BIOL1230

of 51 BIOL1230 MICROBIOLOGY

Northeast State Technical Community College 2005 Updated May 2010

MICROBIOLOGY LABORATORY

MANUAL Updated for fall 2010

Written and edited by Microbiology faculty and staff Student Manual



MANUAL CONTENTS INTRODUCTION TO MICROBIOLOGY LABORATORY

Welcome to the laboratory component of Microbiology! These laboratory sections have been designed to enhance and bring to life the materials that will be covered in lecture by allowing direct observation, experimentation, and application of techniques commonly used when studying the various microorganisms. The student should expect this course to be challenging, informative, and hopefully enjoyable. The latter of the expectations is achieved through preparation (rreeaadd eeaacchh eexxeerrcciissee pprriioorr ttoo yyoouurr sscchheedduulleedd llaabb) and active participation in the laboratory exercises. For the sake of time (and to retain sanity) iitt iiss iimmppeerraattiivvee tthhaatt yyoouu pprreeppaarree bbeeffoorree ccllaassss.. Read the introduction to the scheduled exercise, and familiarize yourself with the steps in each activity process (materials and methods section). It is not expected that you understand everything, just be familiar with the activities. You will be assigned a modified lab write-up as homework for each lab (the format is included in the appendix). The introductory paragraph should include a purpose statement as well as an introduction to the material in the background information. As you will soon realize, experimentation using microbes often requires incubation time for growth in order to make a proper determination from your results. For most exercises results will not be available until the following scheduled class. At that time a record should be made of your results in the given area. Your results sections along with a conclusion paragraph will be assigned as a post-lab. Homework Policy Homework Grade Sheet Microscope Policy EXERCISES Exercise 1 Introduction: Microscopy and Cell types Exercise 2 Basic Procedure in the Microbiology Lab Exercise 3 Aseptic Technique and Media Preparation Exercise 4 Morphological examination: Differential Staining Techniques Introduction to Biochemical/Metabolic Differentiation Exercise 5 Culturing: Media Selection (Defined, Complex, Selective, & Differential) and Inoculation techniques Exercise 6 Media Selection and Metabolic Characterization Continued Laboratory Practicum I - Basic Laboratory Techniques Laboratory Practicum II - Identification of an Unknown Bacterium Exercise 7 Quantification of Microorganisms Bacteria Exercise 8 Control of Microorganisms Testing and Evaluation Techniques Exercise 9 Transformation of E. coli Exercise 10 Parasitology

APPENDIX Possible Organisms Chart (for Identification of Unknown)



HHoommeewwoorrkk PPoolliiccyy Although class work is conducted cooperatively with your lab partners, the design of the

homework is for students to work independently. The purpose of the homework is to get individual students to actively participate in the material covered with each exercise and to allow the instructors to evaluate the level of understanding achieved by each student. For this reason, it is imperative that students complete their homework on their own. Students who are having difficulty with the homework assignments should discuss these problems with their lab instructor.

Plagiarism is a serious concern for all laboratory homework. When writing the introduction section it is important that you write this section in your own words rather than taking phrases, sentences, or paragraphs directly from the lab manual. The best way to avoid plagiarism is to read over the introduction and your notes prior to writing your introduction. Make sure you have a complete understanding of the lab. Then close your lab manual and notebooks and write your conclusion based on your own understanding. This will ensure that you are using your own words. Then go back and double check to make sure you have the information correct. Please see the plagiarism policy in the lab outline for information on the consequences of plagiarizing.

Your instructor may require you to submit your lab write-ups electronically. Be sure and save your file in the following format: a. lastnameE#post.file extension

Homework Grade Sheet

Assignment Your Points Points Possible

Lab Write-ups

Exercise 1

100*

Exercise 2

Exercise 3

Exercise 4

Exercise 5

Exercise 6

Exercise 7

Exercise 8

Exercise 9

Exercise 10

Assessment I: Basic Microbiology Lab procedures 30

Assessment II: Identification of Unknown 30

Assessment III: Covers E7-E10 40

Total Points 200

*Post-labs will be graded the following way: Submitting results pages on time...............................................................................2 points Correct use of grammar, sentence structure, past tense, write-up format and correct use of scientific nomenclature......2 points Well-written introductory paragraph.2 points Correct interpretation of results (drawing conclusions).4 points Each Write-up is worth...10 points Please see individual instructors policies for submission of late work, and how to handle

absences.



Microbiology Microscope PolicyMicrobiology Microscope PolicyMicrobiology Microscope PolicyMicrobiology Microscope Policy

Use ONLY your assigned microscope. Your

assigned # is:

Check the microscope for problems such as: o Slides left behind

o Broken glass on the stage

o Oil residue left on the ocular lens, objective lens or stage areas

o Other obvious problems such as the microscope has been

dropped or damaged

Report any problems detected to your

instructor

Before returning microscope make sure

you havent left any problems (see above)

Each instructor will devise and implement protocol for those students who leave problem microscopes for subsequent classes. Disciplinary actions include subtracting points from

homework, loosing replacement points, etc.



EXERCISE 1 INTRODUCTION: MICROSCOPY AND CELL TYPES

Introduction Microbiology is the study of very small organisms, microorganisms, which can only be viewed with the aid of a microscope. There are several groups of organisms that fit into this category including bacteria, cyanobacteria, fungi, and protists. Within this group there are several species interesting to humans because of their ability to cause disease or their use in the food industry. Many of these microorganisms are unicellular although some are multicellular. These organisms are extremely diverse in cell type, size, color, and reproductive strategy. When working with microorganisms one easy way to classify them is by their cell type. All cells (including plant and animal cells) can be categorized as either prokaryotic or eukaryotic. The primary difference between these two cell types is the presence of a membrane-bound nucleus. All eukaryotic cells have a membrane-bound nucleus that houses the genetic material (DNA) in addition to other membrane-bound organelles such as mitochondria and chloroplasts. Prokaryotic cells lack a membrane-bound nucleus, their genetic material is located in a particular region of the cell called the nucleoid. In addition to this difference, prokaryotic cells are much smaller than eukaryotic cells. Examples of prokaryotic cells include bacteria and cyanobacteria (photosynthetic prokaryotes). Eukaryotic microorganisms include fungi, protozoa, and algae. In this lab, we will be getting some firsthand experience with the microorganisms described above. Since the eukaryotic cells are larger than prokaryotic cells, this will be the best place to start. Once you have a feel for these larger cells you are then ready to begin investigating the smaller prokaryotic cells.

Fungi include unicellular and multicellular eukaryotic organisms. One thing all fungi share is that they are non-motile heterotrophs that absorb dissolved organic material through their cell walls and all but the yeasts metabolize aerobically. We will only be observing yeast in lab. Yeasts are round unicellular microbes that are widely distributed.

Protists are for the most part unicellular, eukaryotic cells consisting of several groups. The two groups of protists under investigation here are algae and protozoa. The primary difference between these two groups is that algae are photosynthetic while protozoa are described as animal-like because they are heterotrophs (consume other protists, bacteria and detritus). There are several protozoa that cause disease including Plasmodium vivax (malaria) and Giardia lamblia (gastroenteritis).

Prokaryotes will be the primary focus of the semester. In todays lab we will observe some of the diversity of this group by looking at both bacterial cells and cyanobacteria. Remember that these cells are much smaller than eukaryotic cells, and lack membrane-bound nuclei.

In order to investigate microorganisms we need to become intimate with our primary tool--the microscope. This invaluable tool allows the viewing of objects/structures that otherwise would go unnoticed by the unaided human eye. The type of microscope shown below is called the light microscope. Light is conducted through curved lenses in such a way that an object may be viewed larger than its actual size. You might want to label the basic structures and take notes as your instructor goes over the microscopes structure and function.



