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  Antibiotic Resistance in E.coli

A laboratory exercise for undergraduate evolutionary biology courses

This laboratory outline includes



 


Instructor Notes

Evolution of Antibiotic Resistance


Suggestions for instructors are in purple
    
This lab will use antibiotic resistance as example on what evolutionary biologists do and how they do it.

Potential pre-lab readings

Brandon, R. N. 1996. Concepts and Methods in Evolutionary Biology.  Cambridge University Press. Read: Chapter 9.
    Theory and experiment in evolutionary biology. Pp. 147-160.

Stearns, S. 1999. Evolution in Health and Disease . Oxford University Press. Read: Chapter 1: Introducing evolutionary
    thinking Pp. 3-15.

Garrett, L. 1994. The Coming Plague. Farrar, Straus, and Giroux, N.Y. Read: Chapter 13: Revenge of the Germs. Pp.
    422-428.

Khachatourians, G. G. 1998. Agricultural use of antibiotics and the evolution and transfer of antibiotic resistant bacteria.
    Canadian Medical Association Journal 159: 1129-1136.

    
Goal of Lab Exercise: To introduce experimental methods for investigating evolution. To understand how evolutionary biologists test hypotheses.
            
Background: Evolutionary biologists ask "why" rather than "how". Causation: ultimate (“why”?) versus proximate (“how”?). A “why” question is one that asks the historical sequence of causation. A “how” question asks about the present day functional sequence.

Lead brief discussion with students about the difference between ultimate and proximate mechanisms in evolutionary biology. Pose a question that asks why a certain trait exists in a modern population. Examples:

Ask how students would test the ultimate explanations generated by the preceding questions.

Talk about difference between “hypothesis generation" and "hypothesis testing".  


The above discussion should stimulate ideas about the following pre-lab question:


Questions to think about before lab:


Now, consider the question: Why is there antibiotic resistance in organism x?  
    Possible responses:
Which of the above responses best addresses the historical events that led to the existence of antibiotic resistance as a trait? (which is a “why” question and which is a “how” question?).

Introduce specific instructions of antibiotic resistance lab (follows)
 
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Instructions for Antibiotic Resistance Lab

Isolation, culture, and transfer techniques for E. coli.

Isolation of Escherichia coli

Background:  E. coli is a common bacterium in the gut of many organisms.  For this lab, we will use sterile cotton swabs to isolate E. coli from ourselves. The swabs will be used to inoculate petri dishes containing a selective and differential medium, eosin-methylene blue (EMB).  Using this medium, we can differentiate E. coli from other bacteria based on two properties.  First, EMB supports the growth of gram-negative organisms and inhibits the growth or gram positive organisms.  Second, E. coli can be differentiated from other coliform bacteria by its metallic green sheen when grown on EMB (many other bacteria will have a pink mucousy appearance).  Once inoculated, the plates are incubated at 37°C (body temperature) to support growth of the bacterial colony.  

Week 1


Objectives:
Isolate up to 18 distinct individual colonies of E. coli for use next week in antibiotic resistance assay.

Day 1

Figure 1

figure1



Day 2


Hopefully today you will have E. coli and possibly other coliform bacterial
colonies on your plate.  E. coli will be metallic green (see below). If you do not have any bacteria, grab another swab and try harder!

 
ecoli colonies

 E. coli colonies - GREEN !!



not ecoli
             

   Not E.coli - PINK !!



Instructors should have a picture or sample of a “good” plate of colonies (see above).

We will now use the following protocol to isolate pure cultures of E. coli. We need to go through these steps to ensure that:
a)    there are no contamination by other bacterial species
b)    our cultures are not mixtures of lines of E.coli

If you have E. coli on your plate, you will need 4 fresh EMB plates.  

You should label these plates A1, A2, B1, and B2.  (Also label the plates with your name and the date and “E. coli”)

To keep track of sequential cultures, make a mark on the bottom of each plate denoting which direction is ‘UP’ on the plate (see Figure 2).  This mark will always be placed pointing away from you.  
 

Figure 2

figure2
Instructors should reproduce a copy of Figure 2 that is the same size as petri dishes for students to use as pattern.

