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Lab 7 Microscopy and Bacteria The cell is the fundamental unit of life; it is the smallest and simplest unit that possesses all the characteristics of the living condition. All living things, including plants, fungi, and animals are made up of cells. Some organisms, such as bacteria, are unicellular, consisting of a single cell. Plants and animals are multicellular, composed of many cells. ALL cells have the following structures in common:

A. Plasma membrane- a flexible barrier made of a phospholipid bilayer with embedded proteins and other molecules that surrounds the entire cell. This is what divides the outside from the inside and controls the movement of materials into and out of the cell.

B. Cytoplasm- a semi-fluid matrix that fills the interior of the cell and surrounds the internal structures of the cell.

C. Cytoskeleton- a rigid support system made of protein tubes that also serve as railways along which materials are carried throughout the cell.

D. DNA- a string of nucleotides that contains all the genetic material of the organism. E. Ribosomes- structures made of proteins and RNA that are used to build proteins.

These similarities lead us to believe that all life evolved once and all species, from bacteria to blue whales to birch trees, inherited these basic structures from that original ancestor. Using their similarities, we can build a tree of life like the one on the right. There are two basic categories of cell: Prokaryotic cells, which were the first types of cells to evolve, lack a membrane-bound nucleus. Bacteria are the best-known prokaryotic organisms; they are unicellular and relatively simple in their components. Eukaryotic cells are larger and more complex, in that they contain a membrane-bound nucleus and organelles. Eukaryotes include fungi, animals, and plants as well as many unicellular organisms like protists.

Prokaryotes are typically much smaller than eukaryotic cells. Notice the size difference (in micrometers).

Here are some bacteria (the small purple lines) on a human cheek cell (the pink outline). What is the dark pink structure in the cheek cell?

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The classic five-kingdom system based on Linnaeus categorization and which contained Monera (prokaryotes), Protists, Fungi, Plants, and Animals lumped together two vastly different groups of single-celled prokaryotes based solely on the absence of membrane-covered organelles (as that is the definition of a prokaryote). In the recent past, Carl Woese and others noticed distinct differences between the two groups of prokaryotes. They studied their evolutionary divergence, and suggested splitting kingdom Monera into two new kingdoms: Bacteria and Archaea. Woese now promotes altering the classification scheme again by adding an eighth level: a Domain. Woeses three-domain system includes Domain Bacteria (in which Kingdom Bacteria would be included), Domain Archaea (which contains Kingdom Archaea), and Domain Eukarya (which includes Kingdoms Protista, Fungi, Plantae, and Animalia). The relationships among these three domains are uncertain. There are several hypotheses trying to understand how these groups evolved. There is even evidence that Archaea and Eubacteria hybridized to form Eukaryota in what has been coined the biological ring of life.

In the next several labs, we will explore the diversity of life by examining several specimens from the major groups. And since most life is microscopic, we will start by learning to use a microscope.

The two figures come from Martin and Embley, Nature 2004

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INTRODUCTION TO THE MICROSCOPE A microscope is a tool used to observe objects that are too small to be viewed by the naked eye. In this lab, we will learn the parts and use of a compound light binocular microscope. Compound means that the scopes have a minimum of two magnifying lenses (the ocular and the objective lenses). Binocular microscopes have two eyepieces, and light refers to the type of illumination used, that is, visible light from a lamp. Parts of the Microscope Here is a diagram of the compound microscopes we will be using:

Each objective lens magnifies the specimen by a certain amount: four times [4x], ten times [10x], forty times [40x] one hundred times [100x]

In addition, the eyepieces or oculars, magnify the magnified image ten times (10x). Therefore, any object under the scope will be magnified twice. If using the 4x objective lens, the eyepiece will magnify that magnified image by 10, thus the object you see in the eyepiece is actually 40 times bigger than the object would be with the naked eye (naked eye x 4 x 10 = 40x). Notice that as the magnification increases, the field of view (the width of space you can see) decreases.

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General rules A. Always cover your very expensive microscope when not in use B. Carry the microscope seldomly. If you have to, support it by putting one

hand on the arm and another under the base. C. Do not adjust the metal screw under the stage! This moves the condenser

off of the light source. D. Always cover your very expensive microscope when not in use

Steps to using your very expensive microscope.

You MUST perform these steps IN THIS ORDER each time you use your very expensive microscope. This includes when changing slides.