The light microscopes used in this lab are binocular and have ocular lenses with a magnification of 10X. In addition to this magnification there are also four different objective lenses to choose from 4X, 10X, 40X, and 100X. Magnification of the object being viewed is the product of the ocular objective multiplied by the lens objective currently in use. For instance when viewing an object on the 4X objective lens, the object is magnified a total of 40X. Even the highest quality light microscope is limited in its magnification abilities. The highest objective for our microscopes is 100X, which has a magnification of 1000X. With this magnification even the slightest distortion of light would greatly reduce the quality of the image. In order to conduct the light properly at this objective, it is necessary to place a drop of oil on top of the slide and immerse the lens. The oil immersion lens

(100X) is specially sealed and iiss tthhee oonnllyy lleennss tthhaatt

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For more information on microscopy visit the URL below. http://biology.clc.uc.edu/fankhauser/Labs/Microscope/Microscope_Features&Care.htm The importance of proper handling and use of the microscope is vital. You will find this to be especially true as beginning microscopists. It is critical that you clean the microscopes before and after use. Please take notes while your instructor goes over this information with you. Materials Needed: Blank Slides/Cover slips Iodine 10% bleach soln. Cultures: Yeast Yogurt Gleocapsa

Oscillatoria Anabaena Volvox Spirogyra Amoeba Paramecium

Activities In order to observe the differences between these cell types and become familiar with the microscope you will be preparing several slides primarily using the wet mount technique as described below. In some cases prepared slides may be provided.

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For each activity below, use the wet mount method to make your slides. Add the drop of iodine as described for those cultures with an (*).

1. Add a drop of live culture 2. Add a cover slip and then add a drop of iodine* beside the coverslip. 3. Observe under the microscope up to 40x and sketch your results in table provided 4. Place slide in 10% bleach solution provided



Activity I: Fungus Yeast Observation*

(http://bugs.bio.usyd.edu.au/learning/resources/CAL/Microconcepts/images/Topics/Diversity/buddingYeastCells.jpg ) Activity II: Protists A) Protozoa

Amoeba * Paramecium *

http://www.hinsdale86.org/staff/kgabric/DIMACS/amoeba.jpg

http://www.educa.madrid.org/web/ies.alonsoquijano.alcala/carpetas/quienes/departamentos/ccnn/web_1_ciclo_ESO/1eso/images/tema-10/paramecium2.jpg

B) Green Algae

Volvox Spirogyra

http://www.lima.ohio-state.edu/biology/images/volvox.jpg

https://www.msu.edu/course/bot/423/Spirogyra.jpg

Activity III: Prokaryotes

A) Cyanobacteria

Gleocapsa Anabaena Oscillatoria

http://www.glerl.noaa.gov/seagrant/GLWL/Algae/Cyanophyta/Images/Gloeocapsa2.jpg

http://www.bom.hik.se/nesch/kac/anabaena.jpg

http://silicasecchidisk.conncoll.edu/Pics/Other%20Algae/Blue_Green%20jpegs/Oscillatoria4.jpg

B) Bacteria Lactobacillus (yogurt) * No photo available



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Eukaryotic Cells Type

Sketch on (40x)

Fungi: Yeast

Protist: Amoeba

Protist: Paramecium

Protist: Volvox

Protist: Spirogyra

Prokaryotic Cells

Type Sketch (40x)

Cyanobacteria: Gleocapsa

Cyanobacteria: Oscillatoria

Cyanobacteria: Anabaena

Bacteria: Lactobacillus (yogurt)

E1 Write-up Introduction paragraph: include a description of the following items from the background information:

Prokaryotic vs. eukaryotic cells

Introduction of types of cells viewed Submit results from lab manual Conclusion:

Draw conclusions about the importance of getting familiar with proper microscope technique (for instance proper handling, focusing, and cleaning of the microscope).

Include a comparison of the prokaryotic and eukaryotic cells observed in this class. This write-up must be typed and be in your own words.



EXERCISE 2 BASIC PROCEDURE IN THE MICROBIOLOGY LABORATORY

Introduction Microbes can serve as either our greatest allies or our worst enemies depending on their type and location. This is why studying these organisms is so vital. Methods for studying microbes are as diverse as the groups themselves. Due to their size and ubiquity, microbes can be a challenge in the laboratory. This unit will focus on the general techniques that are needed in order to achieve good experimental outcomes while protecting the health and safety of everyone. We will be focusing on two important tools in the microbiologists tool boxworking with bacteria cultures and microscopy. The first part of the laboratory will introduce you to the materials and methods we routinely use when working with bacterial cultures. The most important technique to learn today is aseptic technique which will be explored in greater detail in E3. In addition to learning this basic technique we will also focus on a key idea, that our world is full of bacteria present virtually everywhere. Due to the ubiquity of microorganisms we will have to be extremely careful about contamination of our lab materials and the cultures we are working with. You will see that it takes very little exposure to introduce an unwanted organism to your materials. The second part of the laboratory will continue our microscopic investigation into the microbial world by introducing you to the variety of morphologies (shapes) and cellular arrangements that are present among bacterial cells as well as comparing cell size. In addition, we will also focus on using the oil immersion lens which is a critical skill in the microbiology lab.

Cellular morphology and arrangement refers to the cell shape and the association shared between cells (if any). Although bacterial cellular morphology can be very diverse, there are three basic shapes of interest in our lab. They are coccus (pl. cocci), rod (sometimes called bacillus), and spiral. Based on how cocci cells divide they can designated as diplococci (pairs), streptococci (chains), tetrads (groups of 4), or staphylococci (clusters). Rod cellular arrangements can be described as singles, diplobaccilli (pairs), or streptobacilli (chains). When comparing bacterial cell size, we will be using the metric system. The metric system is the standard system of measurement used in the sciences, including microbiology. The system makes measurement and unit conversions much simpler because all units and conversions are based upon the number 10. Three of the most commonly measured properties are length, mass, and volume. The standard metric units for these variables are meter, gram, and liter. Prefixes are placed in front of each units name to designate smaller and larger units. Lets use length as an example to see how metric units work. One meter is equivalent to 39.37 inches, so it is roughly 1 yard in length. Now imagine dividing the meter into 10 equal-sized pieces. Each piece is 1 decimeter (dm) in length (1/10

th of a meter). Imagine dividing the meter into 100 equal-sized pieces. Each piece is 1

centimeter (cm) in length (1/100th of a meter). Finally, divide the meter into 1000 pieces. Each

piece is 1 millimeter (mm) in length (1/1000th of a meter).

of 51

Northeast State Technical Community College

Note the following: 1 m = 10 dm 1 m = 100 cm Most microorganisms are smaller than 1 mm, so we need to intrthe micrometer (m). Imagine dividing 1 mm into 1000 equalextremely small pieces is 1 micrometer in length. A tdiameter and 2-8 m in length.

Morphology/Arrangement

Rods Singles

streptobacilli

Tetrads

staphylococci

Photographs copied from URL:

Materials per pair:

3 TSA plates

2 sterile swabs

AAccttiivviittyy II:: UUbbiiqquuiittyy ooff MMiiThe specific media type used in this lab is providing a wide range of nutrients supporting a diversity of microorganisms.

Ubiquity of Microorganisms:

1. Obtain 3 TSA plateswith your group initials, lab day/time.

2. Label one plate air. Remove the lid and leave it exposed tothe air until the end of the lab period.

3. Add the following informationa. Divide each plate into quadrants

b. Label one plate c. Number each se

would like to and these shouldsurface sample.

Source 1: dirty finger Source 2: clean finger

37oC

25oC

BIOL1230 MICROBIOLOGY


Definition of Fomite:an inanimate object that can transfer disease i.e. door knob

1 m = 1000 mm 1m = 1,000,000 m

Most microorganisms are smaller than 1 mm, so we need to introduce an additional unit called . Imagine dividing 1 mm into 1000 equal-sized pieces. Each of these

extremely small pieces is 1 micrometer in length. A typical bacteria cell is about 0.2

Morphology/Arrangement Species Photo

Escherichia coli

Bacillus subtilis

Micrococcus luteus

Staphylococcus aureus

Photographs copied from URL: http://faculty.mc3.edu/jearl/2.htm

sterile water

prepared bacterial slides

iiccrroooorrggaanniissmmss The specific media type used in this lab is Trypticase Soy Agar (TSA) which is a complex media providing a wide range of nutrients supporting a diversity of microorganisms.

