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.

The general process of performing a serial dilution is shown in the image above. The process we use in this lab is similar to that shown above, but we first perform two dilutions by adding 1 ml of the culture to 99 milliliters of sterile water, followed by the six dilutions shown above. Thus, the final dilution produces is a 10-10 dilution, meaning that it is ten billion (1010) times more dilute than the original culture. The supplies we use in lab for this serial dilution are shown in the image below.

The next step is to pipet 1 ml of the last five dilutions (10-6 through 10-10) to each of two empty Petri plates – ten plates in total. 15 milliliters of melted agar cooled to 50oC is then added to all ten of these plates. The plates are then gently swirled to evenly distribute the bacteria in the media, allowed to cool, and the solidified plates are incubated at 37oC for 18-24 hours. Empty Petri plates and a melted agar deep are shown in the images below.


After an overnight incubation, the plates are visually inspected. Bacteria having landed on the surface of the agar will have produced large, circular colonies, whereas bacteria embedded within the agar will have formed much smaller almond-shaped colonies. You then select the pair of plates with an estimated number of colonies between 40 and 400. Such a pair of plates is shown below.

The colonies on this pair of plates are easily and accurately counted by placing the plates on a colony counter containing a grid and methodically counting the colonies one box at a time. The dilution of the chosen plates and the exact numbers of colonies counted are recorded. You then take the average number of colonies between the two plates and divide this number by the dilution factor (10-6, 10-10, or something in between) to calculate the concentration of bacteria in the original culture. (Note: Dividing a number by 10-10 is the same as multiplying the number by 1010.) The colony counter used is shown in the image below.

Data: For this lab, assume you counted 86 and 97 colonies the two 10-7 dilution plates. Using this data, you should be able to calculate the bacterial concentration in the initial overnight culture in cells per milliliter (cells/ml).


Part II: Detecting Streptomycin-Resistant Mutants

This portion of the lab uses the same broth culture of antibiotic-sensitive E. coli that has been grown overnight, and the goal of this portion of the lab is to find any rare mutants that are resistant to streptomycin.

One milliliter of this overnight culture is transferred into a pair of TSA plates (0.5 ml per plate) containing the antibiotic streptomycin and spread evenly upon the surface of these plates. Only streptomycin-resistant bacteria can survive on these plates. The plates are then incubated at 37oC overnight and inspected shortly thereafter.

Paired plates from thirteen cultures were assessed in the laboratory this semester; eleven of these pairs of plates contained no antibiotic-resistant colonies and two pairs of plates contained colonies of streptomycin-resistant bacteria. One of these pairs of plates contained only a single colony, and the other pair of plates contained six streptomycin-resistant colonies.

From this data and the data in the previous section you should be able to calculate the mutation rate, which is defined as the number of mutations conferring streptomycin-resistance divided by the total number of bacteria. (The total number of bacteria is equal to the total number of cell divisions.)


Part III: Detecting Double Mutants Resistant to both Streptomycin and Ampicillin

In this portion of the lab you will begin with an F (F minus) streptomycin-resistant culture of E. coli (“Strain 1” obtained from the first part of this lab) and an F’ (F prime) E. coli strain that is resistant to ampicillin (“Strain 2”). Each of these strains is resistant to only a single antibiotic. The goal of the experiment is to detect bacteria resistant to both antibiotics – these bacteria are the result of conjugation between the two aforementioned strains.

Each of these strains is inoculated on the surface of three different types of plates – TSA plus streptomycin, TSA plus ampicillin, and TSA plus both streptomycin and ampicillin. The two cultures are then mixed and then allowed to sit at room temperature for 15 minutes. During this time, conjugation should occur between these two strains. The mixture should then be inoculated on the surface of the same three types of plates. These nine plates are then incubated overnight at 37oC and inspected shortly thereafter.

The results of this portion of the experiment are shown below, and all six lab groups obtained the same results (six replications). Strain 1 was inoculated onto the plates of the leftmost column and strain 2 was inoculated onto the plates of the rightmost column. The mixture of the two strains was inoculate into the plates in the middle column. The top row of plates contains streptomycin, the center row of plates contains ampicillin, and the bottom row of plates contains both streptomycin and ampicillin.




Antibiotic Resistance Questions

Please answer the following questions and then submit this completed document to the “Assignments” folder in D2L. Please post only these questions and your answers, not the several pages detailing the lab exercise.

  1. Why did we need to perform a serial dilution in Part I instead of simply inoculating 1 milliliter of the initial culture into pour plates and counting the resulting colonies?
  2. Given the data above, what is the concentration of bacteria in the initial culture (cells/ml)?
  3. In Part II, one pair of plates contained a single colony, whereas the other pair of plates contained six colonies. How many mutations were necessary to produce the six streptomycin-resistant colonies? Explain.
  4. Do you agree of disagree with the following statement. Explain. “Mutations conferring antibiotic resistance occur in order to protect bacteria from antibiotics.”
  5. Which of the nine plates in Part III is/are controls and which are experimental plates? Explain.
  6. Doubly resistant bacteria are present in the center plate in the bottom row. How do we know that these doubly resistant bacteria are the result of conjugation and not the result of new mutations?
  7. Bacteria resistant to multiple types of antibiotics are becoming significant health care issues. In as much detail as possible, describe why this problem has arisen.
  8. I went to a highly regarded public high school, but my biology instructor failed to teach evolution in this course. Studies have shown that this is the case in approximately 1/3 of all high school biology courses in Minnesota. Why do you think so many high school biology instructors fail to cover evolution in their courses? How do you think this contributes to the problem of antibiotic resistance?
  9. Design an experiment to produce bacteria resistant to the antibiotics ampicillin, streptomycin, a d tetracycline.


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