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DNA replication in all organisms is very accurate, it has to be. (What would happen if many mistakes were made during replication?). However, mistakes do occur at a very low rate and this mutation frequency is formally defined as the probability that a particular gene will mutate each time there is cell division. On the average, there is about one chance in a million (10^-6) that a particular gene will mutate when there is DNA replication. Since bacteria can be grown to a very large concentration in artificial culture (10⁹ cells/ml or more), mutants for most genes should be present in a population of microbes larger than a few milliliters. Finding the mutants is the problem.

This experiment introduces the simple technique of direct selection for the detection and isolation of certain mutants. This technique involves plating bacteria on a selective medium on which only the desired mutants can grow. Specifically, the experiment involves taking a culture of microbes at a high density and plating them on a rich medium containing the antibiotic streptomycin. Streptomycin inhibits protein synthesis by binding to the small subunit of the ribosome and blocking entrance of initiator N-formyl methionine tRNA into the ribosome, thus preventing the start of protein synthesis. A single mutation in the S12 protein of the small subunit of the ribosome prevents streptomycin from binding, thus causing the microbe to become resistant to the antibiotic. By plating a strain onto a medium containing streptomycin, it is possible to fish out the microbes present that have a mutation in the S12 gene. Please appreciate the power of this selective technique. By performing a very simple experiment, it is possible to fish out the 100 or so cells, in a mixture containing billions, that have a change in a specific gene. Also, note how easy it is for a microbe to become resistant to an antibiotic. While the drug is killing 99.9999% of the microbes present, 0.0001% are able to survive, and these microbes are now resistant to the antibiotic. This explains why drug resistance in microbes can occur so rapidly and is a constant problem in medicine.

Period 1

Materials

Tube of a concentrated suspension of Staphylococcus epidermidis (approximately 1 X 10¹⁰ CFUs/ml) in saline

1 plate of Nutrient Agar (NA)

1 plate of NA+10 µg streptomycin (SM) per ml of medium

1 plate of NA+100 µg SM per ml of medium

Micropipettes and sterile tips

1. From the S. epidermidis suspension, pipette 0.1 ml onto each plate. Spread the inocula with a sterile hockey stick, proceeding from NA to NA + 10 µg/ml SM to NA + 100 µg/ml
2. Incubate at 37°C for 2-3 days or at 30°C for 4-5 days.

Period 2

Materials

1 plate of NA+100µg SM per ml of medium

Beaker of sterile toothpicks

1. Note the confluent growth on the plate without streptomycin and the colonies on the plates with streptomycin. On the plate containing 10µg SM/ml, there are usually seen two apparently different sizes of colonies. Do you see the smaller type of colony on the plate containing 100µg SM/ml? (If a size difference is not seen, do you at least see a reduction in the number of colonies on the 100µg SM/ml plate?)

What may these differences in colony size and growth characteristics at these streptomycin concentrations mean in terms of the mechanisms of resistance? That is, for the cells in each of the two types of colonies, what structures or functions may have been altered to become resistant (or inhibitory) to the action of streptomycin?

The instructor should pre-run this experiment to determine if lower or higher concentrations of streptomycin should be used. Results of this experiment can vary according to the batch of Nutrient Agar and streptomycin used as well as the particular strain of S. epidermidis. (Twenty µg SM/ml may give better results than 10.) The above method is based on what has worked at UW-Madison (with the use of Difco Nutrient Agar).

2. Taking into account the approximate number of CFUs inoculated onto each plate, determine the mutation frequency of streptomycin-resistant CFUs in the original suspension from the colony counts on each plate containing streptomycin.
3. Mark the bottom of the new plate of NA+100µg SM/ml plate to delineate four sectors:
• one for the colonies on NA+100µg SM/ml.
• one for the largest colonies seen on NA+10µg SM/ml.
• one for the smallest colonies seen on NA+10µg SM/ml.
• one for the growth on the plate of NA without streptomycin.
4. Using the sterile toothpicks and aseptic technique, pick 5 isolated colonies from the plate of NA+100µg SM/ml, and spot-inoculate them onto the appropriate sector of the new plate. Be sure to use a new toothpick for each transfer, and discard them into the disinfectant. Repeat for isolated colonies from the other plates. For the plate without streptomycin, simply pick from 5 sites in the confluent growth.
5. Incubate at 37°C for 2-3 days or at 30°C for 4-5 days.

Period 3

1. Observe and record the number of spot-inoculations showing growth in each sector. Discuss the significance of your results, noting the questions in Period 2, step 1.

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