Ubiquity of Microorganisms: Swab Samples btain 3 TSA plates, flip the plates over, and label the bottom

with your group initials, lab day/time. Label one plate air. Remove the lid and leave it exposed to the air until the end of the lab period.

following information on the remaining 2 plates: Divide each plate into quadrants

Label one plate 37C and the other 25C. Number each section 1-4, and make a key below of the four sampleswould like to swab. You must use the same four sample areas for both plates

these should include one fomite sample, two body samplesurface sample.

Source 3: Source 4:


Updated May 2010

Definition of Fomite: an inanimate object that can transfer disease i.e. door knob

oduce an additional unit called sized pieces. Each of these

0.2-2.0 m in

http://faculty.mc3.edu/jearl/ML/mL-5-

which is a complex media

samples you the same four sample areas for both plates

body samples, and one



4. In order to inoculate sources 1 and 2 start with dirty hands. Moisten your index finger

with water then press your dirty index finger into quadrant 1 of both plates.

5. Now wash your hands following these directions from the Center for Disease Control:

a. Wet your hands with warm running water and apply soap. b. Rub hands together to make lather and scrub all surfaces. c. Continue rubbing hands for 20 seconds. (Imagine singing "Happy Birthday" twice) d. Rinse hands well under running water e. Dry your hands using a paper towel and use your paper towel to turn off the faucet.

6. Moisten your index finger again and press the clean index finger into quadrant 2 of both plates.

7. For sources 3 and 4: obtain two sterile swabs and one micro vial of water from the cart.

8. For source 3, moisten one swab and streak the area of interest thoroughly. Proceed

to streak each of your TSA plates in the designated quadrant for that sample. Be sure to remember aseptic technique! (repeat for source 4)

9. Place the 37 C plate upside down (bottoms up) inside the incubator. Place the 25

C plate upside down on top of the incubator. Since microbes are so small, it is necessary to allow time (24-48 hours) for them to multiply into populations so large we can see their colonies unaided. The plate incubated at room temperature will need a longer incubation (5-7 days).

We will be discussing throughout the semester basic requirements microbes have in order to live and replicate. These requirements include nutrition and environmental conditions such as temperature, pH, moisture, and oxygen. Microbes vary in their nutritional needs and their levels of tolerance to environmental conditions. Since both plates were streaked with the same samples and supplied the same nutritional media, it is interesting to expose each plate to a different variable in this case temperature (either 25

oC or 37

oC). Both plates will be incubated in aerobic

atmospheric conditions.

AAccttiivviittyy IIII:: CCeelllluullaarr MMoorrpphhoollooggyy aanndd AArrrraannggeemmeenntt ((pprreeppaarreedd sslliiddeess)) The purpose of this second activity is 2-fold:

1) introduction to cellular morphology and arrangement 2) proper use of the oil immersion (100x) objective lens.

1. Obtain 3 prepared slides representing different morphologies and arrangements. 2. Begin focusing each slide on the 4x and then move to the 10x and 40x objective lenses. Once you have your image focused clearly on 40x move the turret in between the 40x and 100x objectives.

3. Without moving the stage or changing focus, drop a small, single drop of immersion oil onto the slide.

4. Move the oil immersion (100x) objective lens directly into the spot of oil. 5. View slideyou might have to do some minimal focusing with the fine adjustment knob to clear up your image.

6. Sketch results in table provided. 7. Clean oil off 100x object lens before moving to the next slide.



AIR PLATEyou should have left this plate uncovered on your lab bench the entire lab. At the end of the period be sure to close your dish and invert and incubate at 37

oC.

CClleeaann--uupp aanndd DDiissppoossaall ffoollllooww iinnssttrruuccttoorrss ddiirreeccttiioonnss.. Once all items have been put in their assigned places wipe your work area with ethyl alcohol and wash your hands well.

E2 Results: ACTIVITY I RESULTS: Obtain your two sample plates you swabbed last lab session and look for growth. Use the colony description figure taken from Science Buddies website ( http://www.sciencebuddies.org/mentoring/project_ideas/MicroBio_img_003.gif) to assist you in describing bacterial growth for each quadrant. The results tables provided below are to be used to briefly describe what you observe. List each sample area in the space provided. The lower portion is for growth description. Include color, colony sizes, amount of growth, and colony description for each listed sample area.

Colony Description by Source for 37C Plate Source 1: dirty index finger

Source 2: clean index finger

Source 3: Source 4:

Amount

Description

Amount

Description

Amount

Description

Amount

Description

Colony Description by Source for 25C Plate Source 1: dirty index finger

Source 2: clean index finger

Source 3: Source 4:

Amount

Description

Amount

Description

Amount

Description

Amount

Description



AIR Plate Results: Did you have growth?

ACTIVITY II RESULTS: For each slide sketch enough cells to demonstrate the morphology and arrangement of the bacteria viewed using the oil immersion (100x) objective lens. assigned microscope #:

Slide 1 Slide 2 Slide 3

SKETCH

Describe morphology and

arrangement


Ubiquity of microorganisms and aseptic technique

Discussion of culturing conditions provided including temperatures, media used, and atmospheric requirements

Description of different morphologies and arrangements Submit results from lab manual Conclusion:

Use your data to support use of aseptic technique

Use your data to address the use of optimal temperature during incubation

Discuss your observations of cellular morphology and arrangement. This write-up must be typed and be in your own words.



EXERCISE 3 ASEPTIC TECHNIQUE AND MEDIA PREPARATION

Introduction Asepsis means without contamination. The ability to carry out procedures without the introduction of unwanted organisms, or contamination, is paramount to obtaining correct results/identification. In addition, since some of these microbes are potential pathogens (organisms that cause disease), contamination could expose you and any other person you may come into contact with the possibility of infection. That is why vigilance in proper technique is necessary to reduce risk. Aseptic technique can be further divided into four categories including work area preparation, media preparation/handling, culture transfer, and clean-up and disposal. Work Area Preparation Since microbes are everywhere, even on us, it is necessary to begin to minimize the potential for contamination before we begin to work with the microbes. Prior to beginning each laboratory session:

Remove all you personal items away from the workbench except for your lab notebook and writing instrument.

Wipe down your workbench with ethyl alcohol (ETOH) and paper towels.

Wash your hands using CDC method as described in E2.

Begin gathering materials as instructed and outlined for that particular laboratory. Media Preparation/Handling When culturing bacteria you must provide them with all of the conditions they need for growth including nutrients, temperature, atmospheric requirements and more. Media is the nutrient mixture that is used to grow and keep microbes. Media is usually in broth or solid forms and will vary in content based on the goal of its use. These nutrient mixtures are normally in powdered form to increase their shelf life. When needed, the nutrient mix is added in a specific amount to water, boiled, transferred to containers, and autoclaved to sterilize. As long as the media remains unopened in the original container, it should remain a sterile environment. If the media is transferred from the original container, care should be taken to avoid contamination. In the case of the plastic Petri plates we will be using extensively in this lab section, media must be transferred. Plastic used for these plates are unable to withstand the temperature of the autoclave for sterilization so they are shipped and remain in sterile packaging until their time of use. The agar will be poured hot and quickly into the Petri dish to further avoid contamination. Culture Transfer All of microbiology work involves transferring cultures to different growth media or onto slides. It is critical when transferring bacteria that aseptic technique is maintained. A large portion of todays lab procedure will emphasize how to move bacteria to different media types while preventing contamination. Clean-up and Disposal After EACH Lab (last step of aseptic technique) Unless otherwise instructed items used in the lab session should be treated in the following manner: Glassware - will be autoclaved and cleaned for re-use.

Test tubes/flasks place in the designated test tube rack on the lab cart after the labeling has been removed. If an adhesive label is present, simply peel it off and place in the autoclave bag. If a marker has been used, wipe off with acetone prior to placement in the rack.

Slides If the slide has been heat fixed, wash with sponge and dish soap and place in designated container.