After uncovering the microscope and plugging it in: 1. Lower the stage all the way down using the course focus knob. 2. Cleaning all lenses before each use is highly recommended. Dirty lenses will cause a blurring or fogging

of the image. Always use lens paper for cleaning. Kimwipes or other tissues can scratch the lenses. 3. Make sure that the smallest (4x) objective lens is in the

locked, vertical position above the stage by turning the revolving nosepiece.

4. Lock your slide into the spring-loaded stage clips. The

slide goes between the clips, not under them. 5. Turn on the microscope by flipping the switch in the

back. 6. Bringing the object into focus

a. Look into the eyepieces. USE BOTH EYES! Our ancestors didnt spend millions of years evolving binocular vision just so that you can squint like a pirate into your microscope. You can adjust the distance between the oculars to fit your eyes. To do this, gently push or pull the rectangular metal plate at the base of the eyepiece until you see only one circle as you look through the oculars.

b. Increase the light intensity until you can see light emitted from the lamp. You can do this by adjusting the iris diaphragm which opens and closes the aperture. Or by adjusting the scrolling red button on the side of the scope which controls the intensity of the light source. When looking at small, mostly transparent specimens, often less light is best. At any time, if you are having trouble seeing parts of your specimen, try adjusting your light.

c. Bring the specimen into view using the mechanical stage knobs. These move the stage or specimen horizontally (not up and down). They act a bit like the knobs on an Etch-A-Sketch. The top knob moves the stage back and forth; the bottom moves the specimen left and right. Put the bulk of the specimen in the center of the circle.

d. Slowly bring the slide into focus using the COURSE focus

knobs.

e. Once the specimen is in as much focus as possible you can use the fine focus knobs.

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f. You can also focus each ocular independently. i. Looking through the right eyepiece only, use the fine focus knob to sharpen the image.

ii. Looking through the left eyepiece only, turn the moveable ring on the left eyepiece until the

image is sharply focused. This ring enables you to "custom focus" the microscope to your eyes. You only need to do this once for each lab period.

iii. As you look through the oculars you may see that the image appears grainy. The position of the condenser needs to be adjusted. To do this, raise the condenser to its highest position using the condenser focusing knob. Look through the oculars and slowly lower the condenser until the graininess just disappears.

g. Tips for finding the specimen i. In most cases, everything you are looking for will be

stained a distinctive color. If what you are looking at on the slide is not bright blue, pink, green, etc then you are not looking at the right things.

ii. The specimen is embedded in the slide. If you move the slide via the stage knobs and the dots you are looking at do not move, then you are not looking at the slide.

iii. If you see a lot of brownish-gray dots and squiggles, try

rotating the eye pieces. If the dots move, then you are not looking at the slide. You are looking at the eyepieces.

iv. Once you find the specimen, put it in the center of the view. When you increase the magnification, you reduce the area that you see (look at the trees in the magnification pic above). If the specimen is on the edge of the view at 4x it might be out of sight in the 10x. Note: the pointer is not always at the center of the view.

7. Then and only then can you change objective lenses for a closer view. a. Put the object you want to see closer at the tip of the pointer or in the center of the view.

b. Move the revolving nosepiece clockwise so that you are using the next-sized objective lens (10x).

c. Again use the COURSE focus knobs to bring the specimen into focus.

8. If you want to see it still closer, move the revolving nosepiece clockwise once more to bring the next-

sized objective lens (40x) into use. a. !!!!Only use the FINE focus knobs at this magnification!!!! Otherwise you could ram the stage into

the lens and break your very expensive microscope.

b. DO NOT PUT THE 100x OBJECTIVE LENS DOWN UNLESS YOUVE READ THE RULES BELOW!!!!!!! This lens requires you to apply oil to the slide and other procedures and is easily broken, thus damaging your very expensive microscope.

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Taking good notes 9. When studying from slides, try to focus at the highest magnification that allows you to see the entire

specimen in as much detail as possible.

10. Do not draw dots and do not zoom in so much that you cant see anything.

11. Draw large drawings in as much detail as possible. You need to know what makes each specimen unique.

12. When you are done with this specimen, perform the steps above, but mostly in reverse.

a. Rotate the revolving nosepiece COUNTER clockwise until the smallest objective lens (10x) is held vertically over the stage.

b. Lower the stage completely using the course focusing knobs.

c. Turn off the microscope

d. Remove the slide

e. Cover the very expensive microscope

USING THE 100X LENS Never use the 100x objective lens unless you are given explicit instructions to do so and information on proper technique!!!! Do not use it if you think you know how.

