Production of Antibiotic-Resistant Mutants

This project will encompass two labs in your lab manual – “Production and Isolation of Antibiotic-Resistant Bacterial Mutants” on page 299 and “Production and Isolation of Multiply-Resistant Bacterial Mutants” on page 307. Carefully reading these labs in your lab manual will be important in understanding this process.

This pair of labs will help you learn how to quantify bacteria in a broth culture, identify rare antibiotic-resistant mutants within an otherwise antibiotic-sensitive culture, compute the rate at which this antibiotic-resistant allele arises, understand the process and results of bacterial conjugation, and identify bacteria simultaneously resistant to two different antibiotics.

Part I: Quantification of Bacteria in a Broth Culture

Each student is given a broth culture of antibiotic-sensitive E. coli that has been grown overnight, and the goal of this portion of the lab is to determine the concentration of these bacteria in cells per milliliter (cells/ml). Given the turbidity of the culture, it is apparent that the concentration of bacteria is very high, perhaps millions or billions of cells per milliliter. To determine the concentration, you will produce a series of dilutions of this original culture, after which you will add 1 ml of these dilutions to 15 milliliters of melted nutrient agar.


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