or six dactylozooids surround the 'mouth' and stick through the tiny pores to sting prey -

or divers. Examine the millipora exoskeleton under the dissecting microscope, and view the

demonstration diagram of the zooid types that once covered (and made!) this exoskeleton.

CLASS SCYPHOZOA ("true" jellyfish)

The medusoid (swimming jellyfish} stage of Scyphozoa is dominant and is much more complex than the

medusa in Hydrozoa. These organisms are widespread in oceans and estuaries. Those who frequent the

Chesapeake Bay have probably seen the common "stinging - nettle", Chrysaora.


4

Examine the plaster model of the jellyfish medusa. Note the two-way gastrovascular cavity (GVC). What

is the main disadvantage in having a two-way GVC?

______________________________________________________________________________________

______________________________________________________________________________________

The jellyfish GVC is lined with an epidermal layer called the gastrodermis. Embedded in this layer are

many cnidocytes (stinging cells) with nematocysts to help the animal dispatch prey. The tentacles of the

animal also contain many cnidocytes. Some jellyfish have tentacles 25 meters long!

Can you think of any advantages in having long tentacles (especially filled with cnidocytes) as part of a

body plan?

__________________________________________________________________________________

______________________________________________________________________________________

Jellyfish have the first sense organ in the animal kingdom – rhopalia - located at regular intervals around

the bell of the animal. Rhopalia have two main sensing structures: the ocelli sense light (but do not form an

image) and the statocysts sense gravity.

Think about the ocean environment, and suggest how the jellyfish might use their rhopalia to assist them in

feeding or swimming.

______________________________________________________________________________________

The plaster models and prepared slides available give a visual overview of the jellyfish life cycle. Note

that the life cycle alternates between medusa and polyp forms, but the only haploid stages of the life cycle

are egg and sperm (not illustrated by the plaster model or slides). Jellyfish reproduce sexually when males

shed sperm into the water to fertilize eggs carried by the females. The resulting diploid planula larvae

swim, then attach to a substrate and grow as a polyp form. As the polyp matures, it “buds off” small discs

of itself which grow into genetically identical jellyfish – so, it reproduces asexually as well.

The ephyra larval stage has a relatively simple structure, but the rhopalia appear to be well-developed.

Why do you think this is? _______________________________________________________________

_____________________________________________________________________________________


5

Think a moment about the jellyfish life cycle model and the movie you saw in class. What is the advantage

of sexual reproduction for the jellyfish? _____________________________________________________

______________________________________________________________________________________

What is the advantage of the asexual reproduction in this life cycle? ______________________________

_____________________________________________________________________________________

CLASS ANTHOZOA

There are several subclasses of Anthozoa, including Hexacorallia (anemones, corals, etc.) and Octocorallia

(sea fans, sea whips, sea pansies). These animals exist in polyp form exclusively.

Compare the subclasses Octocorallia and Hexacorallia:

Compare the two subclasses of Anthozoa available in lab today. The Hexacorallia polyps may be solitary

or colonial.

1. Examine the display of anemones. As a group, the anemones have a great diversity in the

number and placement of tentacles, the size of the animal, and the color of the polyps.

2. Coral polyps are similar to those of anemones but secrete calcium carbonate, which provides a

substrate for the polyps to live upon and within. Thousands of generations of corals eventually

yield coral reefs, which are home to a great diversity of sea life. Examine the size, shape and

weight of some of the corals on display.

3. A live coral is available under a dissecting microscope; the polyps may be visible if you are

careful. While you look at this small bit of coral, take a look at the variety of organisms that

live on it. There will likely be small shrimp, hydroids, algae, worms and perhaps a small

mollusk or two that you can see. (Imagine the diversity of protists and bacteria you do not

see!)

4. Now examine the living Octocorals: Leptogorgia (sea whip) and Renilla (sea pansy). The


6

polyps that make up these colonial animals secrete a mucous which helps them trap small

zooplankton for food. The base of the sea pansy is a modified polyp reinforced with mesoglea.

The sea whip’s structure is secreted by the polyps and is made up of protein and calcium

carbontate.

a. How many tentacles do the polyps of these organisms have?

______________________

b. How do the tentacles of the sea whip or sea pansy differ from the tentacles of the

anemones?

_____________________________________________________________

5. Examine a model of seawhip anatomy. The model shows how the polyps are connected and

details of the extracellular axial skeleton. How might a shared gastrovascular cavity be an

advantage for a sessile (non motile) animal? ______________________________________

__________________________________________________________________________

Anemone behavior:

To observe anemone behavior, each pair of students should place a healthy Aiptasia on the stage of a

dissecting microscope and allow it to relax completely. Be sure to keep careful records of what you do

and the results. Each dish may have several anemones; you may wish to use a different one for each

of the exercises below as they take a little time to recover.

1. Coordination: The tentacles of an anemone are covered with cilia that pass water currents

over the animal. Use a paint brush to gently add carmine or carbon particles to various regions

of the tentacles near the oral disc (YOU ONLY NEED A FEW PARTICLES!). You may

repeat this experiment using a few small sand grains.

a. Do you see any evidence of cilia? Describe this.

_____________________________________________________________________


7

b. The creation of gentle water currents by cilia give the anemone an advantage in the

capture of small particles of food. Can you think of another advantage (or two) these

water currents may provide to the animal? (HINT: Think of basic needs!)

_____________________________________________________________________

c. Next use a small brush to gently touch the oral surface and tentacles. Can you trace

the spread of the stimulus? Does the spread of stimulus seem to be in only one

direction, or in both?

______________________________________________________________

d. How does this relate to what you know about nerves in Cnidarians? ______________

_____________________________________________________________________

e. How quickly does the stimulus move?

_______________________________________

2. Muscle movement and hydrostatic skeleton: Recall that the anemone has longitudinal and

circular muscles and that the tentacles are muscular as well. In humans, muscles work against

one another in antagonistic pairs; however, our skeleton is necessary to provide rigidity to hold

the muscles while they work. Anemones (and all cnidarians) have a skeleton, too – it is a

hydrostatic skeleton provided by the water within the animal’s body. The hydrostatic

skeleton, together with the mesoglea (“jelly”), give the cnidarians enough support to

effectively move their antagonistic muscle groups.

a. Using a paintbrush, gently touch or poke your anemone on one side of its base or “stalk.”

Can you see evidence of muscle contraction to one side or another? _________________

b. Now touch your anemone a bit more forcefully, attempting to dislodge its pedal disk from

the glass dish. What does the anemone do?

________________________________________________________________________

c. Did you notice any change in the anemone’s width or length? ______________________


8

________________________________________________________________________

d. Think about the anemone the way you would a water balloon that you are trying to crush.

Does water leak from the balloon easily, or does the balloon change shape to

accommodate the water first? How can you relate this to the hydrostatic skeleton of the

anemone?

________________________________________________________________________

________________________________________________________________________

e. Fortunately for the anemone, it does not have to burst when its hydrostatic skeleton is

under pressure. Instead, its body has perforations that allow water inside the GVC to

escape when the anemone draws within itself.

3. Defense and Nematocysts: Use the anemone from the previous exercise, if possible!

If you’ve really irritated your anemone, it might have blown out acontia through the

perforations in its body as it pulled in its tentacles. Acontia are threadlike structures within the

GVC that are loaded with cnidocytes and can be used as a last line of defense. Use a pipet and

scissors to remove an acontia. Mount it on a slide and examine with a compound microscope.

a. Identify and draw a cnidocyte with an undischarged nematocyst.

b. Add a drop of 10% acetic acid to the slide and record what you see.

________________________________________________________________________

c. The acetic acid is mimicking WHAT GENERAL TYPE of stimulus?

________________________________________________________________________

4. Zooxanthellae: Using scissors and a pipet, carefully snip off a tentacle from Aiptasia or

another suitable cnidarian and place it on a microscope slide. Examine under a compound

microscope.

a. You should be able to see rows of cnidoblasts and lots of brownish cells. The brownish


9

cells are zooxanthellae which are photosynthetic dinoflagellates living endosymbiotically

inside the animal tissue.

b. Zooxanthellae are found in corals as well; in fact, they are essential to the health of a coral

reef. Think about the coral / zooxanthellae symbiosis, and speculate as to what each

organism contributes to the other.

________________________________________________________________________

____________________________________________________________________

________________________________________________________________________

5. Feeding: Attempt to feed your anemone, or another (happier!) one in your dish. Gently touch

a tentacle with a small crustacean or other food. If living food is available you might observe

its capture as directed by your instructor.

a. Record your observations: _______________________________________________

_____________________________________________________________________

b. Is there any evidence of cnidocytes?