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Slide terminology There are some phrases and abbreviations that you need to understand in order to properly orient yourself to slides. The abbreviations especially will be useful throughout the semester. Mount- when a specimen is fixed in glass as in a slide Whole mount (w.m.)- the entire specimen is mounted in the slide. The specimen retains its three-

dimensional shape. You will only see the external parts of the organism unless it is transparent. If it is transparent, you can focus on different planes throughout the organism.

Section this means that the specimen has been cut

in one of the following ways:

Cross section (c.s.)- the specimen is cut the short way across the long axis of the body. This usually results in a thin circular sliver that shows internal structures.

Longitudinal section (l.s.)- the specimen is cut the long way through the short axis of the body. This

usually results in a thin long sliver that shows internal structures.

Smear- a thin layer of a substance (usually a liquid) that is dragged across the surface of the slide in order

to show its component parts. Usually used for blood smears, which show plasma, blood cells, and any parasites living within and fecal smears which will show fecal material and any parasites living within.

Double mount- two individuals are mounted on the same slide. Cover slip- if you set up your own slide (like of pond water or something), you

may need to put a cover slip on it. This is a small square piece of glass or plastic. Be careful not to bend them or they will break. Many of these are disposable when you are through.

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GETTING TO KNOW YOUR SCOPE: I. Finding the Specimen

A. Place the letter e slide on the stage and center it over the stage opening.

NOTE: Slides should always be placed on and removed from the stage when the 4X objective is in place. Removing a slide when one of the higher objectives is in position may result in the lens being scratched.

B. Looking through the oculars, find the letter e and use the stage adjustment knobs to center it in the field of view. Only use the coarse focus knob to focus.

C. Move the slide slowly to the right.

i. In what direction does the image in the oculars move? ____________

ii. Is the image as seen through the oculars inverted relative to the letter on the slide? ____________

II. Determining Spatial Relationships The depth of field is the thickness of the specimen that may be seen in focus at one time. It is necessary to focus up and down in order to clearly view all planes of a specimen. To investigate the depth of field, follow this procedure to study the thread slide provided.

A. Rotate the 4X objective into position, remove the letter slide and place the thread slide on the stage. Center the slide so that the region where the two threads cross is in the center of the stage opening.

B. Focus on the region where the threads cross. Are both threads in focus at the same time?

C. Rotate the 10X objective into position and focus on the cross.

Are both threads in focus at the same time?

D. Determine which thread is on top by using the fine focus knob.

III. Preparing a wet mount

The material studied with a compound light microscope is usually mounted on a slide. To study the living material in the following exercises you will be required to prepare temporary slides called wet mounts. The general procedure for preparing wet mounts follows:

A. Wet mount I: newspaper

i. Using the water dropper bottle, place a drop of water in the center of a clean slide.

ii. Using forceps, add a small piece of newspaper with words to the drop of water.

iii. Place the cover slip at a forty-five degree angle above the slide with one side of the cover slip in contact with the edge of the water droplet as shown:

e

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iv. Gently lower the cover slip onto the drop of water. Be careful not to trap air bubbles underneath. The function of the cover slip is to flatten the preparation, to prevent the material from touching the high power objective, and to help prevent the drop from drying.

B. Wet mount II: Your cells i. Place a drop of water on a clean slide.

ii. Using a toothpick gently scrape your inner cheek surface, and tap

the scraping into the drop of water. Repeat this process two or three times.

IMPORTANT: Discard your toothpick in special biohazard

container

iii. Add a cover slip. Place on the stage with the lowest objective down.

iv. Leaving the slide in place on the stage, add a small drop of methylene blue stain to the edge of the cover slip. Allow the stain to diffuse across the slide under the cover slip for a couple of minutes.

v. At 40X, you should be able to detect the nucleus, organelles, folds in the cytoplasm, and even bacteria on the surface of the cells!!

vi. Methylene blue is a simple stain and should stain everything it contacts. You would not be able to see the clear cheek cells without stain.

vii. Describe the shape and structure of these cells.

viii. Measure their diameter, using the width of the field of view.

ix. Find the following; a. Plasma membrane: flexible edge of each cell b. Nucleus: dark circle inside each cell c. Mitochondria and other organelles: small dots within the cell (however, some of these

might be bacteria) *Please refrain from putting other waste into the biohazard bags, such as paper towels, paper, etc.

CLEAN UP: Used slides go into the sharps container. Toothpicks and anything not glass that has

touched bacteria or other biohazardous waste go into the biohazard bags*!