Figure 3

figure3
Day 3


Day 4

   If you have >10 colonies in pure culture on plates C and D, place plates in refrigerator until the next scheduled lab.  We will use these colonies to assay for antibiotic resistance.


plating colonies

Student plating colonies

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Week 2

Objectives

Testing strains for antibiotic resistance



Assignment for independent projects


Make sure students know what supplies will be available for projects. Advise them to keep projects short and with the possibility of a definitive result by the next scheduled lab meeting. These projects could be turned into semester-long independent research projects if time and facilities permit.

1. Write a paragraph or two describing what you plan to do for your independent projects and discuss project with the laboratory instructor.

2. Do background research and reading on your topic.

Suggested General Reading (before next lab)

Bonten, M., Stobberingh, J. P., Houben, A. 1992. Antibiotic resistance of Escherichia coli in fecal samples of healthy people in     two different areas of an industrialized country. Infection 20: 258-262

Graves, S. R., Kennelly-Merrit, S. A., Tidemann, C. R., Rawlinson, P. A., Harvey, K. J. and Thornton, I. W. B1988.        
    Anti-biotic resistance patterns of enteric bacteria of wild mammals on the Krakatau Islands and West Java, Indonesia. 
    Philosophical Transactions of the Royal Society, London, Series B 322: 339-353.

Levin, B. R., Lipsitch, M., Perrot, V., Schrag, S., Antia, R., Simonsen, L., Moore Walker, N.,
    Stewart, F. M. 1997. The population genetics of antibiotic resistance.  Clinical Infectious
    Diseases
24 (Supplement): S9-S16.

 
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Week 3

Objectives  

Encourage students to question presenters about methods and potential interpretations and pitfalls of proposed projects.

Instructors should provide directions about length and scope of presentations that will be given at next lab period.

Dilution series

Today

Tomorrow

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Week 4

Objectives


Depending upon scope of course or writing assignment, these lab reports could include significant literature review as well as the inclusion of both the initial test of antibiotic resistance that the class did as a whole along with the independent projects.  


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Appendices for Instructors  - Antibiotic Resistance Lab


Student Supplies


Week 1: E. coli  isolation and pure culture

    supplies per student:
        7 EMB plates (for 18 isolates)
        1 sterile swab
        ~75 sterile toothpicks
    supplies at side station:
        Bunsen burner
        loop
        parafilm

Week 2:  antibiotic resistance

    supplies per student:
        2 of each antibiotic EMB plates (we used 6 different antibiotics)
        2 control EMB plates
        ~20 sterile toothpicks
    supplies at station:
        parafilm
    introduction to liquid culture and counting colonies:
        Lab instructor or TA makes 5 bacterial cultures in LB, transfer to MD25
        for counting, students need to dilute and plate:
    supplies per student:
        3 LB plates
        3 dilution tubes with salt solution
    supplies at station:
        P200 pipetteman
        sterile pipette tips
        spreader
        Bunsen burner
        ethanol bath

Week 3: independent projects

    Varies based on independent projects

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Growth Media Recipes

Luria-Bertant (LB) Medium

    10g bacto-tryptone
    5g bacto-yeast extract
    10g NaCl
    15g agar
    1 L deionized water
    adjust pH to 7.0 with  ~250 ml 1N NaOH
    autoclave 20 minutes @ 15lb/sq inch on liquid cycle

Eosin-methylene blue (EMB) agar, Levine (pH 7.2)

    10g peptone
    5g lactose
    2g potassium phosphate dibasic
    15g agar
    0.4g eosin-Y
    0.065g methylene blue
    1 L deionozed water
    adjust pH to 7.2
    autoclave 20 minutes @ 15lb/sq
        inch on liquid cycle
Dilution tubes
    9.9 mL 2% NaCl solution


antibiotic solutions
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Sample Results

Class resistance totals – UVA 2000

Antibiotics tested

1.    ampicillin    (Amp)
2.    chloramphenicol    (Chl)
3.    kanamycin     (Kan)
4.    rifampicin (Rif)
5.    streptomycin (Strep)    
6.    tetracycline (Tet)

Class size of 15

12 people with no resistance to any antibiotics
    (total 216 lines with no resistance)

3 people with resistance
    person A
        17  lines resistant to Amp, Strep, and Kan
        1 line resistant to Kan only
    person B
        1 line resistant to Amp, Chl, Kan, and Strep
        16 lines resistant to Amp and Strep
    person C
        11 lines resistant to Tet

Total number of lines grown = 262


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