_____________________________________________________________________

c. Pay particular attention to how the food gets down the gullet.

STUDY GUIDE: You should be able to:

Explain the scyphozoan life cycle using the model

Identify the major parts and their functions on the jellyfish model

Understand the difference in life style between colonial and single animals.

Know the function and advantage of zooxanthellae on an anemone

Explain simple anemone or jellyfish anatomy

Identify and know the function of nematocysts and cnidoblasts

REFERENCES: Hickman et al. (2008) pp. 260-288 Radiate Animals (Phyla Cnidaria and

Ctenophora)


10


Name of Specimen: __________________________ Date Observed: _____________ Preparation: ________________________________ Magnification: _____________ Natural Environment: _________________________ Comments and Observations: ____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

____________________________________________________________________________________

1

Exercise 14:

Platyhelminths, Nematodes and Annelids


Unlike cnidarians, worm bodies develop from three primary germ layers (cnidarians develop from only

two):

• The endoderm (innermost) layer gives rise to the gut and gut-related organs.

• The ectoderm (outermost) layer establishes the outside layers of the body (like cuticle,

skin, etc.), sensory organs and nervous system.

• The mesoderm (in between) layer develops into internal organs, muscle, and the heart and

circulatory system.

The three worm phyla you will examine today illustrate three different levels of overall structural

complexity. These levels are defined by the organization and location of the mesoderm tissue within the

animals. Platyhelminths have solid mesoderm tissue (called parenchyma) between the endoderm and

ectoderm layers, and thus have no body cavity (acoelmate). Members of the phylum Nematoda have a

“pseudocoelom”, or a cavity lined by mesoderm on one side and endoderm on the other; these are

pseudocoelomates. Finally, Annelida have a true, fully mesoderm-lined body cavity (eucoelomates).

Each phylum has undergone considerable speciation and has representatives frequenting a number of

habitats and lifestyles. Human parasites are represented in each phylum and have been objects of medical

research.

EXERCISES:

PLATYHELMINTHS (Flatworms)

The acoelomate flatworms are usually divided into three distinct classes. The basic features of the group

are visible in the class Turbellaria (free-living flatworms). The two parasitic classes, Trematoda (flukes)

and Cestoda (tapeworms) are highly modified for parasitic life and bear little resemblance to the free-living

Exercise 14: The Worms

2

forms (or to each other).

Trematodes (flukes):

You have seen a film on the "Biology and Control of Schistosomiasis in Puerto Rico" which reviewed the

various stages in the life cycle of the trematode causing this disease. The film also illustrated how a

thorough understanding of the life cycle is essential to effective disease control.

Cestodes (tapeworms):

These Platyhelminths live as parasites in the intestines of vertebrates. Their bodies are ribbon like and

divided into short reproductive units called proglottids. These units grow and mature from a “head”

region, the scolex. In the pre-lab discussion, your instructor identified the parts of a tapeworm. Examine

the demonstration slides, diagrams and life cycles. Use this information to answer the questions below,

which focus on the adult stage of the life cycle (as an intestinal parasite).

1. Observe a prepared slide of the anterior portion of Taenia. Note the somewhat enlarged scolex

bearing suckers and a circle of hooks. Recall that this adult tapeworm lives in intestinal tracts.

a. What is the function of hooks and suckers? ___________________________________

______________________________________________________________________

b. What can you infer about the external covering of the tapeworm, given its preferred

environment? _________________________________________________________

c. Note the immature proglottids extending from the scolex. As the worm grows, it adds

proglottids at the scolex end. Mature proglottids can be found in the middle of the

worm, and gravid proglottids (full of fertilized eggs) at the end.

2. Now view slides of mature and gravid proglottids. Use the diagrams alongside the

microscopes to assist you in identifying structures.

a. The tapeworm is hermaphroditic, and the mature proglottids contain mature ova and sperm

but an empty uterus. These proglottids may shed sperm as well as receive sperm from


3

another tapeworm to fertilize its eggs. If another tapeworm is not present, the animal will

self-fertilize.

b. The gravid proglottids toward the end of the worm have uteri stuffed with egg capsules;

the uterus occupies most of the proglottid and the rest of the male and female reproductive

systems have degenerated. These proglottids are ready to be shed from the organism.

3. How could you confirm the presence of a tapeworm in a human or any other animal?

_____________________________________________________________________________

_____________________________________________________________________________

4. Note that the proglottids have ample reproductive structures but no visible structures for a

number of other basic needs. Why are there no digestive system organs present?

____________________________________________________________________________

____________________________________________________________________________

5. The tapeworm lives in an anaerobic environment, and has no respiratory organs. What does

this tell you about the tapeworm’s energy needs, as related to its lifestyle? ________________

____________________________________________________________________________

6. There are nervous system components in a tapeworm; it has concentrations of sensory cells

(chemoreceptors) on the scolex region and around the genital pores of each proglottid. What

would be the advantage of these sensory cells for an organism that lives in an intestine?

____________________________________________________________________________

____________________________________________________________________________


4

Figure 14.1 – Dugesia, a free-living flatworm.


5

Turbellarians (free-living flatworms):

By examining living Dugesia (often called planaria) we can see many of the basic features of the phylum

Platyhelminthes without the extreme adaptations exhibited by the parasitic classes. The definitive bilateral

symmetry and the anterior to posterior differentiation may be taken for granted, but these are major

advances over the cnidarian radial symmetry. In Turbellarians, we see the beginnings of cephalization – a

concentration of nerve ganglia and sensory organs at the anterior end of the animal. In Dugesia, ocelli

detect light, and on the auricles there are chemoreceptor cells to detect chemical signals and tactile cells

to detect mechanical stimulus. One-way nerve cords transmit sensory information throughout the body.

Obtain a sample of Dugesia in a depression slide and observe its movement and behavior using a dissecting

microscope. Use Figure 14.1 to help you locate structures, and make a drawing of this organism.

1. Locate the ocelli and auricles on the anterior end of the animal.

2. Note that there is a definite anterior and posterior end to the animal; the anterior leads the way

as the animal moves. There is also a dorsal (back, upper) and ventral (front, lower) surface as

well.

3. Observe the gliding movement. Cilia propel the animal on a mucus path secreted by cells in

the epidermis. This ciliary-mucoid system of locomotion is quite like the one that moves

particles on the oral disc of anemones, only here it is used to move the whole animal. (This

same system allows anemones to slowly creep along on their pedal discs). Is the motion due to

cilia alone? Is there evidence of muscles? Explain.

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

2. Design simple experiments to observe the response of Dugesia to tactile stimuli and to light.

Record your experiments and observations in Table 14.1.


6

Table 14.1 – Tactile and phototaxic responses in Dugesia.

Experiment Observation

Light

Touch

3. The ventral side of Dugesia has a pharynx (retractable tube-like structure) that leads to the

mouth of the animal. The mouth is in the center of the body, and leads to a two-way

gastrovascular cavity. Introduce a small amount of liver or cooked egg to the animal’s dish

and allow the animal to approach the food. Is there any evidence of the animal “sensing” the

food? Describe any feeding behavior you may see, including any structures involved.

___________________________________________________________________________

___________________________________________________________________________

4. Most nitrogenous waste is diffused from the body of the worm in the form of ammonia, but

some is released by flame cells near the excretory canals. The main job of flame cells,

however, is to maintain osmotic balance in this freshwater animal. Try to locate flame cells

along the margin of the worm’s body. (This may be difficult if the worm is moving!)

5. Think about the anemones (Aiptasia) from last week’s lab. Compare the increased overall

mobility and coordination of Dugesia to anemones on Table 14.2. Review the advances in

muscular and nervous organization that make this possible.


7

Table 14.2 – A comparison of coordination and mobility in Dugesia and Aiptasia.

Animal

Nervous system (include type of

nerves, arrangement, sense organs

and locations)

Structures used for mobility

Planaria

(Dugesia)

Anemone

(Aiptasia)

NEMATODES (roundworms)

The nematodes constitute by far the largest and most important of the eight pseudocoelomate phyla. In

general animals in these phyla are round and unsegmented. Almost all members have a complete (“one

way”) digestive tract and a stiff outer cuticle covering the epidermis. Recall that these animals have a

cavity surrounding the digestive tract, but it is not a true coelom because it is not completely lined in

mesoderm. This pseudocoelom within the animal is maintained at high turgor pressure so the worm’s

longitudinal muscles can effectively move against this “hydrostatic skeleton” and cuticle. Although some


8

nematodes may exceed a foot in length, such as the internal parasites of dogs, horses, pigs and humans (i.e.,

Ascaris), most are minute animals, some less than 1 mm in length. Millions of tiny nematodes are found in

each square meter of soil where they usually feed on bacteria and fungi. However, some nematodes cause

root damage to garden crops.