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EXPLORING THE DIVERSITY OF LIFE Once again, the next several labs will take you through the diversity of life and point out several features that make each group successful and unique.

This lab will focus primarily on the bacteria. We will not see any Archaea. This does not mean members of Domain Archaea are not important; they are among the strangest of all species, able to inhabit a variety of environments, including the most inhospitable places on Earth. THE EXTREME LIVES OF THE ARCHAEA Archaea can be classified into three general subcategories: Methanogens, Extremophiles, and Nonextreme archaea.

Methanogens live in anaerobic environments (small amounts of oxygen are toxic to them) and use hydrogen gas (H2) to reduce CO2 to CH4 (methane gas).

Extremophiles are capable of living in environments that are very hot

(thermophiles), very salty (halophiles), highly acidic or basic (pH-tolerant), and under great amounts of pressure (pressure-tolerant).

Nonextreme archaea grow side-by-side with bacteria but differ, as all archaea, in

some key attributes.

Archaea are different from bacteria because peptidoglycan (discussed later in this lab) is absent in archaeas cell walls and the archaea possess a very unique set of lipids. The genetic material of archaea is also different from bacteria; Archaea have intron sequences whereas bacteria do not. Members of Domain Archaea can also be identified through unique ribosomal RNA sequences. Scientists study these weird creatures to figure out what life might have looked like in the beginning or what lif might look like on other planets.

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BACTERIA ARE EVERYWHERE! Bacteria can be found anywhere you look for them; they are ubiquitous. They are on your skin, in your mouth, your intestines, the lab bench, in water, in snow in Alaska, in the hot springs in Yellowstone National Park, and most predominantly on your cell phone. Some bacteria can grow without oxygen and others thrive in extreme pH or salinity. Bacteria are typically too small to see (unless they bunch up into a film, like plaque on your teeth). Bacteria are so small that their size is measured in micrometers. There are 10,000 micrometers (m) in 1 centimeter (cm). To give you an idea of how small that is think about this: The periods in this document are about 500 m in diameter. A eukaryotic cell is about 50 m.

Bacteria average about 5 m. A virus is about 0.05 m. DNA has a width less than 0.0005 m. Since bacteria can be found anywhere, it is important to your health to understand there are good bacteria and harmful bacteria. Pathogens are organisms that make people sick, such as Streptococcus pyogenes, which causes strep throat. Most people think that all bacteria are pathogens. This is NOT true. Only about 1% of all known bacteria actually make people sick. They others just move around doing their own bacteria stuff. The majority of bacteria actually are beneficial to life as we know it. Bacteria are responsible for humans being able to digest food, make yogurt and some cheeses, cleaning up wastewater, creating certain antibiotics and some organic pesticides. Bacterial Structures Bacteria have three structures that separate their internal contents from the outside world:

1. The capsule is the outer most structure. It is a polysaccharide structure that protects the bacterial cell.

2. Just inside the capsule is the cell wall. The cell wall is stiffened by peptidoglycan (a protein and sugar molecule), and serves as a barrier that provides structural support for the bacteria.

3. Inside the cell wall is the plasma membrane, the same structure as all cells. It is a semi-permeable phospholipid bilayer.

Another unique feature of bacteria is the way its DNA is structured. Rather than being made of long lines of chromosomes, like ours is, bacterial DNA is one piece that is circular. Bacteria also do not have a nucleus in which their bacteria is housed, rather their DNA forms a dense ball called a nucleoid.

Bacteria on the head of a pin

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Since most bacteria reproduce asexually, in a process called binary fission (see image at right), they do not combine genes with any other individual. However, bacteria often have other pieces of circular DNA called plasmids. Bacteria can share these plasmids between individuals and can even pick them up from dead bacteria. In this way, bacteria can evolve much quicker than expected. This is sometimes why anti-biotic resistance can spread so quickly. Bacterial cells also often have long tail-like structures called flagella (singular flagellum), that are used for locomotion and smaller, hair-like structures called pili (singular pilus) which bacteria used to attach to each other and share genes (a process called conjugation).

Binary fission Conjugation Naming Bacteria Bacteria are generally unicellular prokaryotes. It is important to say generally here because some bacteria exist primarily as a colony of unicellular individuals. Some bacteria can be named using their shape and how they hang out with each other. All bacteria have shape, but not all are named in this way.

If they form colonies, add a prefix:

strepto- long chains

staphylo- clusters

diplo- pairs

Shapes:

bacilli- rod shaped

cocci- round and ball-like

spirilla and spirochetes- cork-screw-shaped

For example, bacteria shaped and organized like this:

Would be called diplobacilli.