Disposable Items items intended for one time use and items that cannot maintain their form and function after being autoclaved should be placed in the autoclave bag. These items will be sterilized prior to their disposal to avoid contamination during and after waste collection. These items include but are not limited to Petri plates and any paper or plastic items that have come into contact with microbes. Goggles clean with diluted dish soap, dry and then put away. Once all items have been put in their assigned places wipe your work area with ETOH and wash your hands.

Activities Materials: 4 test tubes of TSB 1 TSA plate plates from E2 stirrer/hotplate magnetic stirrer

hot pads flasks with filtered water powdered media thermometers

Aseptic Transfers A.) Sterile Broth to Sterile Broth: 1. Obtain two sterile Trypticase Soy Broth (TSB) test tubes.

Label one tube A and one tube B and label both with your group initials and lab day/time. 2. Flame loop until the loop and some of the wire is red hot then let the loop cool. 3. Open test tube A using the pinky finger techniquehold the lid of the test tube with your

pinky finger of your dominant hand, and use your other hand to twist the test tube away from the lid.

4. Flame the lip of the test tube 5. Get loopful of broth; make sure that only the sterilized portion of the loop makes contact

with the broth. 6. Flame and close test tube. 7. Open test tube labeled B using the same technique as above. 8. Flame the lip of the test tube. 9. Inoculate test tube B with the loopful of sterile broth from tube A. Be sure that only the

sterilized portion of the loop makes contact with the broth. 10. Flame and close test tube B. 11. Flame loop red hot. 12. Place both test tubes in a rack in the incubator.



B.) Sterile Broth to Sterile Plate: 1. Obtain one sterile TSB test tube and a sterile Trypticase Soy Agar plate (TSA). Label both

with your group initials and lab day/time. Label the test tube C and the plate D. Be sure to put your labels on the bottom of the Petri dish.

2. Flame loop until the loop and some of the wire is red hot then let the loop cool. 3. Open the test tube using the pinky finger technique (see above) 4. Flame the lip of the test tube 5. Get loopful of broth; make sure that only the sterilized portion of the loop makes

contact with the broth. 6. Flame and close test tube. 7. Open the Petri dish just enough to get the loop in. 8. Very gently move the loop across the Petri dish. Be careful not to dig into or make any

breaks in the agar bottom. 9. Close the Petri dish. 10. Flame loop red hot. 11. Invert the Petri dish and add it to your class stack in the incubator. Put the test tube in the

incubator rack too.

C.) Colony to Broth Transfer 1. Obtain one sterile TSB test tube and a plate with growth from E2. Label with your group

initials, lab day/time and E on the TSB tube. 2. Flame loop until the loop and some of the wire is red hot. Allow loop to cool. 3. Open the Petri dish just enough to get the loop in and very gently use your loop to pick up an

isolated colony of bacteria. Be sure not to disturb the agar or any neighboring colonies.

4. Close the Petri dish. 5. Open the test tube using the pinky finger technique (see above). 6. Flame the lip of the test tube 7. Inoculate the broth; make sure that only the sterilized portion of the loop makes contact with

the broth. 8. Flame and close test tube. 9. Flame loop red hot. 10. Put the test tube in the incubator rack.



Media Preparation Each group will be asked to prepare media. The type of media and the specific instructions will be assigned by the instructor. It will take just a few minutes to get the media measured and added to the flask. It will take about 20-30 minutes for the media to reach boiling before it is ready to pour into test tubes. Instructors will need to make sure that each class tubes are autoclaved as soon as possible and then stored in the refrigerator as room allows. The basic procedure to be used by all classes is as follows: 1. Measure out the proper amount of media powder for L of media using the electronic

balance. 2. Fill up your flask with the proper volume of filtered water (1/2 L) and place on the

stirrer/hotplate. Drop in a magnetic stirrer and turn the stirrer on 6-8. 3. Slowly pour in the media powder into the water. 4. Turn the hotplate on high. Agar can superheat so it is important to keep an eye on your

boiling agar. Also agar will not dissolve in water that is hot, so DONT heat the water until after adding the powder.

5. Once the agar or broth has reached boiling point (use thermometers to register 100

oC). You

will notice that the liquid is clear rather than cloudy indicating that the media has dissolved. Also there is often a frothy head that forms on the top of the boiling media (when TSA and TSB start to look like beerthey are ready).

6. Follow instructors directions on how to pour the media into the test tubes given. Loosely cap

the test tubes and put autoclave indicator tape on the rack. Be sure to provide a label on the rack with the type of media, date made, and group initials. Your instructor will help you get your rack autoclaved with the rest of the class tubes.

7. Test tubes need to be autoclaved as soon as possible after being poured. Once sterilized

test tubes can be stored in the refrigerator.



E3 Results For each item record whether or not you had growth. If you have growth in TSB then the test tube will be cloudy. You might need to vortex your TSB to see growth as the bacteria can settle.

Procedure Materials Growth? Yes or No

Sterile TSB Sterile TSB Tube A

Tube B

Sterile TSB Sterile TSA

Tube C

TSA plate D

Isolated colony sterile TSB Tube E


Aseptic technique define, state purpose, give some examples of Submit results from lab manual Conclusion:

Did your results turn out as expected? Explain.

Describe some procedural errors that could negatively affect your results. This write-up must be typed and be in your own words. .



EXERCISE 4 DIFFERENTIAL STAINING TECHNIQUES GRAM STAIN AND SPECIAL

STRUCTURE STAINS

Introduction As we have seen in the previous exercises, light microscopy and the use of a stain are valuable tools for viewing bacteria. This allows us to see the morphology of a microbe of interest. A differential stain technique allows additional discrimination and helps to narrow down the list of possibilities with regard to its identity. A differential stain would be one that allows determination of differences between species having similar morphologies based on their ability to take the stain. In multiple stain procedures, the initial stain is retained by cells that are termed positive while the negative cells loose the initial stain. This necessitates the addition of a secondary or counter stain, to make the negative cells visible under the microscope. Differential stains show a difference in chemical composition, metabolism, and/or the presence of a special structure. The first step in making a slide is to smear the bacteria onto the slide. Preparation procedures for the smear will vary according to cell concentration in the culture. Generally a broth culture will be less concentrated than surface cultures and can be smeared directly onto the surface of a clean slide and allowed to air dry. Surface cultures (ones growing on agar) need to be diluted in order to discriminate single cell shapes once stained. A loop of water is added to the slide and the loop of sample is mixed in and distributed on the surface of the slide and allowed to dry. The second step in making most differential stains is to heat fix the slide. A heat fixed slide is one where the organisms are placed on the slide and the slide is allowed to dry. After the slide is free of visible moisture, it is passed through an open flame 2-3 times before the staining begins. It is imperative that the slide is completely dry before the heat-fix step; otherwise the bacterial proteins can be denatured during the heat-fix step and cause a distortion in the cell morphology. The purpose of the heat-fix process is to affix them to the surface of the slide so that they are not washed off during the staining process. When done correctly, heat-fixing will also kill the organisms.

The most well-known and used differential stain is the Gram stain. This stain process determines differences in peptidoglycan (a chemical found only in bacteria) on the outside of the cell wall. Bacteria can be divided into one of two categories based on their gram reaction, either gram positive or gram negative. Gram positive cells have a thicker peptidoglycan cell wall than gram-negative cells, and retain the initial stain, crystal violet (purple), in the gram stain procedure. Gram negative cells are decolorized during the gram stain procedure which requires the application of a counter stain, Safranin (pink), so that these cells can be easily viewed.

The acid-fast stain is used in a limited number of cases to stain organisms that have an addition of mycolic acid in their cell walls which prevents them from Gram staining. The Mycobacterium species require this type of stain process in order to view them. Due to the nature of the waxy mycolic acid, these cells must first be steamed in order for the stain to penetrate the cell wall. In this procedure acid-fast cells retain the initial stain, carbolfuchsin (reddish-purple), while negative cells are stained by the secondary stain, Methylene blue (blue). Some species have cellular structures that are uncommon and can be used in identification. Endospores are examples of specialized cellular structures produced by some Bacillus and Clostridium species in response to stressful environmental conditions. Endospores are dormant cells able to survive hazardous conditions that later germinate to form vegetative (metabolically

active) cells when conditions improve. The endospore stain, employs a multiple stain procedure that stains both endospores and vegetative cells. The endospore stain involves steam similar to the acid-fast stain. Without steam the tough spore coat prevents the endospore from taking up stain. During this procedure, endospores keep the initial stain, Malachite green (green), while the vegetative cells retain the secondary stain, Safranin (pink).