One especially important member of the Nematodes is Caenorhabditis elegans, a roundworm that has been

the subject of much genetic and developmental study. C. elegans was the first multicellular organism to

have its DNA completely sequenced and mapped. A complete developmental map has also been

constructed, so the fate of each cell is known. This small worm is a “model organism” for eukaryotic

animals and has provided invaluable genetic and developmental information that is useful in understanding

the biology of more complex animals.

Use a dissecting microscope to look at living "vinegar-eels" that inhabit fermenting fruit. Their lashing

motion is a distinctive feature of this phylum and readily distinguishes Nematodes from the Annelida and

Platyhelminthes phyla. You may also view the larger preserved specimens of some of the parasitic

nematodes.

ANNELIDS (segmented worms)

The Annelids consist of earthworms, leeches, and polychaetes. The most striking feature of annelids is the

condition of metamerism, i.e., the division of the body into similar segments which run linearly along the

anterior-posterior axis. Annelids are eucoelomates; they have a true body cavity lined completely with

mesoderm. One of the advantages of a true body cavity (coelom) is that it allows greater internal

specialization. Here the coelom is partitioned by internal septa to form many individual hydrostatic units

with a rather complex system of muscles in each. This allows a very characteristic "peristaltic" type of

movement as each segment can move separately.

Some annelid classes are established by the presence or absence of setae - bristle-like hairs on their

external surface. These hairs can serve as “anchors” to help the animal move more effectively.

Hirudineans (leeches):

These animals live in streams and only adults attach as ectoparasites. While their anterior and posterior


9

ends are modified for attachment, leeches still retain evidence of segmentation and exhibit the characteristic

peristaltic movement of annelids. Leeches have no setae but they have suckers which are used for

locomotion and attachment to hosts while sucking blood.

Observe the swimming and “looping” behavior of living leeches. If liver is available, you may place a

piece in one end of the container. Some species of leech move towards the liver. (Please remove the liver

when you are finished!)

What general class of sense organ might a leech use to locate food? _______________________________

Oligochaetes (earthworms):

Observe some living earthworms. These animals have “few” setae. Consult Figure 14.2 for internal

details of the worm’s body plan.

1. Stroke an earthworm in one direction, then in the other (or view a worm in a dissecting scope).

Can you detect the setae? _____________________________________________________

2. These worms are modified for burrowing in soil. Watch to see how this is accomplished.

___________________________________________________________________________

___________________________________________________________________________

3. How do you think the “one-way” direction of the setae assist the worm in this task?

__________________________________________________________________________

4. Annelids such as earthworms also support a population of nematodes. If a section is cut from

an earthworm and incubated on nutrient agar, nematodes will be found on the agar.

Polychaetes (example is Nereis, the clamworm):

Each pair of students should obtain and examine one lively specimen of Nereis, a worm with “many setae.”

In polychaetes, the setae are attached to parapodia, fleshy extensions along either side of almost every

segment of the worm. Use an observation sheet to draw and identify the worm’s structures and record any

observations.


10

Figure 14.2 – Lumbricus, an earthworm.


11

External anatomy: Carefully examine the "head" region, using a dissecting microscope. Use the

illustration in Hickman to help identify all of the structures you see and keep a record of what you observe

on your observation sheets. In contrast to the Oligochaetes and Hirudineans, there is a visible concentration

of sense organs at Nereis’ anterior end.

1. Locomotion: Carefully watch the normal crawling motion and the coordination of the

parapodia. This motion is diagrammed in Figure 14.3.

a. Do parapodia on the same segment move alternately or together?

_____________________________________________________________________

b. Does the size or shape of the individual segments change?

_____________________________________________________________________

c. Now gently prod your specimen to increase its speed and compare this motion to

slower motion. Look at the parapodia carefully.

1) What structures can you see?

______________________________________________________________

2) How are they used in locomotion?

______________________________________________________________

3) Watch a demonstration of Nereis swimming. How is this done?

______________________________________________________________


12

Figure 14.3 – A polychaete worm.


13

2. Circulation and Gas Exchange: Increased size and activity are facilitated by two circulatory

advances of annelids: a closed circulatory system with a pump and respiratory pigments to

greatly increase oxygen carrying capacity. In Nereis the dorsal contractile blood vessel serves

as a pump. Blood with hemoglobin-containing red blood cells can be seen pulsing along the

body.

When your worm stops for a moment, increase the magnification and examine a parapodium

again. Trace the flow of blood here and describe or illustrate it on an observation sheet.

Can you determine in which direction it moves? Oxygen must enter the animal by diffusion.

The numerous thin parapodia provide increased surface area for gas exchange and are highly

vascularized. During this semester, and in Bio 105, you will examine many different

respiratory structures that exhibit increased surface area plus vascularization. The primitive

parapodial system in Nereis is simple but adequate for highly-mobile polychaetes.

Polychaete diversity:

Observe the living and preserved marine polychaetes available. Record in your notebook the features of

their anatomy that suit them to their different lifestyles. Pay particular attention to elaborations of the

anterior end and to special respiratory structures.

1. Sabella, a feather duster worm, lives in a parchment-like tube. This organism quickly

responds to light or shadows by pulling itself into its tube for safety. The feathery crown of

tentacles may break off in response to being attacked by a fish but will regenerate.

2. Chaetopterus, the parchment worm, is described and pictured in your text and a preserved

specimen is available in a glass tube. Its bizarre anatomy reflects considerable modification

for life in a tube. A commensal crab usually lives inside the tube with the worm.


14

ASSIGNMENT:

Although the three phyla examined today are wormlike, they reflect three quite difference levels of

structural complexity (Figure 14.4). Some of this is reflected in their locomotion. In Table 14.3, compare

the locomotion of Turbellarians, Nematodes and Annelids. How are the arrangement of the nervous

system, sense organs and muscular system involved in the specific type of locomotion you see in each

group? What are the limitations or advantages of the skeletal systems (or lack thereof) in each

group?

Figure 14.4 – Worm body plans

STUDY GUIDE: Complete Table 14.3, summarizing body organization and features of locomotion in the major phyla of worms reviewed in this lab. You should be able to:

• recognize the different phyla of worms examined in class • understand the differences in body plans (acoelomate, pseudocoelomate, eucoelomate) withiand

between worm phyla • understand how different body plans are responsible for different types of worm locomotion • understand the general Cestode body plan and life cycle • understand the method of gas exchange for Nereis • know the two advances in circulation exhibited by annelids

READINGS: Hickman et al. (2008)

Ch. 14 Ph. Platyhelminthes (Flatworms) and Ph. Nemertea (Ribbon Worms) only Ch. 18 Ph. Nematoda (roundworms) only Ch. 17 Ph. Annelida (segmented worms) only


15

BIOLOGY 104 LABORATORY: OBSERVATION SHEET Name of Specimen: ____________________________ Date Observed: _____________ Preparation: __________________________________ Magnification: _____________ Natural Environment: _________________________ Comments and Observations: ____________________________________________________________

_____________________________________________________________________________________

_____________________________________________________________________________________

16

Table 14.3 Worm body organization and types of locomotion, including systems involved in locomotion. Phylum / Class Body Organization Type of Locomotion or

Movement Systems Involved in Locomotion

Turbellarians (free-living flatworms)

Nematodes (roundworms)

Hirudineans (leeches)

Polychaetes (clamworm Nereis)

1

Exercise 15:

The Arthropods


The arthropods are the largest phylum of animals. Some of the features responsible for their success include

the impermeable exoskeleton, jointed appendages, flight, unique respiratory system, complex life cycles,

and sophisticated nervous and sensory systems and behaviors. The arthropods have undergone fantastic

adaptive radiation with many diverse forms that are found in virtually every possible habitat.

Compared to annelids, arthropods in general exhibit a number of features that show the natural evolution of

the bilaterally symmetric body plan. Cephalization is much more pronounced in arthropods and they have a

higher concentration of sense organs in this area. These organs are, in some cases, uniquely suited for the

particular arthropod’s lifestyle (for example, the eyes of flies). Rather than repeated segments that serve

many functions as seen in annelids, arthropod segments are less redundant and serve particular functions for

the entire animal. This type of organization allows for much greater efficiency.

In this laboratory you will have the opportunity to study a very few of the almost one million species of

arthropods. You will see living representatives from each of the three subphyla.

1. The chelicerates include horseshoe crabs, spiders, and ticks. They are characterized by

pinching mouth parts called chelicerae, five more pairs of appendages, and no antennae or

mandibles.

