Laboratory Investigations in Microbiology
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Chapter 21: Microbial genetics

We have recently discussed several ways by which bacteria change - altering gene expression, genetic recombination, and mutation. One such area of change that has a tremendous impact on society is the emergence of antibiotic-resistant bacterial strains. Antibiotic resistance can arise in several ways; most prominently, bacteria can mutate or obtain plasmids that carry resistance genes. Today we will investigate the emergence of Streptomycin resistance in Staphylococcus by mutation.

Although bacteria, like animals and plants, have a good system for proofreading DNA and repairing DNA damage, mutations occur quite frequently in bacterial populations. One reason for this is that bacteria have only one chromosome, and hence, only one copy of each gene. (Many eukaryotes are diploid or polyploid, having two or more copies of each chromosome). What this means to the bacteria is that any mutation in a gene will affect the organism immediately, since there is no "backup copy" of that gene available. A second reason for the rapid emergence of mutants is that bacteria replicate so rapidly. Within 24 hours, some bacteria may have gone through nearly 100 generations! With several 100 million bacteria all dividing, the chance of a mutation arising is actually quite high.

Streptomycin is an antibiotic that binds to the ribosomes of bacteria. Since it easily penetrates bacterial membranes and cell walls, it is a broad spectrum antibiotic. However, its effectiveness depends on the shape of the bacterial ribosome. A normal bacterial ribosome will be inhibited by streptomycin, whereas mutant ribosomes may just be shaped differently enough to escape the effect of this antibiotic. Although these mutant ribosomes likely do not work as well, the slower growth is offset by the advantage these bacteria have when in the presence of streptomycin. Such mutations that change the ribosome shape occur randomly and spontaneously, as replication errors. Thus, any population of Staphylococcus will contain some of these mutants. However, because they grow less efficiently, they usually do not flourish. However, under selective pressure (e.g. the presence of streptomycin), the mutants are now the only bacteria that can grow at all. Thus, the presence of streptomycin selects for any mutants that are already in the population. 

The purpose of this exercise is to examine a Staphylococcus population and to isolate any spontaneous mutants resistant to streptomycin.

Materials

Per Lab group

• 1 sterile Petri plate
• 2 test tubes of melted TSA (keep in water bath until needed)
• Streptomycin solution (10 mg/ml)
• Culture of Staphylococcus aureus
• 6 TSB tubes

Procedure

1. Prepare a streptomycin gradient agar plate as follows:
   • Pour one melted TSA deep into a sterile Petri plate that is raised up on one side so that the agar hardens at an angle
   • Add 100 µl of Streptomycin to a second agar deep
   • When the bottom agar is hard, set the plate on a level surface. Pour the streptomycin agar on top. Let it harden
   • Be sure to label which side of the plate is the high-Streptomycin side
2. Inoculate your gradient plate with Staphylococcus:
   • Swab the entire plate with a sterile cotton swab soaked in the S. aureus culture
3. Label your plate and incubate at 35°C

Next Lab period

1. Examine your spread plates for growth. Describe the growth on your plate.
2. Select some colonies of S. aureus closest to the Streptomycin end of the plate for further study (circle them on the back of the plate)
3. Obtain 6 TSB broths.
   • Add 100 µl of streptomycin (10 mg/ml) to two of these tubes. Label these tubes "hi-S"
   • Add 10 µl of streptomycin (10 mg/ml) to two of these tubes. Label these tubes "lo-S"
   • Label the last two tubes "CON"
4. Inoculate one hi-S, one lo-S and one CON broth with a colony from the Streptomycin end of the gradient agar plate. Label these tubes as "Resistant"
5. Inoculate the remaining broths with a loop full of the original SA broth. Label these tubes "Sensitive"
6. Incubate at 35C.

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