Other important structures that can be detected with stains are flagella. These structures are responsible for the motility of an organism. While the presence of flagella alone can be important in characterizing bacteria, the ability to describe the arrangement of the flagella can be even more useful. For direct observation of flagella a tricky staining procedure is used. Motility media is an alternative method to staining that allows for indirect observation of flagella. Due to the dilute nature of this media microbes are able to travel in the media if they are mobile. This takes time and incubation will be necessary. In addition the media has a chemical added, TCC, which microbes take into their cells. When metabolized, the TCC changes from being colorless to a pink color allowing easier viewing of the placement of the growth. There are plenty of resources on the internet that can help you visualize these processes a little better. Check out the following links:

Gram Stain: http://faculty.mc3.edu/jearl/ML/mL-5.htm

Acid-fast Stain: http://faculty.mc3.edu/jearl/ML/mL-6.htm

Endospore stain: http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.htmL

Activity Materials: Per Class: Blank slides Hotplates/beakers

Per team: M. smegmatis (pathogenic) B. subtilis E. coli

K. pneumonia S. epidermidis P. aeruginosa 3 motility media test tubes

Students will have 2 lab periods to complete E4.

Each group should start by inoculating the 3 motility tubes 1. Obtain 3 test tubes of motility media and label 1-3, group initials, and class period.

1. Escherichia coli 2. Pseudomonas aeruginosa 3. Klebsiella pneumoniae

2. Using aseptic technique, stick the sterile inoculation needle into the broth culture (be sure to flame the entire length of the needle).

3. Insert the needle straight down into the motility media and pull the needle straight

back out.

4. Fire the tube and quickly replace the lid. Fire the needle

5. Place in the test tube rack in the incubator for 48 hours.

Each student should obtain a total of 8 slides and label slides a-h (see list of bacteria below). Make all 8 slides using the heat-fix procedure. After heat-fixing all 8 slides begin staining slides starting with the 4 gram stain slides.

Slide label Organism Stain Technique

a and f Staphylococcus epidermidis Gram stain and acid-fast stain

b Pseudomonas aeruginosa Gram stain

c and h Escherichia coli Gram stain and endospore stain

d and g Bacillus subtilis Gram stain and endospore stain

e Mycobacterium smegmatis (PATHOGENIC) Acid-fast stain

i MIXED slide of S. epidermidis and E. coli Gram stain



For All slidesmake smear using heat-fix procedure: From Solid Sample:

1. Clean and label bottom of slide 2. Add loopful of water 3. Flame loop red hot 4. Aseptically obtain bacteria (isolated

colony if possible) 5. Smear (spread-out) bacteria on

slide 6. Flame loop red hot

7. AIR DRY COMPLETELY 8. Heat-fix (pass slide through flame) 9. Proceed to directions below for the

specific differential stain.

From Liquid Sample: 1. Clean and label bottom of slide 2. Flame loop red hot 3. Vortex Sample 4. Flame test tube opening 5. Aseptically obtain bacteria 6. Flame test tube opening 7. Smear (spread-out) bacteria on

slide 8. Flame loop red hot

9. AIR DRY COMPLETELY 10. Heat-fix (pass slide through flame) 11. Proceed to directions below for the

specific differential stain.

Under oil immersion, observe your slides--remember you are looking for differences in morphology, size and arrangement in addition to whether the organism is positive or negative for the differential stain. Then sketch your observation in the results section. (Dont forget to clean your microscope BEFORE & AFTER use.) Procedure I

Gram Stain Procedure Organisms: a. Staphylococcus epidermidis b. Pseudomonas aeruginosa c. Escherichia coli d. Bacillus subtilis

i. MIXED slide of S. epidermidis and E. coli

1. Prepare slides for Gram stain as instructed above. 2. Attach a clothespin to the end of the slide and hold it over the sink.

3. Cover the slide with Crystal Violet stain and allow it to react for 1 minute 4. Rinse the slide with a small amount of running water and remove the excess water by gently

shaking the slide. 5. Cover the slide with Grams Iodine--allow it to react for 1 minute. 6. Rinse the slide with water as in Step 4. 7. Hold the slide at an angle and apply 1 drop of decolorizer 8. Immediately rinse the slide thoroughly and remove any excess water as in Step 4. 9. Cover the slide with Safranin for 1 minute. 10. Rinse the slide thoroughly. 11. Blot the slide dry by placing it in between the sheets of the bibulous paper and lightly pat

with your hand. Apply immersion oil when ready to view & record in Results section



Procedure II

Acid-fast Stain Procedure Organisms: e. Mycobacterium smegmatis (pathogenic) f. Staphylococcus epidermidis

1. Prepare slides as instructed above. 2. Cover the slides with a piece of paper towel the same size

as the smear. 3. Place clothespins at both ends of the slide to form a rack

and place it on top of the steaming water beaker. 4. Flood the slide with Carbolfuchsin stain. 5. Gently steam the slide for 10 minutes, reapplying the stain

as needed to prevent the slide from drying out. 6. Remove the paper towel carefully with forceps and place in trash. 7. Rinse the slide with a small amount of running water until the excess stain is removed. 8. Hold the slide at an angle and apply acid alcohol by making drops at the highest part of the slide

(near the clothespin handle) and allow it to drip down the slide. Do this for 25-30 seconds. 9. Rinse the slide. 10. Apply several drops of Methylene Blue stain and leave for 45 seconds. 11. Rinse the slide thoroughly and blot dry.

Apply immersion oil when ready to view & record in Results section

Steam

of 51

Northeast State Technical Community College

Procedure III

Endospore Stain ProcedureOrganisms:

g. Bacillus subtilis

h. Eshericia coli

1. Prepare slides as instructed above 2. Apply clothespins to each end of the slide and place

over a steaming beaker of water 3. Apply a piece of paper towel cut

slide on the surface of the slide. 4. Flood the paper towel with Malachite Green stain. 5. Allow the slide to steam for

prevent the paper towel from drying out. 6. Remove the paper towel gently using forceps and remove one clothespin. 7. Rinse the slide with a small amount 8. Hold the slide over the sink and apply Safranin and allow it to react for 1 minute.

9. Rinse and blot dry. Apply immersion oil when ready to view & record in Results section

http://a-s.clayton.edu/furlong/BIOL2250LAB/Reviews/mediastudy.htm


Gram stain, endospore stain, acidSubmit results from lab manualConclusion:

Use your results to describe the cellular This write-up must be typed and be in your own words.

Motile N



http://www.sp.uconn.edu/~terry/229sp03/lectures/structure.htmL

Vegetative (A); Endospores (B)

Endospore Stain Procedure

Prepare slides as instructed above.

Apply clothespins to each end of the slide and place over a steaming beaker of water (like acid-fast).

Apply a piece of paper towel cut to the size of the slide on the surface of the slide.

Flood the paper towel with Malachite Green stain.

Allow the slide to steam for 5 minutes and reapply the stain as needed to prevent the paper towel from drying out.

Remove the paper towel gently using forceps and remove one clothespin.

Rinse the slide with a small amount of running water.

Hold the slide over the sink and apply Safranin and allow it to react for 1 minute.

Apply immersion oil when ready to view & record in Results section

s.clayton.edu/furlong/BIOL2250LAB/Reviews/mediastudy.htm

include a description of the following items from the background

Gram stain, endospore stain, acid-fast, and motility media Submit results from lab manual

Use your results to describe the cellular characteristics of each organism tested

up must be typed and be in your own words.