2. The crustaceans are arthropods with mandibles and two pairs of antennae. This group

includes a vast array of critters ranging from tiny planktonic forms to barnacles, crabs and

lobsters.

3. The uniramians, which include the insects, centipedes and millipedes, have mandibles, one

pair of antennae and, as their name implies, unbranched appendages.

Exercise 15: The Arthropods

2

You will divide your time between arthropods found in aquatic habitats and those that occur on land.

EXERCISES:

AQUATIC ARTHROPODS - CRUSTACEANS: BRINE SHRIMP, CRAYFISH AND CRABS

The crustaceans are ancient creatures and have had ample time to become highly specialized although some

quite primitive forms have survived. You will observe living and preserved animals that illustrate some

trends in crustacean evolution.

Brine Shrimp (Artemia salina):

Using a depression slide observe some brine shrimps under a dissecting scope. The brine shrimp are

among the most primitive of living crustaceans. You may wish to draw the shrimp and note your

observations.

1. Note the following features of this group:

a. Large number of body segments but little specialization of body regions

b. Very simple nervous system

c. A series of similar and unspecialized trunk (thoracic) appendages.

2. Examine the large head of Artemia.

a. Note the stalked compound eyes. The median eye seems to be responsible for the general

orientation to light.

b. The first pair of antennae is greatly reduced but the second antennae are large and may be

modified in males to function as claspers to hold the females for mating. (You will

probably see a few mating shrimp in your preparation.)

3. Observe the general structure of the appendages in Artemia and their use in swimming and

feeding. Although the abdomen lacks appendages, the thorax bears 11 pairs of appendages that

are soft and flattened. These lack the joints so typical of other arthropod appendages. In

Artemia the same appendages serve four functions:


3

a. Locomotion

b. Filter feeding

c. Food manipulation

d. Respiration

4. If possible, also observe the shrimp’s internal structure and circulation.

5. Design an experiment to test the response of brine shrimp to light.

a. Do they move toward or away from light?

_________________________________________________________________________

b. Is their behavior or swimming affected by light?

_________________________________________________________________________

Brine shrimp are denizens of temporary freshwater ponds or salt lakes that are usually devoid of carnivorous

fishes. In these ephemeral ponds conditions change from week to week. Structurally simple Artemia have

evolved impressive physiological mechanisms to cope with wide ranges of pH and salinity. They can

regulate their internal osmotic concentration in water as dilute as tapwater or two times as salty as seawater!

Brine shrimp eggs are extremely resistant to desiccation. When ponds dry up, the eggs may remain viable

for years. When submerged again the eggs hatch within 48 hours.

If available, observe the eggs and newly hatched nauplius larvae on display. The nauplius is the

characteristic larval stage of Crustacea. In more advanced groups, the nauplius may give rise to

successively more complex larval stages before the adult form is attained.


4

BIOLOGY 104 LABORATORY: OBSERVATION SHEET Name of Specimen: ____________________________ Date Observed: _____________ Preparation: __________________________________ Magnification: _____________ Natural Environment: _________________________ Comments and Observations: ____________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________


5

The crayfish (Cambarus):

The most advanced group of crustceans is the Decapoda. Decapods include most of the large, familiar

crustcea such as shrimp, lobsters, crayfish, and crabs. As you dissect the crayfish, compare its overall

organization to both the more primitive Artemia and to the polychaete annelids studied last week.

Work in pairs to dissect a preserved crayfish. Use Figures 15.1, 15.2 and 15.3 to help you identify

structures.

1. External Anatomy:

a. Identify the two major body regions: cephalothorax and abdomen. Note that the

carapace covers the cephalothorax dorsally and extends laterally to cover the gill

chambers.

b. Identify the eyes, antennae and mouth, and locate the excretory openings from the

green glands (or antennal glands) on little papillae at the base of the antennae.

c. Use Figure 15.2 to help you determine the sex of your crayfish. Is it male or female?

d. Cut away the part of the carapace that covers the gill chamber on one side. Note the

general structure and attachment of the gills. What is their relationship to the legs?

_____________________________________________________________________

f. Beginning with the fifth walking leg and working anteriorally, remove each appendage

on the side you have exposed until all thorax and head appendages, including antennae,

have been removed.

1) Arrange the appendages on a sheet of paper in the order of their occurrence in the

animal as shown in Fig 15.3. Notice the wide variety of modification of the

primitive biramous form.


6

2) Note the structure and function of each appendage.

2. Internal Anatomy:

a. Thorax: Carefully remove the entire carapace up to the rostrum to expose the heart

lying in the pericardium in the posteior part of the thorax.

1) If the heart is intact, look for the ostia (valves) on its dorsal surface.

2) What is the function of the ostia and pericardium in this open circulatory system?

__________________________________________________________________

3) Locate the stomach and digestive glands that lie beneath the heart.

b. Abdomen: Remove the exoskeleton from the abdomen to expose the ventral nerve

cord and its segmental ganglia. Trace this nerve cord toward the brain.

3. When you have completed the dissection, please dispose of the crayfish parts.


7

Figure 15.1 - External and internal structures of a male crayfish


8

Figure 15.2 - External structures of a male and a female crayfish; ventral views


9

Figure 15.3 - Crayfish appendages


10

Figure 15.4 - Limulus, dorsal and ventral views


11

AQUATIC ARTHROPODS - CHELICERATES: LIMULUS, THE HORSESHOE "CRAB"

Limulus is often called a living fossil because it has remained virtually unchanged for some 300 million

years (from about the time dinosaurs first appeared). The distribution of horseshoe crabs is puzzling

because they occur only on the east coasts of continents.

Examine Limulus and identify the following parts (use Figure 15.4 to help you):

1. the pincher like chelicerae,

2. the other paired appendages,

3. the book gills

4. the simple and compound eyes.

How do you suppose this animal could use its telson, the long tailpiece? ____________________________

______________________________________________________________________________________

TERRESTRIAL ARTHROPODS - UNIRAMIANS: CENTIPEDES, MILLIPEDES AND INSECTS

Centipedes and Millipedes:

These are of uncertain affinity to the other arthropods. Look at the representatives available.

What feature(s) allow(s) you to readily identify each? __________________________________________

Insects:

The fantastic success of the insects is closely associated with adaptations in reproduction, excretion,

respiration and locomotion (i.e., flight).

Examine some living cockroaches to gain an understanding of their respiratory and excretory systems. Use

Figure 15.5 to identify important structures.


12

Figure 15.5 - External anatomy of Periplaneta


13

The truly unique tracheal system of insects (and some arachnids) allows the cuticle to be covered with an

impermeable waxy layer. The tracheal system can be seen readily in the common cockroach, Periplaneta

americana. Observe the living specimen under a demonstration dissecting microscope. Adjust the

orientation of the insect so that it is on its side but with the ventral surface facing up at about a 45° angle.

Take a few moments to look at the overall structure of this typical insect.

1) Examine the thoracic region carefully and locate the large flap-like spiracles and observe

any movements they exhibit.

2) What is their function?

_________________________________________________________________________

3) There are other spiracles and tracheae in the body but those in the thorax are by far the

largest. Why?

_________________________________________________________________________

4) The spiracles lead to the large chitin lined tracheae which progressively divide to form the

much smaller trachioles where gas exchange occurs. Both tracheae and trachioles can be

seen in the femur or tibia of the leg if you adjust the light carefully. Transmitted light may

work better than reflected light.

In your notebook include illustrations or precise descriptions of spiracles, tracheae and trachioles

along with any other observations you make.

Most animals that excrete ammonia or urea (including humans) must keep these toxic substances diluted.

Water lost in the urine is significant for most terrestrial animals but not for insects, arachnids, birds or

reptiles because they convert their nitrogenous wastes into insoluble uric acid.

Insects and arachnids accomplish this via the Malpighian tubules which take in body fluids and actively

remove water. Uric acid crystals are deposited in the hindgut. Frequently the hindgut also actively

reabsorbs water. Cockroaches will be dissected to reveal the Malpighian tubules. The tubules are blind sacs

that empty into the digestive system at the juncture of the midgut and the hindgut. They are transparent and


14

look like cellophane if empty. However, if uric acid crystals are inside they will reflect the light so that the

tubules have a bright, yellowish appearance that is unmistakable.

1) Examine a dissected cockroach and observe the Malpighian tubules. Malpighian tubules

are also somewhat flattened in cross section compared to the cylindrical trachioles.

2) Are the tubules moving? ___________________________________________________

3) Now examine a sample of Malpighian tubule under the compound scope, stained with

neutral red dye (which mimics ammonia waste in the insect body). What do you notice

within a couple of minutes after dye application to the tubule?