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The Gram Stain Bacteria are classified as either Gram-negative or Gram-positive via the use of a Gram stain developed by Hans Christian Gram in 1884. Gram-positive bacteria have high levels of peptidoglycan, a polymer of modified sugars that is cross-linked by short polypeptides specific to each species of bacteria, which traps violet dye. Gram-negative bacteria still possess peptidoglycan, though the amount of the polymer and organization of the cell wall (the peptidoglycan layer is sandwiched between an outer membrane and the cells plasma membrane) keeps the violet color from fixing to the cell. The tartar on your teeth contains both Gram-positive and Gram-negative bacteria. Using the following directions (taken in part from the ScholAR Chemistry instructions and Scully 2009) and a toothpick to make a Gram stain.

1. Put on goggles. 2. Use a toothpick to make a wet mount of tartar from your teeth or scraping of cheek cells

Or use a sterile loop to scrap just the bacteriafrom the Petri dishes provided (try not to dig into the agar).

3. Allow the slide to dry by attaching it to the clothespin or if available, pass slide through flame for

one second. Do not torch the slide, just pass it through to dry it off. This fixes the bacteria to the slide.

4. Cover smear with several drops of crystal violet. Let stand for one minute. This is a dye that

diffuses under the cell wall and hangs out in the peptidoglycan layer. It turns the cells dark.

5. Pour off excess stain. Dip slide into container of tap water to rinse or use squirt bottle, but do

not squirt too hard or you will remove all the good stuff.

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6. Drain excess water. Cover smear with iodine solution. Let stand for one minute. The iodine is a large molecule that binds to the crystal violet, preventing it from diffusing back across the cell wall.

7. Pour off excess iodine. Wash lightly as before. Drain excess water.

8. Tip slide and add several drops of 95% denatured ethanol (EtOH) to upper end of slide.

Allow alcohol to flow over smear. After several seconds, wash slide again, as before. Do not leave alcohol on too long. The alcohol removes some of the peptidoglycan from the bacterias cell wall. In Gram-negative cells, the entire outer wall is washed away, allowing the violet stain to leak out, discoloring it. In Gram-positive cells, the peptidoglycan layer is thicker and so not all of the wall is removed. What would happen if you leave the alcohol on too long?

9. Drain excess water. Cover smear with Safranin O stain. Let stand one minute. This stain gives the Gram-negative cells some color so that they can be seen under the scope (otherwise theyd be transparent). The Gram-positive ones are colored by the crystal violet left under their cell walls, so they remain darker.

10. Wash slide again, as before. Blot away excess moisture and allow it to dry. 11. Add a cover slip and put the slide on slide stage. 12. Use the steps and rules of using the microscope above to view the specimens. The 40x objective

(400 times magnification) is all that is needed. Look for colors, shapes and groupings.

Gram-positive bacteria appear dark purple while Gram-negative bacteria are pink.

GRAM NEGATIVE = PINK

GRAM POSITIVE = PURPLE

CLEAN UP: Used slides go into the sharps container. Toothpicks and anything not glass that has touched

bacteria or other biohazardous waste go into the biohazard bags!

Empty excess stain into the chemical bucket under the fume hood.

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And why do we care? Gram staining is used to identify unknown bacteria. When you go to the doctor with a cold, he or she may scrape your throat to take a sample of bacteria and perform a Gram stain to see how to treat you.

Gram-negative bacteria tend to be more pathogenic while Gram-positive ones are less so. The outer membrane can be toxic and prevents both built-in defenses of the infected organism and outside treatment (e.g. antibiotics) from penetrating the bacteria. In fact, penicillin works by preventing growing bacteria from building those peptidoglycan layers. (FORM = FUNCTION!)

Although it seems simple, to perform it reliably requires practice. Bacterial diversity Although I am trying to structure these labs phylogenetically, and phylogenetics these days are based mostly on DNA, bacteria are still hard to put into categories that make sense consistently. This is because they can exchange genes easily via conjugation, even between bacteria that are distantly related. Here we will look at some bacteria that exemplify their shapes and relationships.

You are responsible for looking at a variety of slides of each type of bacterium and knowing the information given. It is recommended that you copy the information and draw examples of what you see when looking at the slide. I also recommend drawing these at 40x and in as much detail as possible. Make large drawings. Otherwise your notes will say that they all look like little dots. There may be more or fewer slides than listed here.