Motile Non-motile Motile


Updated May 2010

http://www.sp.uconn.edu/~terry/229sp03mL

Vegetative (A); Endospores (B)

Hold the slide over the sink and apply Safranin and allow it to react for 1 minute.

s.clayton.edu/furlong/BIOL2250LAB/Reviews/mediastudy.htm

include a description of the following items from the background

characteristics of each organism tested



E4 Results Procedure I-Gram Stain assigned microscope #:

Slide Label Organism Sketch & Indicate Color

a. Staphylococcus epidermidis

b. Pseudomonas aeruginosa

c. Escherichia coli

d. Bacillus subtilis

i. MIXED S. epidermidis and E. coli

Procedure II-Acid-fast assigned microscope #:

Slide Label Organism Sketch

e. Mycobacterium smegmatis (pathogenic) (pathogenic)

f. Staphylococcus epidermidis

Procedure III-Endospore Stain assigned microscope #:

Slide Label Organism Sketch & Color

g. Bacillus subtilis

h. Escherichia coli

Procedure IV-Motility Results: Draw the regions where growth was observed.

Organism 1.) Escherichia coli

2.) Pseudomonas aeruginosa

3.) Klebsiella pneumoniae

Sketch

See previous page for E4 write-up assignment.



EXERCISE 5

CULTURING: MEDIA SELECTION AND INOCULATION

TECHNIQUES Introduction In the previous four exercises we became familiar with visual techniques used in microbiology. You learned to describe various colony characteristics that, although not often, may aid in the determination of a particular microbe. We also examined various types of bacteria under the microscope to record cell morphology and any biochemical/structural differences that could be obtained through the use of staining procedures. Although helpful, determination of cell type and structure is limited when it comes to correctly identifying a microorganism. There are many species that, for example, are Gram negative rods with motility. When studying a bacterium or when trying to diagnose a disease for proper treatment, the exact species (or even strain of that species) must first be determined. For further characterization we look to various metabolic differences that are specific to that type of bacteria. All living organisms must acquire energy from their environment. There are differences in energy sources and metabolic pathways between groups of bacteria. There are also ranges of environmental conditions that are particular to groups of bacteria (pH, temperature, atmospheric requirements, etc.) where they best function (refer to E2 for temperature differences). Knowing and manipulating these conditions provide excellent tools for correctly identifying bacteria. The next two exercises will focus on various procedures that have been developed to manipulate differences in energy needs and environmental condition ranges. Exercise 5 will be focusing on the use of media. For solid media, agar base (solidifying agent) is added to a broth containing nutrients that provide energy for microbes so that they may metabolize and replicate. The ingredients that are present in the broth will determine what groups of microbes are able to metabolize and grow. There are some metabolic differences among similar microbes that can be used for identification (see differential media below). Metabolic differences are often observed using colorimetric tests which typically incorporate either a pH indicator that will change the color of the media if there is a change in pH or a chemical that attaches to the metabolite of interest and concentrates in the microbes changing the normal colony color. In general there are three different groups of media type used in this lab:

Complex media is comprised of partially digested chemical compounds from organic substances such as yeast, meats, dairy products, tissues, or vegetable materials. The amount of each cannot be known due to differences between the organic compounds and the amount of digestion that has occurred. This knowledge is not necessary when culturing most types of microbes. This media type is useful when trying to grow out bacteria, or when culturing a mixed diverse sample (see E2). An example of a complex media is Trypticase Soy Agar (TSA).

Selective media is used to select, or isolate, specific groups of bacteria. This is usually based on a known environmental condition range they can tolerate that most groups cannot.

Differential provides chemical compounds that bacteria metabolize differently. This difference is observed by colony or media color change once the microbe has been introduced to the media and allowed to grow and metabolize. Often this type of media is used to differentiate between similar groups of organisms with the same morphology and biochemical resemblances.



T-Streak

A B C

Determination of Oxygen

Requirements

There are several medias that are both selective and differential these include Eosin Methylene Blue and Mannitol Salt Agar.

Eosin Methylene Blue (EMB) is used to differentiate between gram-negative, enteric, rods because this media primarily supports (or selects for) the growth of these organisms. Growth on EMB can be used to differentiate organisms based on their ability to ferment lactose. During the fermentation process, acid is produced as a waste product. When grown on EMB this acid production results in a colony color change ranging from pink to purple. In the case of E. coli and Klebsiella pneumoniae, colonies can appear dark purple with a metallic green sheen. One exception to EMB selectively growing gram-negative rods is the gram-positive cocci Enterococcus faecalis. E. faecalis is able to grow on this media because it is an enteric bacterium.

Mannitol Salt Agar (MSA) contains salt that most organisms cannot tolerate due to their osmolarity ranges. Microbes that normally exist in an environment with this condition, such as microbes of the skin, are able to grow. For those halotolerant organisms, MSA is also used to differentiate species based on their ability to ferment mannitol. Organisms that can ferment mannitol produce an acid by-product causing the pH indicator in the media to turn from pink to yellow.

In addition to using color changes to differentiate bacteria,

location of growth in a special media can be useful as we have already seen with motility media (E4). Media can be used to determine whether a microbe is an obligate aerobe (A), obligate anaerobe (B), or a facultative anaerobe (C) species by observing location of growth in special media (see figure). An oxygen gradient is formed in the oxygen requirement media once the test tube is boiled. An aerobic environment remains in the top 1-2 cm of the test tube, while anaerobic conditions are present below this point.

When working with media it is important that you place the bacteria on the media in an informative way. When trying

to simply grow bacteria on a media the best method to use is a streak-to-grow technique. With this method you are simply trying to get the bacteria on the media and you can either use a back-and-forth motion on the plate with your loop or one distinct streak. If you want to obtain isolated colonies for use in working with a pure culture, than a T-streak method would be more appropriate. The idea is to dilute or disperse the cells so

when incubated, they will form isolated colonies that are separated to such a degree the colonies do not touch. This will allow viewing of the individual colonies for any

distinguishing culture characteristics as well as for creating and working with a pure culture.

Activities Materials Per team: 4 TSA plates 2 EMB plate 2 MSA plates 4 Oxygen Requirement tubes

S. aureus S. epidermidis


Northeast State Technical Community College 2005 Updated August 2010

Per table: Test tube rack containing Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Enterococcus faecalis, Klebsiella pneumoniae, Clostridium perfringens, and Micrococcus luteus

Activity I: Inoculation and Isolation of Colonies

Isolation of a Colony: T-Streak Technique 1. Obtain a TSA plate and label on the back your groups initials, YOUR NAME, class

time, 1-4, and divide your plate by drawing a T (see below). 1. Staphylococcus epidermidis 2. Pseudomonas aeruginosa 3. Escherichia coli 4. Enterobacter aerogenes

2. Sterilize your transfer loop and allow it to cool. 3. Aseptically obtain a loop of broth from one of the cultures (see box below). 4. Streak the top section with closely spaced streaks that DO NOT overlap 5. FLAME loop 6. Drag bacteria from top section into right hand section, streak as in step 4 7. FLAME loop 8. Drag bacteria from right section to left section, streak as in step 4 9. FLAME loop 10. Invert and Incubate at 37

oC

Activity II: Media Selection (MSA & EMB) 1. For each type of media plate (2 MSA & 2 EMB) label with your initials, class day/time,

and label one set of MSA and EMB plates as a-d (see figure) and the second set of plates e-h.

2. For each plate aseptically streak to grow with the appropriate culture. Remember to

always practice good aseptic technique, especially between the transfers of the different species.

a. Staphylococcus epidermidis b. Staphylococcus aureus c. Micrococcus luteus d. Enterococcus faecalis

e. Pseudomonas aeruginosa f. Escherichia coli g. Enterobacter aerogenes. h. Klebsiella pneumoniae

3. Invert and incubate all 4 plates 37oC.

Flame loop Flame loop

1

3 2 2

1 1

A B

D C

E F

H G



Activity III using media to determine O2 requirements:

1. Obtain 4 Oxygen Requirement test tubes. Label with initials, day/time, and 5-8.

2. Allow the oxygen requirement tube to cool to body temperature (it will feel comfortably warm to the touch). DO NOT allow your media to solidify before inoculation. Be sure to sterilize the full length of the loop and wire to prevent cross contamination of the tubes.

