You have a culture of Staphylococcus epidermidis cells at a concentration of 2.5

Question

You have a culture of Staphylococcus epidermidis cells at a concentration of 2.5x10^9/ml. You know that this organism is normally sensitive to streptomycin, yet you would like to check to see if there are mutants present in the culture that happen to be resistant streptomycin. You plate 0.1 ml of this culture on a plate containing Nutrient Agar plus Streptomycin.

a. After incubation, 31 colonies appear on the plate. What is the mutation frequency?

b. What specifically happened to cause these mutants to appear?

c. What were the differences in types of colonies that grew under the different concentrations of streptomycin?

Explanation / Answer

A) determine whether the agar plate assay is suitable for mycobacteria, we plated Mycobacterium smegmatis on quinolone-containing agar and measured colony accumulation over a 2-week period. To test for mutant induction, we blocked the induction of the mutagenic SOS response with a recA mutation and measured the effect on the accumulation of ciprofloxacin-resistant colonies. The sensitivity of the assay to fluoroquinolone structure was then examined with four commercially available compounds, and assay sensitivity to mutator mutants was assessed with a spontaneous mutator. When we applied the agar plate assay to M. tuberculosis using ciprofloxacin, a fluoroquinolone known to enrich resistant mutants (65), mutant induction was readily observed. We expect the assay to be useful for comparing anti-TB agents and for assessing the mutator status of bacterial isolates.

c)

Originally many attempts were made to demonstrate the presence of strepto- mycin-dependent variants in the parent streptomycin-susceptible cultures.

We have shown previously (Yegian and Vanderlinde, 1948) that in a large popu- lation of cells there area few naturally occurring variants that are able to grow in the presence of 100 and 1,000 ,&g of streptomycin per ml of medium.

In the course of our studies more than 200 colonies growing in the presence of 100 or 1,000 Mug of streptomycin per ml have been analyzed to demonstrate the presence of streptomycin-dependent bacteria. Thus far

no organisms have been found which grow only in the presence of streptomycin even though the 200 colonies represent resistant organisms obtained from vast numbers of the parent-type cells. It is evident that the incidence of streptomycin-dependent variants is very low.

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