________________________________________________________________________

________________________________________________________________________

TERRESTRIAL ARTHROPODS--CHELICERATES: ARACHNIDS

(SPIDERS, TICKS, MITES AND SCORPIONS)

This eight-legged class of chelicerates is also extremely well adapted to land. In fact, many live in the

harshest desert conditions where few other animals survive. This is possible because most have tracheae

and Malpighian tubules.

Spiders are certainly the most successful of the arachnids.

1. Observe the tarantula on display. Note its general body form and body regions.

2. As predators, the spiders have evolved rather sophisticated sense organs and behaviors to

match. Perhaps you will be able to watch the tarantula capture a cricket for a meal.


15

BIOLOGY 104 LABORATORY: OBSERVATION SHEET Name of Specimen: ____________________________ Date Observed: _____________ Preparation: __________________________________ Magnification: _____________ Natural Environment: _________________________ Comments and Observations: ____________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________


16

ASSIGNMENT:

To review and summarize the most important aspects of today’s lab, fill out Table 15.1 below.

STUDY GUIDE:

You should be able to understand:

• The functions of arthropod appendages, using Artemia and Cambarus as examples

• The functions of the structures in bold for the Cambarus dissection and Limulus

• The functions of Malpighian tubules, trachioles, spiracles, and tracheae; and how the thick cuticle

layer surrounding an insect relates to these structures

• Adaptations insects exhibit that make them so successful

• Differences between the three subphyla of Arthropods observed in lab

REFERENCES:

Hickman, et al. (2008):

Ch. 19 Subphyla Chelicerata and Myriapoda (centipedes, millipedes only)

Ch. 20 Subphylum Crustacea

Ch. 21 Subphylum Uniramians (Hexapods): Insects


17

Table 15.1 – A summary of arthropod adaptations which meet basic needs Organism

Gas Exchange

Waste Removal

(including product)

Locomotion

Appendage

Specialization

Artemia salina (brine shrimp)

Cambarus (crayfish)

Periplaneta (cockroach)


18

ARTHROPOD MOUTHPARTS: A SIMULATION OF DIVERSITY AND NATURAL SELECTION In this experiment, you will simulate the effects of natural selection on a population of crabs on an island. After the experiment, answer the questions below; we encourage you to discuss these! BACKGROUND: Your class has been transformed into individuals of the terrestrial crab species Uca goucherensis. You inhabit a tropical island and exhibit variation in mouthpart morphology, which is heritable. There are three varieties of mouthpart (clothespin mouth, toothpick mouth and spoon mouth), all occurring in the same frequency. While this variation results in different feeding strategies, you are all able to feed efficiently on the most abundant food on the island, the white berries of the Mallow Bush. EXERCISES:

1. Your instructor will assign you “mouthparts:” two spoons, two toothpicks or one clothespin. You will also receive a paper plate with 50 mini marshmallows and a cup.

2. Your goal is to pick up as many mini marshmallows as you can in 30 seconds with your mouthparts. The rules:

You must NOT stab the food You may only take one marshmallow “berry” at a time, and You must put the food into the cup to consider it “eaten.”

3. When your instructor gives the signal, “eat” as many mallow berries as you can for 30

seconds. Your instructor will collect all data.

a. What was the median number of berries collected? _____________

b. Identify any crabs that collected less than the median number of berries. Does there seem to be a disadvantage associated with any particular mouthpart? If so, which?

_______________________________________________________

c. Does there seem to be an advantage for one type of mouthpart over another

when collecting mallow berries? Explain.

______________________________________________________

4. There is a flood, and the Mallow Bushes on the island now produce fewer berries. Fortunately, the wet weather causes an explosion in growth of the Mottled Mud Tree, and those berries are abundant.


19

a. Each group of three students (all with different mouthparts) will receive a plate

with both mallow and mottled mud berries.

b. When your instructor gives the signal, “eat” as many berries as you can in 30 seconds.

c. Your instructor will collect all data and place it on a chart on the board. Use

this data to answer the following questions:

1) What was the median number of berries collected? _________

2) Identify the crabs that collected fewer berries than the median. Is there a particular mouthpart associated with collecting fewer total berries?

__________________________________________________

3) Was any type of mouthpart the most “successful” at collecting berries (total number of berries)? If so, which? __________________________________________________

4) Do you notice any correlation between the crab mouthparts and the type of berries collected? ___________________________________________________ ___________________________________________________

5. One day, a hurricane hits the island and destroys all plants except for the Mottled Mud Tree. Thus, the only food available is Mottled Mud berries.

a. Each student group will receive a plate with lots of Mottled Mud berries.

b. When your instructor gives the signal, “eat” as many berries as you can in 30

seconds.

c. Your instructor will collect all data on the board. Use it to answer the following questions:

1) What was the median number of berries collected? _________

2) Identify the crabs that collected fewer berries than the median. Is there a

particular mouthpart associated with collecting fewer total berries?

__________________________________________________


20

3) Which mouthparts were the most successful at collecting berries?

__________________________________________________

d. Unfortunately, all of the crabs that collected fewer than the median number of

berries cannot survive. However, the students who represent these crabs can re-enter the game as the offspring of the most successful crabs, and should be issued the appropriate mouthparts.

e. Once again, when the instructor gives the signal, “eat” as many berries as you can,

and repeat the steps in c) above. Continue the experiment as necessary.

QUESTIONS: Think about the survival of the crabs with different mouthparts, and answer these questions.

1. Which crabs survived in the final exercise? Which did not, and why? _______________________________________________________________ _______________________________________________________________

2. The final exercise was an example of what evolutionary principle?

_______________________________________________________________

3. In the first exercise, only one food source was available to the crabs: Mallow berries. Although all of the crabs were able to eat the berries, can you think of a disadvantage in having only one food source available?

_________________________________________________________________

4. Consider the results of your entire experiment. Can you draw any conclusions regarding

species diversity in plants (or food sources) and species diversity in animals? Explain.

_______________________________________________________________ _______________________________________________________________

1

Exercise 16:

The Molluscs


The phylum Mollusca is a large and diverse phylum. It contains such familiar organisms as snails, slugs,

clams, oysters, scallops, squids and octopi. Although most of the molluscs are marine, some snails and

clams live in fresh water and many snails and slugs are terrestrial.

EXERCISES:

GASTROPODS: (Snails and Slugs)

There are three subclasses of gastropods, which are the most diverse class of Molluscs. These molluscs are

the only class that undergo torsion, which is a developmental “twisting” of the organism. In torsion, the

mantle cavity is twisted from the posterior of the organism to the anterior, so the now-anterior anus and

mantle cavity open above the mouth and head. Despite the fouling that may take place above the

gastropod’s mouth, this arrangement is beneficial to the animal in that the head may retreat into the mantle

cavity under the protective shell.

A. The Prosobranchs include most of the familiar shelled marine and fresh water snails.

1. Carefully observe the living representatives available.

2. Note the diversity of forms and habits.

B. The Opistobranchs, commonly known as sea slugs, consist of marine gastropods in which the

shell is reduced or absent. They have external gills that are not true ctenidia, and they are

hermaphroditic. Observe any living opistobranch available and consider this question: How

can such slow, exposed, soft-bodied creatures avoid predation?

_______________________________________________________________________

Exercise 16: The Molluscs

2

Figure 16.1 - Mercenaria with left valve and mantle removed.


3

C. The Pulmonates are primarily terrestrial snails and slugs. In these hermaphrodites, the ctenidia

have been ditched in favor of a highly vascularized mantle cavity which acts as a crude lung.

Preserved specimens show the life cycle of a snail.

BIVALVES: (Clams, Mussels, Oysters and Scallops)

A. The common Atlantic clam, Mercenaria mercenaria, is often called a quahog or cherrystone

clam. Look at the clams in a bowl of sea water to observe opening and closing of the valves

and the functioning of the siphons.

B. Morphology

1. Each pair of students should obtain a living clam. The dorsal bulge in the shell near the

hinge is the umbo, located slightly toward the clam's anterior. The instructor will

carefully use the "clam opener" to remove the left valve.

2. Immerse your clam in a finger bowl of fresh sea water; see Hickman for illustrations of

clam anatomy. Gently try to locate and label on Figure 16.1:

a. Fleshy mantle tissue underlying the shell

b. Pericardium (dorsal in the visceral mass, thin and delicate). Look for a slow

heart beat.

c. Incurrent and excurrent siphons; which is dorsal?

d. Adductor muscles which close the valves

e. Head region and labial palps

C. Feeding - Observe the respiratory current and movement of food as follows:

1. Place the fingerbowl containing the clam on the stage of a dissecting microscope and

fold or remove the mantle so that the ctenidia are exposed.