3. Inoculate the test tube with the appropriate microbe. To do this, put the loop with inoculate straight down to the bottom of the tube and pull out. Repeat for all 4 species.

1. Klebsiella pneumoniae 2. Pseudomonas aeruginosa 3. Escherichia coli 4. Clostridium perfringens

E5 Results: Activity I: Inoculation and Isolation of Colonies Obtain your best T-Streak plate and observe the growth. Sketch your results in the diagram below, and then count the total number of isolated colonies you observe. Total Isolated colony count: _____

Dont forget you will be using these T-streaks for your E6 procedure. Activity II: Media Selection (TSA, MSA, & EMB) For each microbe - record the presence of growth on each media. Also record the appearance of colony and/or media. The highlighted organisms are gram () rods, and the remaining are gram (+) cocci.

Label Organism

MSA EMB

Growth Yes or No?

Media Appearance

Notes Growth

Yes or No? Colony

Appearance Notes

a. Staphylococcus epidermidis

b. Staphylococcus aureus

c. Micrococcus luteus

d. Enterococcus faecalis

e. Pseudomonas aeruginosa

f. Escherichia coli

g. Enterobacter aerogenes

h. Klebsiella pneumoniae



Activity III using media to determine O2 requirements: For each tube, observe the areas of growth and sketch your results in the tubes provided below.

Organism 1. Klebsiella pneumoniae

2. Pseudomonas aeruginosa

3.Escherichia coli 4.Clostridium perfringens

Sketch


EMB, MSA, and oxygen requirements

Obligate aerobes, obligate anaerobes, and facultative anaerobes Submit results from lab manual Conclusion:

Use your results to describe the cellular characteristics of each organism tested This write-up must be typed and be in your own words.



EXERCISE 6

MEDIA SELECTION & METABOLIC CHARACTERIZATION

CONTINUED Introduction As described in the previous exercise, identification of bacteria is limited to visual observation. Knowledge of individual energy requirements and specific environmental condition ranges were introduced in Exercise 5 with the various media types. Within these there are a variety of ways by which groups of microbes metabolize. These differences will be the focus of this exercise. There are numerous procedures presently practiced, however for the purposes of time, we will be conducting only a couple of tests that focus on the ability to use a certain carbon source, the waste products produced, and the enzymes present.

1. Carbohydrate fermentation is a test often used to help with the identification of enteric bacteria. Fermentation is a common practice of enteric organisms (as well as many others) allowing them the ability to still metabolize even in the absence of oxygen. For this test, tubes contain a sugar source, a pH indicator, and an inverted Durham tube in the solution. Once the tube has been inoculated it will be incubated at body temperature and observed for any color change and/or gas production. A phenol red (pH indicator) change from red to yellow indicates an acid has been produced as an end product of fermenting the sugar source. For a color change to be visible with phenol red pH indicator, the pH must be below 4.3. The inverted Durham tube allows us to observe whether a gas has been formed during catalysis of the sugar. Gas will be trapped in the tube and appear as a bubble. As pressure of gas increases, this tube will travel upward. Any Carbohydrate can be used to analyze metabolic activity of a microbe. We will be testing the disaccharide lactose. Not all organisms can metabolize this sugar. Only organisms with a special enzyme called -galactosidase are able to break the glucose-galactose bond.

2. Citrate Utilization Test will indicate whether or not the microbe in question is able to use citrate as its carbon source. Simmons Citrate agar is used for this test. This differential agar contains a pH indicator, bromothymol blue, which will turn from green to dark blue if there is a rise in pH. The premise behind this test is that citrate is the only carbon source in the media and only microbes that have the enzyme citrate permease will be able to transport citrate inside the cell for catalysis. The end product of citrate breakdown is carbon dioxide gas. This gas will be released and combine with salts and water in the media to form sodium carbonate, a base (Stukus,1997).

Enzymatic indicators are chemicals that allow determination of how a microbe of interest respires. The following two tests we will be observing today are mainly for bacteria that can respire aerobically.

3. Oxidase Test- Aerobic organisms must have an enzyme that is able to carry electrons to oxygen in order to produce a usable energy source through aerobic respiration. One of the four different enzymes that have this ability is cytochrome c. The Oxidase test uses a reagent, Tetramethyl-p-phenylenediamine dihydrochloride, to determine the presence of this enzyme. A positive test result would be a dark color change in the area of the colony where the test was conducted.

4. Catalase Test- Most cells that respire aerobically have an enzyme to combat hydrogen peroxide, a by-product of aerobic respiration. A few but important anaerobes also produce catalase. The catalase test may be used for differentiating among aerobes



(e.g., Staphylococcus from Streptococcus and Enterococcus species) and among anaerobes (e.g., Propionbacterium acnes from Actinomyces meyeri). Catalase breaks down hydrogen peroxide into water and oxygen gas. To test for the presence of catalase hydrogen peroxide is dropped on a colony. Bubbling indicates presence of catalase.

Materials Per Team: 4 Durham tubes Phenol Red lactose broth (black lids) 4 Simmons Citrate agar slants T-streak plates made in E5 (Pseudomonas aeruginosa, Enterobacter aerogenes, Escherichia coli & S. epidermidis) Test tube rack containing: Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, Escherichia coli) Per class: TSA plates of (Klebsiella pneumoniae, B. subtilis, & E. faecalis) Oxidase reagent, filter paper, wooden sticks Hydrogen peroxide (H2O2)

Activities I. Carbohydrate Fermentation Test (Phenol Red & Durham Tubes)

1. Label 4 Phenol Red and Lactose tubes (black lids) a-d, groups initials, and class day/time.

2. Aseptically inoculate each of the tubes with a loop of designated bacteria.

a. Pseudomonas aeruginosa b. Enterobacter aerogenes c. Klebsiella pneumoniae d. Escherichia coli

3. Place in the test tube rack in the incubator.

II. Citrate Utilization Test (Simmons Citrate agar)

1. Label 4 Simmons citrate slants a-d, groups initials, and class day/time. 2. Aseptically streak-to-grow with the microbe that corresponds with the label and

incubate. a. Pseudomonas aeruginosa b. Enterobacter aerogenes c. Klebsiella pneumoniae d. Escherichia coli

III. Oxidase Test: TSA streak plates will be located up front with pure cultures of several known organisms.

1. Place a drop of oxidase reagent on an piece of filter paper located in a disposable Petri dish.

2. Obtain a colony with a wooden applicator and rub onto filter paper. 3. Record any purple color change within the first 10-15 seconds. After 60 seconds

color changes are NOT considered positive. The color change might be faint so look carefully.

IV. Catalase Test:



Hydrogen peroxide and droppers will be available for the catalase determination. 1. Place a drop of hydrogen peroxide on an area of growth on the TSA streak plates provided.

2. Record whether or not bubbles are present for each.

E6 Results Organism Results by organism

Lactose Fermentation Simmons Citrate a. P. aeruginosa

b. E. aerogenes

c. K. pneumoniae

d. E. coli

Catalase and Oxidase Determination Source of sample Organism Catalase

Reaction Oxidase

Determination

E5 T-streak #3 E. coli

E5 T-streak #4 E. aerogenes

E5 T-streak #2 P. aeruginosa

Demo plate K. pneumoniae

Demo plate B. subtilis

E5 T-streak #1 S. epidermidis

Demo plate E. faecalis


Carbohydrate fermentation test, Simmons citrate test, oxidase test, catalase test Submit results from lab manual Conclusion:

Use your results to describe the cellular characteristics of each organism tested This write-up must be typed and be in your own words.



