Quantitating a Mutagen

LAB #9

Quatificating a Mutagen

Focused Reading: "Most cancers..." pp 388 Stop at end of page.

Goals for This Lab:
This week, you will count the colonies on each spot on each plate. These results will be posted on the board and discussed as a group. We will determine which stains and YFPM suggest that a compound is mutagenic. Then each group will design a new experiment to quantify the potential mutagen.

I. Introduction

We will start this week by looking at each group's data from last week. Each group should list their results on the chalkboard. List the number of colonies observed for each strain and test chemical on the chalkboard. We will discuss this together, then each lab group will determine which substance will be quantitatively analyze today.

II. Plate Incorporation Protocol

This method is different from last week's, though the principle is the same.

1. Each group will need eight plates that you poured last week. Label them with your group name, and what condition will be tested on it. I recommend writing small, and around the perimeter of the bottom. You will perform two experiments, each experiment will use four plates, and each experiment will use one plate for an appropriate control.

2. Locate the tubes of the soft-agar overlay tubes (the volume will be 5 ml instead of last week's volume of 1 ml) in the water bath on your table - each group will have 8 of these. These tubes have been autoclaved to sterilize them and put at 47° C to keep them liquified.

3. Locate the tubes containing the different appropriate strain of Salmonella which will be kept at room temperature on the lab bench. You will only test one strain that was the most sensitive one based on your results from last week.

4. If you will be touching tubes, pipets, or plates that contain bacteria, you must PUT ON GLOVES.

5. Locate your chemical to be assayed and any solvents you may be adding to the overlay soft agar tubes.

NOTE: Now you must work quickly to make sure the soft agar overlay does not harden in the tube. Make sure you understand all aspects of step 6 before proceeding.

Step 6 is not the same as it was last week.

6. Sterilely add your test chemical to the overlay tube.

a) Set your pipet to the appropriate volume of test chemical. Open the box of sterile pipet
tips, aseptically put one on, and take an aliquot of your test chemical (or solution being
used as a control) up into the pipet.

b) Remove the cap from the overlay tube, and discharge your aliquot of the test chemical into the overlay tube.

c) Sterilely add 300 µl of the proper strain of Salmonella typhimurium to the
appropriate overlay tube. Mix the bacteria, chemical and agar by thumping the tube for 3
seconds or so.

d) Lift the top of the petri plate and quickly but gently pour the entire contents of the overlay
tube onto the agar surface (try to do it gently and prevent the introduction of bubbles).

e) Gently swirl the plate with the soft agar overlay until it completely covers the surface of the minimal agar base layer.

f) Put the plate down on a flat surface and allow to harden at least 5 minutes before moving the plate again.

g) Repeat steps a-f until you have tested duplicates of 4 concentrations of your mutagen on a single strain of bacteria.

h) Invert the plates (i.e., store with the lid on the bottom) and place in a 37° C incubator for 48-72 hours.

III. Analysis of Results

After the appropriate incubation period, count the number of visible colonies on each plate. An exact size definition of a colony is less important than each group being consistent in counting colonies from one plate to another.

Written Report: Full Scientific Paper Due in one week

Be sure to include results from the control conditions.

Study questions:

1. Each group tested different concentrations of a sodium azide. Based on your results, was the mutagenicity of sodium azide proportional to the amount present? Explain your answer.

2. In your experiment testing the mutagenicity of your unknown substance, please explain the ingredients and the purpose of the positive and negative controls.

3. a) If you added a known mutagen such as sodium azide to the rich media that contained histidine, what would you expect to see? Explain.

b) If you knew that 5 µM of sodium azide was highly mutagenic, what would you expect to see if you added 50 mM sodium azide? The exact amount is not the critical issue here. The main point is that you are added a whole lot more.

4. Fact #1: Davis Minimal Agar (DMA) has none of the 20 amino acids in it.
Fact #2: Sodium azide causes base substitution mutations at locations all over the bacterial DNA, not only at the single nucleotide that is wrong in the his- gene of this mutant bacteria.

Question: What if sodium azide caused a base substitution mutation in a gene coding for an enzyme needed to make a different amino acid, such as leucine, instead of the base substitution for the wrong nucleotide in the gene for the enzyme needed to make histidine? What would you see on your petri dish of mutant bacteria on DMA and why?

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