4

2. Use a dropper to add some carmine or Aquadag (carbon) suspension to the water near

the clam's posterior. Trace the direction of water flow and the movement of "food"

particles toward the mouth and labial palps. This requires care in adding the

suspension plus a little time and patience.

3. Describe your results and indicate what you see on the diagram.

4. Now observe the ciliary movement in the ctenidium under a compound microscope

(Demonstration). How does the coordination you see compare with the cilia of

Paramecium you saw earlier in the semester?

__________________________________________________________________

__________________________________________________________________

D. Break off a piece of the shell (valve) you previously removed and note:

1. The thin, outer proteinaceous periostracum (peri, around; ostracum, a shell). The

same material forms the hinge.

2. The middle prismatic layer of calcium carbonate.

3. The smooth nacreous (mother of pearl) inner layers.

E. Other bivalves: Some bivalves are adapted for burrowing into hard materials like wood or

rocks. Shipworms are capable of causing extensive damage to wooden ships, wharfs and

pilings.

1. There is a piece of wood on display that has been attacked by the shipworm Teredo.

Look for the remnants of its rasping shells in the holes in the wood.

2. Observe other demonstration material including shells from a variety of bivalves.


5

CEPHALOPODS: (Squid, Octopus, and Nautilus)

A. Each group of students should obtain a squid. Use Figure 16.2 as a guide in studying its

structure. Carefully dissect to find the major structures. You should know the function of each.

B. Make sure you understand how these animals can swim backwards or forwards using jet

propulsion.

C. Observe preserved cephalopods on display. If possible there will be a living octopus in the

laboratory.

CHITONS

The chitons are a small and fairly specialized class of molluscs rather common on rocky shorelines where

they scrape algae from the rocks with their radulas.

A. Observe the living chiton on display. The chiton shell consists of eight hinged plates

surrounded by a stiff but flexible girdle. The shell and girdle can conform to irregular rock

surfaces - a necessity to avoid being washed away in the pounding surf. Try to dislodge the

chiton.

B. How is this adaptation effective?

________________________________________________________________________


6

Figure 16.2 - Loligo male and female


7

ASSIGNMENT: Fill in Table 16.1 below based upon what you have observed in the laboratory.

REFERENCES: Hickman, et al. (2008) Ch. 16, Ph. Mollusca


8

Table 16.1 - Comparison of major classes of Mollusca.

Gastropoda Bivalvia Cephalopoda

Type of shell

Prosobranchia Opistobranchia Pulmonata

Type of foot and locomotion

Type of feeding

Type of “gills”

Appendix A: Laboratory Rules

1

LABORATORY SAFETY RULES 1. No smoking, eating or drinking. A table outside the lab will be provided for drinks and food. 2. Wear proper protective clothing:

• No open-toe shoes • Keep clothes away from flames • Pull long hair away from face and fasten • Wear proper protective gear, such as lab coats, gloves and goggles, when suggested

3. NEVER mouth pipet. 4. NEVER use a piece of equipment unless you are knowledgeable regarding its proper use 5. Notify the instructor if you must leave the room. 6. Report any injury at once. 7. Clean up spills immediately. 8. ALWAYS consider the safety of yourself and those around you when performing experiments. 9. CLEAN UP after lab classes:

• Dispose of paper/plastic trash in the trash can • Dispose of glass ONLY in glass disposal bins • Rinse all glassware thoroughly and place in a designated area • Wipe down your work station

10. Before leaving lab, ALWAYS wash your hands. 11. ALWAYS be aware of the possible hazards of the chemicals or equipment you are using. _____________________________________________________________________________________ For Biology 104 lab, Fall 2010, hazard rankings are as follows:

• Dissection labs are ranked Low/Medium Hazard • All other labs are ranked No Hazard or Low Hazard

I, _______________________________ have read the Goucher College Bio 104 Lab Rules and agree to (print student name) abide by them. _________________________________ __________________ (signature) (date)

Appendix A: Laboratory Rules

2

No Hazard procedures are those lab procedures which pose no hazard to a student. Examples include: entering data into a computer, reviewing documents or preparing reports, viewing samples using a microscope, etc. No protective equipment is required for such procedures. Low Hazard procedures only pose a hazard if a significant amount of material is splashed or spilled. Examples include: working with solutions of low-hazard materials like salt solutions or buffers, working with media in culture tubes, etc.

Protective equipment for low hazard procedures:

• Chemical fume hoods, safety cabinets, etc. (optional) • Lab coat (optional) • Gloves (recommended) • Protective eyewear (recommended)

Low/Medium Hazard procedures are a special level for dissection of biological specimens. Such procedures require special attention, as preservatives may create fumes and biological specimens require special handling upon disposal.

Protective equipment for low/medium hazard procedures:

• Chemical fume hoods should be at maximum ventilation when preserved specimens are being dissected. Doors should remain closed and air filters may be used to control fumes.

• Lab coat (recommended) • Gloves (recommended) • Protective eyewear (required)

Appendix B: The Research Paper

1

HRIII, 1/07

THE RESEARCH PAPER To be of lasting value, the results of any scientific endeavor must be communicated to others. This may be done orally or via posters at meetings or symposia, but the primary forum in science is the published journal article. Journals in the biological sciences differ markedly in both scope and quality, but the basic style (and format) of research papers is surprisingly similar. Although there is no firm rule many scientific authors follow this order of preparation: 1. Consider what the results show, 2. Select the kinds of figures and tables that would best illustrate the results, 3. Write the text of the Results section to go with these figures and tables, 4. Write the Materials and Methods section to go with these results, 5. Write the Discussion section where you describe your interpretation, conclusions and significance of this work, 6. Write the Introduction that sets the stage for this experiment, 7. List the cited sources in the Literature Cited section, 8. Prepare the Abstract. With practice you will find that this approach and style is an effective and concise way to convey experimental results. [Notes: 1.) All information enclosed in these brackets [ ] is directed specifically to students at Goucher College. 2.) Students should remember that although data are often collected with lab partners and discussion of the results between them is encouraged, written reports must be prepared individually. This includes preparation of all figures and tables, unless otherwise directed. 3.) You should be taking a similar approach in a student research paper as you would for writing a preliminary manuscript for publication. Therefore use: a 12 point font, double space, staple in the top left corner only, and do not use any additional folder or binding apparatus. We are acting as though we are a legitimate research group, therefore in your manuscript do not mention, “ the lab,” “my lab partner,” “the class,” etc. 4.) All student papers at Goucher must be prepared on a computer. Therefore, students are urged to take full advantage of the accuracy/editing features of your word processing program. And, do not neglect the most basic step in any kind of writing: proofread! - a printed copy, not just the computer screen! 5.) Before writing your research paper the biology department also recommends: McMillan, V.E. 2001. Writing papers in the biological sciences. Third Ed. Bedford/St. Martin’s Press, Boston. (Available from the bookstore and the library.)


2

This is an easy to read “How-to-do-it” book with helpful suggestions and examples for beginning biologists. The Goucher Biology Department strongly encourages prospective biology majors to buy and use this book. 6.) Also see Goucher College’s on-line guide to science writing: http://faculty.goucher.edu/writingprogram/how_to_write_for_the_sciences.htm ] The following is a brief introduction to the basic organization and writing of a research paper, and is intended to help students manage the significant differences between scientific writing and other forms of writing.

TITLE/NAME PAGE (SECTION)* These two features appear together on the title page. 1.) A “descriptive” title. By which we mean a title that states (using correct scientific terminology) what you have measured or observed, what animal you are using (including both common and scientific name when possible), a hint of your major result so that the reader will get, from just the title alone, a real idea of what this research can reveal to him. 2.) Your name, institution, [course, lab section and the names of any lab partners], and the date, should be centered beneath the title. Sample Title/Name:

Nembutal is an Effective and Safe Anesthetic for Little Brown Bats Myotis lucifugus

Sven Speleologist

Goucher College Bio 105, Thurs PM Sally Salamander

Holly Hagfish Jan 1, 1997

Space these elements out to use the whole Title Page most effectively. [See the posted sample in the intro lab.] *Note: The “Title/Name” heading should not appear on the title page, but the headings for the remaining sections, as stated below (in bold), should appear in your paper.

ABSTRACT The abstract is a brief summary of your experiment (experimental approach, major findings and conclusions). It usually appears at the beginning of the paper and is often published alone. The most common error here is to try to put too much into this section (think about half to three quarters of a page), however, you should specifically state your main result in a quantitative fashion if possible. (i.e. state “. . . after treatment with the hormone, the metabolic rate went up 50% compared to control


3

animals.” Don’t just say, “the rate increased.”). Because abstarcts are often published separately, citations are not normally included.