Microbiology Lab Practical Assessment I: Basic Microbiology Lab

Procedures (30 points)

STUDENT STUDY SHEET:

Your instructor will ask you to perform a series of tasks and will verify the proficiency with which you conduct each task. Tasks will be assessed by a combination of watching students perform the task, and/or looking at the students results from each task. You should be prepared to do the following procedures:

1. Aseptic Techniqueaseptically transfer sterile broth

2. Gram Stainsuccessfully stain a known organism and interpret result (you will NOT need to memorize the gram stain procedure, but you will need to know how to make a smear from memory)

3. Microscopysuccessfully focus and maintain a microscope

4. T-streaksuccessfully isolate a colony

Each item of the test will be worth 7.5 points for a total of 30 points.



PRACTICAL ASSESSMENT II:

IDENTIFICATION OF AN UNKNOWN ORGANISM (30 points) Each student will select an unknown culture and use the lab procedures and data collected from exercises 1-6 to correctly identify the culture. Students will be able to use their lab manual, lab notes, textbook, and Internet research materials to help with this project. Students will need to review the Possible Organisms Chart (see Appendix ) in order to help with the identification of the unknown cultures. Students will be given time in 3 class periods to complete this project. Students must attend and be prepared to use the time provided in class. **If possible bring a digital camera (or use your cell phone) to take photographs of your results as you get them. Include these images in your write-up.** Class 1 (1 hour 50 minutes):

1. Obtain numbered culture: 2. Describe the color of the growth on TSA 3. Complete Gram stain and record results below

assigned microscope #: 4. Use chart in Appendix to help you narrow down your possible organisms. 5. Based on gram reaction and research inoculate the minimum number of tests to be able to confidently identify your organism:

Motility Media (E4) EMB slant or pour plate (E5) MSA pour plate (E5) Oxygen Requirement (E5) Simmons Citrate (E6) Phenol Red and Lactose (E6) Oxidase (E6) Catalase (E6)

Class 2 (30 minutes):

1. Obtain results from Class 1, record in a results table and draw conclusions 2. Inoculate additional tests as needed.

Class 3 (10-15 minutes):

1. Get results from tests inoculated in class 2. 2. Write-up due following class periodsee instructor directions.

RESULTS TABLE:

Date Test Completed Test Results Date Result Recorded

Pour Plate Directions:

1. Boil to melt media 2. Once melted

aseptically pour into sterile plate

3. Allow media to cool and solidify

4. Inoculate plate



EXERCISE 7

QUANTIFICATION OF MICROORGANISMS Introduction Determination of a microbial population is an important aspect in microbiology. It is used in industrial applications, municipal and federal standards for our water and food, and the health and safety fields. Prior to this exercise, the majority of microbes we have been working with in the laboratory have employed pure cultures purchased by suppliers and reanimated when needed. In the real world, microbes are not only abundant, they also cohabitate and function in mixed populations. The dynamics for many of these groups are not well understood. Studies that have been conducted on some of these populations have indicated that some species may be dependent on others for metabolic activities, growth factors, etc{For example, the US Army Corps of Engineers is currently researching biofilm dynamics and their potential applications.} In order to study these environmental microbes it is necessary to take a sample of the environmental media. Samples can be obtained from virtually anywhere. Environmental samples may include those from the soil, air, and water. They may also be obtained from surfaces, inanimate objects, food, drink, and any other products. It is also important to understand that not all microbes can live in the same places. Their presence in a certain system is dictated by their metabolic requirements and environmental ranges. Sampling is necessary for a variety of reasons:

a. To identify an infectious agent b. To monitor the health status of an ecosystem c. To monitor the status of bioremediation activities d. For Quality Assurance/Quality Control (QA/QC) activities in industry and

food/beverage production processes e. To find new microbes and/or their metabolic products (novel antibiotics, for

example). A sample is only as good as the techniques employed. It is vital that some sort of aseptic technique is utilized throughout the entire process. This includes but is not limited to the use of sterile containers, proper sampling strategies, and handling. Prior to this lab, you were asked to bring in a water sample. You will need to collect 1/2 Liter of untreated, recreational water from a source of your choice. Be sure to make sure the container you collect the water in is clean. Quantification methods for can be direct or indirect. Direct methods count cells. Microscopic counts and electronic counters such as the flow cytometer count every cell. Other direct methods are concerned only with viable cells cells capable of multiplying. Methods used for this include the viable plate count method (serial dilution), most probable number method (MPN) and filtration. Some samples may have so many viable organisms that it would be impossible to determine individual colony forming units (CFUs). If this is suspected to be the case, serial dilutions are necessary to obtain a countable plate. A serial dilution is a series of 1:10 (or 1:100 if highly concentrated) dilutions in hopes of obtaining a countable number of CFUs. Since these counts will be derived from a known amount of the original sample it will be easy to establish the number of organisms in the original sample. Usually the last three to four tubes will be plated and incubated to increase the odds of obtaining a plate where it is possible to count CFUs in an amount that is considered statistically significant (25-250 colonies). The most probable number (MPN) method can be used in water testing and uses a series of phenol red and lactose tubes with a certain amount of sample in each series. Evidence of lactose



fermentation, as indicated by color change and gas production, in each series of tubes is observed and recorded. The results are then compared to a chart to determine the MPN. Some samples, like air or well water, may have such a dilute amount that the CFU results would be indeterminate. To remedy this, a known amount of sample is taken and with the aid of vacuum, pulled through a membrane filter, this method is referred to as membrane filtration. The pores are small enough to trap cells while the water passes through. Once complete, the sample microbial population, if present will be concentrated onto a membrane that can be removed and placed on growth media and incubated. Colonies may then be counted to establish the number of microbes present in the original sample amount. In this exercise, both MPN and Membrane filtration methods are testing for indicator organisms designated coliforms or total coliforms that may indicate fecal contamination. Water contaminated with fecal matter may contain pathogens, including bacteria, viruses, and parasites. Total coliforms or other indicators are easier to detect than pathogens and therefore are used to screen for possible contamination. Coliforms are lactose fermenting gram negative rods of environmental or fecal origin, including among others: Enterobacter aerogenes, E. coli, and Klebsiella pneumoniae. E. coli, and Klebsiella pneumoniae are also designated as fecal coliforms based on their ability to grow at 44.5 C and are more specific for fecal contamination than total coliforms. If drinking water is found to contain total coliforms it should be tested for fecal coliforms and/or E. coli, which is the most specific indicator of fecal contamination since it is found in the intestine of most humans and many warm-blooded animals. Other indicator organisms which will not be tested in this lab include fecal streptococci, enterococci, and/or bacteriophage. Lactose broth with added phenol red indicator is used in the MPN test and EMB in the membrane filtration test to detect total coliforms in our lab. Lactose broth is an alternative to the standard media (lauryl tryptose broth, which is more selective) in the first (presumptive) step of a three step procedure for detecting total coliforms. Tubes demonstrating gas are normally subjected to confirmed and completed steps to rule out false positives caused by other lactose fermenting bacteria, but will not be performed in our lab due to time and material restraints. Total coliforms (lactose fermenters) on EMB are pink to purple with or without a green metallic sheen. The standard media for testing by membrane filtration for total coliforms is mEndo media at 35 C where they detected as red colonies with a green metallic sheen. References http://www.epa.gov/volunteer/stream/vms511.html http://www.norweco.com/html/lab/WhatTests.htm http://www.epa.gov/safewater/disinfection/tcr/index.html http://www.water-research.net/coliform.htm http://filebox.vt.edu/users/chagedor/biol_4684/water.html http://www.bd.com/ds/technicalCenter/inserts/m_Endo_Broth_MF.pdf http://www.bd.com/ds/technicalCenter/inserts/Eosin_Methylene_Blue_Agar.pdf

Activity I Serial Dilution E. coli culture 4 9.9 mL tubes 8 pipettes

1 pipetter 4 sterile Petri plates 4 TSA deeps

1. Label your 4 sterile water blanks as 10

-2 ,10

-4 ,10

-6 ,and ,10

-8

2. Change pipettes with each transfer:

a. Vortex E. coli culture

b. Transfer 0.1 mL of E. coli culture into the 9.9 mL test tube labeled 10-2

and vortex.



10-8

10-6

10-4

10-2 E.

coli

0.1 mL 0.1 mL 0.1 mL

10-9 10

-8

10-7 10

-

BIOL1230

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