Day 1: Syllabus, Safety, and Movie introduction to the microbiological laboratory




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THE BUGS

Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis


EXERCISE

1)Gram stain Enterococcus faecalis and Staphylococcus aureus

2)View your plate from last week.

3)Carry out catalase test on Staphylococcus epidermidis, E. coli, and E. faecalis

4)Inoculate a tri-sectored mannitol salt plate with S. epidermidis, S. aureus, and E. coli (3 bugs/plate).

5)Innoculate MacConkey's with E. coli, E. faecalis, and S. aureus.

6)Inoculate tri-sectored Bile Esculin plate with S. epidermidis, Staphylococcus aureus, and Enterococcus faecalis

7)Look up properties of the bugs - Gram's character, where do they normally live, and their medical significance.

DEMONSTRATION


Tri-sectoring plates

QUESTIONS


1)Look up expected results.


2)How (exactly) are genes involved are biochemical pathways?

3)In the cell, how is catalase formed?

4) Which diseases are cause by Staphylococcus aureus? Enterococcus faecalis?

5)Are Staphylococci strict aerobes or facultative anaerobes?

6)Look up and know the selective and differential properties of each of the test media above. This means, know the chemicals and reactions that allow differentiation and selction.

7) Know definition of terms ‘selective’ and ‘differential’.


LABORATORY HELPERS

1)NA plate cultures of Micrococcus, S. epidermidis, E. faecalis, and E. coli - one plate per bug.

2)Fresh hydrogen peroxide from Safeway

3)5 beakers with autoclaved tootpicks.


3)48 mannitol salt agar plates

4)48 MacKonkey's agar plates

48 Bile esculin plates


REFERENCES

Introduction to Diagnostic Microbiology: A Text and Workbook, Maria Dannessa Delost, Mosby, Inc., St. Louis, © 1997, pages 105, 274-276, and 284.


Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 125-126 and 141-143.


Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, page 159.


Microbiology Experiments: A Health Science Perspective, 3rd Edition, Kleyn & Bicknell, McGraw-Hill Companies, Inc., Boston, © 2001, pages 200-202.

Day 8: Bacteriophage


BACTERIOPHAGES
Bacteriophage are viruses that infect eubacteria and archaebacteria. The T even phage infect and kill the common intestinal inhabitant, Escherichia coli. They enter the bacterial cell by ‘landing’ on the cell wall and injecting their DNA into the bacterial cytoplasm. After entry, the phage DNA acts as a template for production of phage proteins. These proteins replicate the phage and subjugate the cell, eventually causing lysis and death of the host cell.


PLAQUE ASSAY

In the interest of controlled experimentation or accurate diagnosis, we often need to quantify the number of bacteriophage (or other viruses) in a stock. The method employed is called the plaque assay.

The principle: If you have a tube of bacteria and plate them out onto an agar plate, what do you expect to happen? Now if you mix a bunch of bacteriophage in with these bacteria, what do you expect to happen? If there are enough viruses, all of the bacterial cells will become infected and nothing will grow on the plate. If there are only a few phage, what will happen?


DILUTIONS
Since microbes may grow in large numbers, it may be necessary to reduce the number of microbes within your sample in order to actually isolate individuals on a plate. You use the process of dilution to accomplish this. Sometimes, as is often the case with virus preparations, the sample must be diluted extensively by a process called serial dilution. Mathematically, a dilution equals the volume of the sub-sample divided by the volume of the sub-sample plus the diluent.


DEMONSTRATION: Use of micropipettors– be careful these are expensive. Use of hockey stick for spread plate.


EXERCISES

1) Plaque Assay- Your task is to figure out how many bacteriophage are in this tube.

Serial dilution

a)Add 90ul sterile H2O to sterile microcentrifuge tubes. Remove 10ul of virus stock and add it to tube (#2) containing 90ul H20. Mix well. Remove 10ul from tube 2 and add to next tube containing 90ul H20. Mix well. Repeat until all tubes contain a phage dilution. Label all tubes and plates. MIX WELL AT EACH STEP!!

b)Add 100ul of Escherichia coli B overnight broth culture to each dilution. Incubate for 5 minutes at room temperature.

c)Distribute entire contents of each tube onto a labeled Nutrient Agar plate using 200ul micropipettor. Distribute with hockey stick. Incubate lid up.

2)View selective and differential plates from last week and Gram stain Micrococcus luteus.



THE BUGS


T even series bacterophage. T2, T4. Escherichia coli B = phage host.


QUESTIONS:

1)How does a plaque form?

2)What is a bacteriophage?

3)Are viruses cells?

4)What is a serial dilution?

5)Describe differences in cell entry strategies of bacterial versus animal viruses.

6)Why is it necessary to mix the solution at each dilution?

HELPERS

  1. 6 tubes - E. coli B phage host 48 hour culture in 10 ml of nutrient broth.