INTRODUCTION The introduction serves to set the stage for thinking about the topics and concepts that will be dealt with in this research report. Here, you: 1.) Provide relevant context. How is this specific line of research related to general biological phenomena and the state of knowledge in the field. Move from general issues to points of specific interest (e.g. human health ⇒ cancer ⇒ anticancer drugs ⇒ testing taxol effects on tumors in mice). In other words, provide enough biological background so that your reader will understand how your work fits into the larger picture. An investigator would first conduct an exhaustive literature search to be sure that previous work is not duplicated needlessly and to ensure that they are well versed on the current state of that field. Accordingly, the introduction of the research report would mention specific relevant papers and tell how the present study differs from and/or extends previous results. [Note: Your instructor will establish the depth of background and perhaps the number of references (citations) required for each student report.] 2.) You must let the later sections of your report be your guide as to what must be in the introduction. For example if your research addresses cellular transport mechanisms in mouse intestinal epithelium, then this section must introduce cellular transport, specifically the types found in intestinal epithelium and the reasons why mice are useful experimental models in which to study this process. 3.) State clearly what your rationale was in performing this investigation by explaining what your objectives were or what question you proposed to address. You should also include an explanation of any existing controversy in this area. Sample sentences in introduction: Systemic lupus erythematosus (SLE) is a disease process characterized by the frequent presence of serum autoantibodies and cutaneous manifestations (Ratrie & Provost, 1990). We examined the sera from 23 female SLE patients for the presence of antinuclear antibodies (ANA) and present a correlation of the presence of antibody with the severity of cutaneous symptoms. In-Text Citations: Responsible scientific writing always acknowledges previous work. Therefore, all statements about outside information used in your paper must be followed by a literature citation. Any section of the paper may require citations. Research papers most commonly cite journal articles, but books [e.g. your textbook], internet sources, or even unpublished work from other


4

investigators may also be cited to establish background or support arguments. Footnotes are very rarely used in scientific publications. Direct quotes are also rarely used in scientific publications, but instead a relevant point from another source is paraphrased or summarized and then cited. Take care to keep careful track of all sources because, even in this altered form, they must still be referenced. The names of the author(s) and date (year) in parentheses should be given (see samples below). If there are three or more authors, give the last name of the first author followed by the Latin term "et al." (and others) followed by the date. If no author, use the publishing organization (e.g. Department of Agriculture) and the date in the text. The complete reference for each source will be listed at the end of the manuscript in the Literature Cited section. Note: With the rare exception of some specialized procedures laboratory manuals are not usually considered an acceptable source for a citation [unless your instructor approves]. [Note: Internet sources have become increasingly popular as a way of locating a convenient lead to the major workers or the hot new topics in a particular field. However, students must keep in mind that in an academic field, to maintain confidence in the reliability of any particular claim, peer-refereed journals remain the citation of choice. Therefore, when possible, attempt to cite internet sources only as extensions of other sources of information. It is also important to be discriminating in your choice of sites: choose sources that are likely to produce some confidence in the reader (i.e. the home page for the national “Center for Disease Control” might be more appropriate than “Paul’s Pelagic Parasite Page”).] Sample Citation In Text: Nation and Marin (1991) reported a decline in the incidence of UFO sighting during the period of prohibition. or an alternative form: A decline in the incidence of UFO sightings during the period of prohibition has been reported (Nation and Marin, 1991).

MATERIALS AND METHODS A reader who is reasonably experienced in basic laboratory techniques should be able to repeat your experiments by using the information in this section alone. Note that a cookbook-like list of individual steps or reagents is not acceptable here. You do not tell the reader what to do, like a lab manual would; instead, describe the relevant and unique things that you did. For common procedures a citation referring the reader to a previously published method [or the lab manual] may be sufficient. However, any modifications or special procedures should be specified, as well as such culture conditions as temperature, light, medium, etc.


5

The trick is to use some discretion and report all novel aspects of your procedure, without the trivial details. For example, if you used a reasonably specialized piece of equipment such as a spectrophotometer your results were determined in part by its quality, and you would report the make and model number. On the other hand, details of more standard equipment, for instance the make of the balance used to weigh chemicals, are not usually relevant. It is assumed that normal operating techniques are followed for all equipment utilized, therefore, the steps for blanking the spectrophotometer, boiling samples in a water bath, or other standard procedures need not be detailed. That, “observations were made,” and “measurements were recorded,” etc., is understood by all investigators and should not be mentioned. You should state exactly what organisms were utilized (common and scientific name if possible), and what concentrations of reagents or drug treatments were used. [In this regard, try to avoid two traps to which beginning biology students commonly fall prey: First, before you write this section, it is well worth taking the necessary time to be sure that you understand the difference between the absolute amount and the concentration of a reagent. And second, understand the metric system terms used to describe concentration: e.g. that a 1 µM solution is a million fold less concentrated than a 1M solution, etc. A little effort here will pay off rapidly in your level of scientific literacy.] Mention and reference any statistical analysis procedures that were applied to your data. Sample sentence in materials and methods section: Livers were excised, and a 20% homogenate prepared, as described by Kupffer et al. (1982), except that the final storage buffer was adjusted to pH 7.5 to minimize RNA degradation. Look at several published research articles to get a feel for how this section is handled.

RESULTS Having performed your experiment, the results section presents the real story of what you have learned. You will want to communicate your findings to others, so this will be the heart of your paper. Usually the writing is in the third person, past tense. In recent years a trend towards accepting scientific writing in the first person has appeared, especially in fields such as ecology. Be sure to check the preferred style for the journal to which you plan to submit your manuscript [or check with your instructor]. Present your data here and nowhere else. The results section includes your statement in text form of the facts of what happened or what you observed. There must also be specific references in the text to illustrations of your data in the form of figures and tables. There must be at least one figure or table in every research paper and there must be at least one reference to each figure or table in the text of the Results section.


6

Note: While these illustrations are technically part of your Results section, for a preliminary manuscript such as this, the figures and the tables should not be incorporated into the text of the paper, but should be attached, each on its own page, just after the Literature Cited section. This allows the journal editors to adjust the size and presentation of these illustrations in ways most appropriate to their particular publication. Remember, however, that even though they are at the back of the manuscript the figures and tables are critical components of the body of a research paper (the results section) – they are not part of the Appendix. Preparing the Text of Results section: The text of the Results section points out the particular features of your work that you want to emphasize and report. Begin with the most important, general and obvious findings, then move on to more subtle or minor effects. Be sure to refer to specific aspects of your figures and tables as you make your points. A step from the Materials and Methods section may briefly be restated here in the general form of, “When this was done, these results were seen (see Fig. 1).” Try to avoid saying, “In fig 1 we see . . .” Remember that the focus should be on your result and the figure or table serves as merely an illustration of that result. Therefore, you would normally state a result clearly and quantitatively (when possible) in words first, and then refer the reader to the figure or table that serves to illustrate that result. Sample section in the results section: When male praying mantises were introduced into the cage and mated with female praying mantises, the heads of 98% of the males were eaten during the act of copulation (see fig 1). You may find more than one result to refer to in a single graph. Therefore, along with pointing out the basic increases or decreases (do not fail to point out even the most obvious results) look for trends, subsections of the curve that are revealing, indications of exceptional error etc. and point those out as well – referring each time a major point is made to its illustration. [Remember, you will be writing this paper as if you were part of a research group submitting a paper for publication – therefore, do not refer to, “Our class,” “our lab,” etc. Along the same line of reasoning, because we are submitting a research report, it really goes without saying that we have performed an experiment. Therefore, do not say, “In our experiment we studied respiration rate . . .” Instead, jump right into a statement of what was done – for example: “When the respiration rate was studied . . .”] You may comment briefly here on the validity of your results, but you should not comment on why your results did or did not match your expectations. You must intentionally save all mention of expectations, surprise, interpretations and conclusions for the Discussion section. Some indication of the variability of your data is necessary. Average values of replicates are impossible to interpret without some indication of the overall range, standard error or other measures of variability. This information is often included in the figures. If any data are discarded, explain the reason for this decision.