Domain Bacteria. Kingdom Eubacteria A. GRAM-POSITIVE

1. Round-shaped a. Staphylococcus- These cluster-forming bacteria are non-

motile and are commonly found in the human mouth and skin. When they get into food, they can release toxins that can cause food poisoning. They can also cause infection of wounds in the skin.

b. Streptococcus- These chain-forming bacteria (sometimes

found in pairs) are commonly found in the mouths of humans, but can become a human respiratory pathogen that can cause strep throat and bacterial pneumonia. They are also responsible for Scarlet Fever.

2. Rod-shaped

a. Bacillus spp- Species in this genus can be found almost everywhere and live as nonpathenogenic and pathogenic forms. They are used in molecular research because of their relatively large size and ease of use. Some species (B. subtilis, B. cereus, and B. coangulans) are known to cause food to spoil. Anthrax, a disease of humans and other animals with many symptoms, is caused by inhaling or ingesting the spores of B. anthracis.

PURPLE

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B. GRAM-NEGATIVE 1. Spiral-shaped

a. Spirillum volutans- These spirally bacteria do not really cause any major disease, but they are spirally-shaped, so thats neat.

2. Rod-shaped

a. Escherichia coli-This chain-forming bacterium lives a happy, friendly life in human intestines, but certain strains are responsible for most food-borne illnesses in the US.

b. Pseudomonas aeruginosa- Because it thrives on most surfaces, this bacterium is often

found on and in medical equipment, including catheters, causing cross-infections in hospitals and clinics. It is implicated in hot-tub rash. It is also able to decompose hydrocarbons and has been used to break down tarballs and oil from oil spills

C. CYANOBACTERIA

Also known as blue-green algae, these bacteria are amazing, because they invented photosynthesis. They take energy from the sun, a few inorganic materials (water and carbon dioxide) and convert them into fuel and materials for lifes processes. In the meantime, they release oxygen. Billions of years ago, the Earths atmosphere contained very little oxygen and lots of carbon dioxide. The evolution of photosynthesis in the cyanobacteria changed all that. Scientists are now looking to cyanobacteria to create fuel to replace gasoline.

We have many types of cyanobacteria to see in lab today: Nostoc, Saprolegnia, Oscillatori, Gleocapsa

D. NITROGEN-FIXERS All organisms need nitrogen to build their proteins and DNA. Fortunately, nitrogen is the most common element in the air. Unfortunately however, the nitrogen in the air is not in a form that most organisms can use. Instead, plants often rely on nitrogen-fixing bacteria to convert the nitrogen in the soil to a form that they can use. Some plants even have special chambers on their roots (root nodules) to house these bacteria; thats why plants like peanuts and soy beans have a lot of protein. In turn, animals get their nitrogen from the plants they eat. In lab today, you can see Rhizobium, a root nodule nitrogen-fixer and the nitrogen-fixing Anabaena (also a cyanobacterium).

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BIOL 1108K Lab 7 Microscopy and Bacteria Name_______________________________ 1. What is the eighth taxonomic level proposed

by Woese? A. Kingdom B. Domain C. Planet D. Kingdom E. Malebolge

2. Say you want to look at a known specimen,

like a protist (a single-celled eukaryote). Which objective lens is most appropriate to use when you start looking for it on our scopes?

A. 4X B. 10X C. 40X D. 100X

3. When looking at a specimen using the 10x

objective lens, how much total magnification is it under?

A. Double B. 10 times C. 40 times D. 100 times E. 1000 times

4. Which of the following lives peacefully in

your body, most of the time? A. Rhizobium B. Plamsodium falciparum C. Trypanosoma D. Escherichia coli

5. All Gram positive bacteria are pathogenic.

A. TRUE B. FALSE

6. What does it mean when a slide has c.s. written on it?

7. For the table below, put a check in the box if the group has the structure. Prokaryotes Eukaryotes

Cell membrane Cytoplasm

Cell wall Asexual reproduction

DNA Nucleus

Chloroplasts Ribosomes

Unicellular species Peptidoglycan

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8. Draw the cells from your cheek. Is this prokaryotic or eukaryotic? Label all the visible structures.

9. Draw Anabaena and explain why it is important.

10. Why is it important to know if bacteria are Gram positive or Gram Negative before a doctor prescribes a medicine?

11. Name the following bacteria based on their shapes and groupings:

A.

B.

12. Describe the type(s) of bacteria you saw in your Gram Stain.

A. Is it Negative or Positive? B. Name them using the same nomenclature as above.