  2. 90 nutrient agar plates

  3. 50 tubes each with 10 mls of sterile deionized water

  4. 500 ml Beaker (cover beaker with aluminum foil while autoclaving) full of autoclaved microcentrifuge tubes (for dilutions).

  5. Bring micropipettors to lab. Sterilize micropipettor tips.

6) Virus microfish slides and viewer.


REFERENCES

Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 411-414.


Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, page 275.


Microbiology Experiments: A Health Science Perspective, 3rd Edition, Kleyn & Bicknell, McGraw-Hill Companies, Inc., Boston, © 2001, pages 183-190.


Microbiology: A Human Perspective, 2nd Edition, Nester, Roberts, Pearsall, Anderson, & Nester, McGraw-Hill Companies, Inc., Boston, © 1998, pages 169, 299, 305-306, and 311-313.


Microbiology: An Introduction, 6th Edition, Tortora, Funke, & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 1998, pages 234, 284, 367, and 370-375.

Day 9 - Unknown Analysis and Antibiotics

1)Read Plates from last week


2)Discuss bioinformatics search


3)Unknowns

The unknown preparation will contain one of the organisms you have studied this term. Your approach needs to include a flow chart or table outlining your plan of attack. When you are finished with your plan, review with instructor to ensure that you are on the right track. You will have one lab period to inoculate plates and carry out Gram stain on your unknown. You will come in the next day to read your plates. Then, with data, write up the lab report as described below


This report of your unknowns will be graded 80% on how you rationalize you approach and the accuracy with which you interpret the results of your tests. The report format is described below.


FOR LABORATORY HELPERS TO DO

    1. Streak each unknown onto each plate type, three bugs per plate

    2. 50 mannitol salt plates

    3. 50 tomato juice agar plates

    4. 50 MacConkey's

    5. 50 NA plates

    6. 50 Bile esculin plates



ANTIBIOTICS

ANTIBIOTICS

Antimicrobial agents are chemotherapeutic agents used to control microbial infections in humans, animals, and even plants. Antibiotics are antimicrobial agents that have a biological origin. Penicillin, doxycycline, and neomycin are examples of antibiotics that are commonly used in modern antibiotic therapy regimes. Ampicillin is used commonly in the research laboratory.


EXERCISES

1)In groups of two, one partner spread plate E. coli and the other S. epidermidis onto nutrient agar plate.

2)Overlay the provided antibiotics and/or antibiotic discs onto plate and incubate lid side down at 37C. Look up target and source of each antibiotic. What category of antibiotic is ampicillin? Doxycycline?

3)Inoculate the API 20 E systems identification with E. coli and Staphylococcus epidermidis.


4)Record results of the plaque assay and answer the question ’How many viruses were in the original tube of virus?’


QUESTIONS

1)Why do bacteria and fungi produce antibiotics?

2)Do you expect G+ and G- bacteria to respond differently to antibiotics used in this exercise? Why?

3)Why are API test strips useful?

4)Is neomycin administered internally? Why or why not?


HELPERS

1)Nutrient agar plates with E. coli and Staphylococcus epidermidis.

2)48 Nutrient Agar plates

3)1 pair of forceps for each table

4)antibiotics discs from the refrigerator.

5)Broth cultures of E. coli and S. epidermidis.


REFERENCES

Exercises for the Microbiology Laboratory, Burton E. Pierce & Michael J. Leboffe, Morton Publishing Company, Colorado, © 1999, pages 51-98.


Introduction to Diagnostic Microbiology: A Text and Workbook, Maria Dannessa Delost, Mosby, Inc., St. Louis, © 1997, pages 28-31.


Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 93-96.


Microbiology: A Human Perspective, 2nd Edition, Nester, Roberts, Pearsall, Anderson, & Nester, McGraw-Hill Companies, Inc., Boston, © 1998, pages 99-100.


Microbiology: An Introduction, 6th Edition, Tortora, Funke, & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 1998, page 166.


Microbiology Experiments: A Health Science Perspective, 3rd Edition, Kleyn & Bicknell, McGraw-Hill Companies, Inc., Boston, © 2001, pages 57-60.


DAY 10

Unknown report

The report should contain the following headings.


Introduction

  1. Description of all organisms that could be in your sample

  2. Principles of all test media and chemical tests you used

Methods

  1. List reagents used

  2. Describe experimental protocols employed

Results

  1. Describe the results of each test performed (don’t discuss results in this section; results are discussed in the conclusion section)

Conclusion

  1. List the name of each positive identification

  2. Beside each suspect, summarize the results that led to the identification. Negative results are valid evidence.


Scoring Breakdown Table

Section Headings

3points

Principles of tests

8 points

Descriptions of microbes

5 points

Results Section

4 points

Results Table

2 points

Conclusion and arguments

8 points

Italicize or underline scientific names

1 point

Tell me which tube you had

2 points

Correct ID

3 points

Handing in the report

3 points






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