7

Note that the measurements that you recorded in your notebook as you performed the experiment are known as your "raw data." Do not present raw data as a figure in the results section! This information is almost always processed, by taking averages of replicates or by other statistical manipulations to simplify and summarize the results, before presentation in figures and tables. If you feel that the raw data, as you originally recorded it in your notebook, has information that the reader would find useful [or if it is required for a student paper], include it as an Appendix at the end of the paper, after the attached figures and tables. Preparing the Illustrations of the Results: You are charged with selecting the most effective figures and tables to present your data. Figures: A “figure” includes everything that is not a table. For instance graphs of all types, photographs, maps, drawings of an apparatus etc. are all figures, and are referred to as they are mentioned in the text as figure 1, figure 2, . . .etc. When graphs are used, make sure that the axes are labeled to show what is measured, and include the units employed in that measurement (see sample below). Always include the line that connects the points on a graph (unless you have some specific reason [or instruction] not to). Do not ask a computer program to fit a curve or straight line to the points on your graph unless you have a compelling reason [or specific instruction] to do so. Every figure must be labeled at the bottom with its appropriate number and include a descriptive legend. Figures do not have titles at the top. “Descriptive” means a statement of what you have measured or observed, what animal you are using (including both common and scientific name when possible), how many animals are represented in this illustration, what the error bars mean, etc. This information should be complete enough to allow this illustration to “stand alone” (i.e. the reader will know what is going on without reference to the text). Therefore, just stating, "Absorbance versus Concentration" is not enough! In your figure legend do not state, "This is a graph . . ." or, "This graph shows . . ." It is obvious that this is a graph. Unlike the descriptive title of the manuscript itself, do not describe your results in the descriptive legend, e.g. do not say, ". . . over time the girls grew taller." The figure that you will have selected will be such an effective visual aid that the reader will instantly see for himself the increase in height. Instead, make these statements of findings in your Results section, and then refer the reader to the appropriate illustrative figure. See the sample figure with descriptive legend below.


8

Sample figure:

Tables: Tables can be a very effective way to organize and summarize data, so take time to design them to best advantage. Tables must be labeled with their appropriate number, and a descriptive title at the top. Tables may include additional explanatory statements at the top, or sometimes these may be given in their own footnotes at the bottom, to clarify individual entries. The labeling and information presented in the table should make it understandable without consulting the text, i.e. it should be able to "stand alone." The table along with its number and descriptive title should all fit together as part of a single construct (i.e. If possible, avoid a pieced-together look). See the sample table given below, with descriptive title on top.


9

Sample table: Table 1. Comparison of published estimates for RNA molecular weights with those determined by the glyoxal method. RNA molecular weights were determined after gel electrophoresis from standard curves derived using denatured SV40 DNA restriction endonuclease fragments of known size (Wallace, 1990). Each determination reflects the average of at least five separate determinations using different gel systems. Molecular Weight X 10-6 RNA Glyoxal Gel Published Reference 16S E. coli 0.55±0.03 0.54 Smith, 1972 23S E. coli 1.03±0.03 1.07 Jones, 1977 18S Mouse 0.68±0.02 0.68 Filipowitz, 1981 28S Mouse 1.75±0.05 1.74 Eager, 1966 18S Human 0.67±0.02 0.68 Ernst, 1988 28S Human 1.75±0.05 1.76 Ernst, 1988 19S Physarum 0.70±0.02 0.76* Sampson, 1990 26S Physarum 1.29±0.0s 1.37* Sampson, 1990 *Determined under denaturing conditions

DISCUSSION Your discussion explains what the results show and/or interprets what they mean in light of the question or controversy which motivated your experiment. Open the discussion by briefly restating the question addressed by your experiment. For example, if you did an experiment to determine if petroleum products could make hair grow on hairless (nude) mice, your opening sentence could restate the question or topic like this: Sample discussion opening: We studied the effects of petroleum compounds on hair growth in nude mice. When compound X was tested we found . . . etc., which adds support to our hypothesis that these classes of compounds can be effective in . . . etc. Match each paragraph in the discussion section with a paragraph in the results section. Remember that interpretation and conclusion is what you want here, not just a rehash of the results. Open each paragraph with a one-sentence summary of the method used and the result obtained. When conclusions follow directly without interpretation from the results, use words


10

and phrases like, “therefore” and, “this result shows that. . ." Use, “this result suggests that . . .,” and, “this result supports the conclusion that . . .,” when the results are not sufficient in themselves to confirm conclusions. Justify your conclusions. Feel free to discuss alternative explanations for your findings. This is where you may elaborate on, and analyze how, your findings did or did not match expectations you may have had based on previously published research or other preliminary work. Frequently, new and interesting questions may arise from your work. If so, these should also be discussed (you may also briefly suggest follow-up experiments and improvements in the experimental protocol). If you found articles in the scientific literature that support or contradict your findings, mention and explain (and cite!) their implications here. [Consider a sentence that should never appear at the beginning of the discussion section of a student paper. “The results were as expected . . .” This statement reveals immediately that very little thought has gone into the meaning of your findings. Instead, concentrate on interpreting why the result was seen, what mechanisms were involved, and what this tells us about the usefulness of the system we are studying. After that, if it has not yet become obvious, you might reveal that the observation was consistent with our expectations, and why. A second sentence to be avoided is, “For figure 1 the results were . . .” Here we see that the student has forgotten that the result itself is more important than the illustration called figure 1.] [Note: Your interpretations, analysis and conclusions should be the greatest proportion of this section. Discussions of error should be in a short paragraph just before a summary statement. If you find that statements of possible sources of error or suggested improvements comprise the bulk of your Discussion section go back and think some more about what you have learned from your results!]

LITERATURE CITED All sources cited (referred to) in the text (and no others) should be listed here, with the first author’s last names in alphabetical order, as shown in these samples. The exact format varies slightly among journals. Your instructor may specify a journal to use as a guide. In any case, be consistent. Note: In science writing this section is not called: Work Cited Sample citations: 1. From a journal: Smith, A.B. and C.D. Jones. 1986. Growth of Euglena on autotrophic media in light and dark. American Journal of Botany. 73:104-110. [Many journals that used abbreviations in the literature cited in the past have switched to using full journal names.] 2. An article or chapter from a book:


11

Touched, I. B. 1991. Unidentified flying objects during modern history. Pages 115-119 in UFOs in Our Society. New York, ABC publishers. 3. A book: Hickman, C.P., Roberts, L.S., and Larson, A. 2001. Integrated Principles of Zoology. 11th Edition. New York, McGraw Hill. 4. From the Internet: Umble, M. The Student Arrives at the Door. Online. Internet. 7 October 1996. Available http://www.peacecorps.gov/www/essay/ If there are multiple citations from the same author list them chronologically (earliest first).

APPENDIX In some cases, where it is useful, [or if required by your instructor] detailed explanations of techniques, formulas or “raw data” may go in an appendix at the end of the paper. Note: the material in the appendix is extra - it is not really needed to write the paper.


12

Appendix C: Spectrophotometer and O2 Meter Operating Instructions

1

DIRECTIONS FOR THE SEQUOIA-TURNER MODEL 340 SPECTROPHOTOMETER 1. Make sure the machine is plugged into the outlet. 2. Turn the MODE switch from OFF to TRANS and allow the machine to warm up for five

minutes. 3. Set the wavelength with the thumbwheel knob (590 for BPB, or 525 for Euglena). 4. Place the appropriate “blank” in the tube holder and cover the tube with the cap. 5. Press and hold the ZERO SET button while adjusting the ZERO knob until the display

indicates 0.0. Release the ZERO SET button. 6. With the MODE switch still set at TRANS, adjust the 100% T/OA COARSE knob to

approximately 100. Adjust the T/OA FINE knob to exactly 100.0. 7. Set the MODE switch to ABS. The blank should now read 0.000. 8. Replace the blank with a tube of well-suspended cells or other solution. Replace the cap and

read the absorbance from the digital display. It may take a few seconds for the digital display to stabilize.

9. When finished, turn the machine OFF and unplug it from the outlet. NOTES:

1. Follow the directions given in lab for careful and thorough suspension of cells before reading.

2. Make sure you have wiped off the outside of each tube. Fingerprints will affect your

readings. 3. For accurate readings place your tubes in the tube holder in the same direction each

time.

Appendix C: Spectrophotometer and O2 Meter Operating Instructions

2

DIRECTIONS FOR THE OXYGEN METER 1. The oxygen meter requires about an hour or longer to warm up once plugged in and turned

on. The oxygen meters should already be plugged in and warmed up when you come into the lab.

2. Hold the probe so the membrane end is exposed to air. NEVER touch the probe membrane! 3. Turn the function switch to “%”. 4. Turn the “O2 Calibration” knob until the display reads 100% and varies no more than about

0.1%. 5. The meter is now calibrated. Place the probe into the Euglena culture in the BOD bottle and

take readings as instructed. 6. Upon completion of the exercise, rinse the probe with distilled water, shake off excess water,

and place the probe into the BOD storage bottle with about a half-inch of water inside. If you find it impossible to calibrate the oxygen meter, please tell the instructor. The membranes on the probes are prone to damage, and often simply changing the membrane resolves the problem.

Bio 104 Lab Manual 2010

Documents

Transcript of Bio 104 Lab Manual 2010