MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #13: GENETICS; SPONTANEOUS AND UV INDUCED MUTATIONS

NAME, ID #:_______________________________________________.

TA Name __________________________

REVISED: 08/04/99

INTRODUCTION

A mutation is any change in base pairing in genomic material.

As you learned in the lecture section on #evolution, spontaneous mutations provide the RAW MATERIAL for evolution by providing random genetic variation. Spontaneous mutations are those mutations whose CAUSE IS NOT IDENTIFIED. It is known that the earth is constantly exposed to a rain of mutation-producing cosmic radiation from space, as-well-as to earth-bound natural radiation sources (e.g. radon & uranium in the soil) and to natural mutagenic chemicals. In the modern world we are also exposed to a variety of man-made mutagens. You have seen how DNA #replicates. Although there are elaborate correctional system built into the DNA replication process, #mistakes do occur and mutations become fixed in the newly replicated DNA. In general, mutations are RANDOM, in that every base pair on a DNA or RNA genome has approximately the SAME CHANCE of being changed (mutated) in any given time period.

Most mutations are HARMFUL in that they destroy, or otherwise perturb, a function that is PRECISELY INTEGRATED into a series of interdependent living processes. Much like damaging or removing a part from a computer would more likely damage it than it would make it run better. However, many mutations are neutral in the environment of the creatures they occur in; they may only add innocuous or minor VARIETY, like different eye or hair coloring. In a few cases they are clearly BENEFICIAL, like the mutation recently discovered in a small group of Italians that renders cholesterol harmless to their circulatory system. However, when the "cold winds of natural selection" blow over a species a once "NEUTRAL VARIATION" may suddenly mean the difference between SURVIVAL or EXTINCTION.

Specific mutations can not be INDUCED on demand, they can only be SELECTED FOR by the environment if they are present in a population. This point was illustrated by the appearance of ANTIBIOTIC RESISTANCE in bacteria. Soon after antibiotics became commonly used it was observed that if you plated several millions of bacteria on a medium containing a lethal dose of an antibiotic, an occasional colony grew (see pg. 91 in the Atlas). When tested, cells from such colonies were found to no longer be SUSCEPTIBLE to that antibiotic; they were now RESISTANT to it. They retained this resistance even when cultivated for many generations away from the antibiotic; that is, the resistance had become a genetic characteristic of that MUTANT. The argument raged over whether the antibiotic itself somehow "INDUCED" mutations causing the resistance or if the resistant microbe already EXISTED PRIOR to exposure to the antibiotic. After a series of brilliant experiments it was proven that the latter was the case. The only role of the antibiotic was to SELECT out antibiotic resistant mutant cells that, BY CHANCE, happened to be present in the large population of cells introduced to the plate.

You will attempt to demonstrate the selection of antibiotic resistant mutants to an antibiotic using the GRADIENT PLATE TECHNIQUE. In this procedure two tubes of melted/cooled agar media are prepared. To one of them is added the antibiotic, while nothing is added to the other. A petri dish is tilted at an angle and the medium from the tube WITHOUT the antibiotic is poured into the petri dish and allowed to solidify. Then the plate is placed flat and the antibiotic-containing medium is poured on top of the first medium. After solidification bacteria are swabbed over the surface of the plate and it is incubated. Because of the varying thickness of the antibiotic-medium a gradient of antibiotic concentration is formed from near 0 to the maximum concentration. If antibiotic resistant mutants are present a few colonies will often develop in areas of higher antibiotic concentration.

The single major cause of mutation on earth and, therefore of CANCER, is UV light. Lethal doses of UV radiation from the sun baths the earth, yet life did develop. It developed because of the evolution of photosynthetic bacteria that produced free oxygen as a waste product of photosynthesis. This oxygen not only makes the AEROBIC FORM OF LIFE we enjoy possible, but because it is converted to OZONE (O₃) in the upper atmosphere and because ozone ABSORBS the bulk of the UV radiation that impacts on our planet, life on land is possible. Elimination of the ozone would likely destroy all land life on earth. Do you know what the "Ozone Hole" is and why people are worried about it?

In this exercise you will see how UV light causes mutations. You will expose a pigmented bacterium to high doses of UV light. You will plate the UV-treated bacterium on medium and examine it for the present of nonpigmented mutants.

PURPOSE OF LABORATORY:

1. To introduce you to the concept of spontaneous mutation.
2. To demonstrate spontaneous mutation.
3. To demonstrate the mutagenic effect of UV light.

RELATIONSHIP TO LECTURE MATERIAL

• NetText101/102: CHAP. VIII , Fundamental of genetics, Mutations

GENERAL INSTRUCTIONS:

1. Please place all appropriately labeled drawings on the back of the manual so the instructor can identify them.

MATERIALS:

1. Cultures of E. coli and Serratia marcesens.
2. Melted agar in tubes with and without antibiotic in water bath. One of each/student.
3. Empty sterile petri dish. One/student
4. UV lamp and eye protection.
5. Sterile swab. One/student.

PROCEDURE:

SPONTANEOUS MUTATION

1. Please read pg. 98-90 in A Photographic Atlas for the Microbiology Laboratory before proceeding and examine the fig. below carefully.
2. Obtain a sterile petri dish. Label it with your name. Draw an arrow in the center of the bottom of the plate.
3. Place the plate on a pencil or other object to tilt one end up with the arrow is at a right angle to the object the plate is sitting on.
4. Pour a tube of the MELTED-COOLED medium without antibiotic into the plate and allow it to harden. The tilt of the plate should be such that the liquid doesn't quite reach to the top edge of the angled plate.

5. When the agar has hardened (2-5 minutes), set the plate flat on the desk and add the medium containing the antibiotic. The TA will add the appropriate quantity of antibiotic immediately before you pick up the medium.

6. Allow it to harden for 15 to 20 minutes. The TA may allow you to place it in the refrigerator to speed hardening up.

7. Using a sterile swab gently spread the E. coli culture evenly over the surface of the medium, being careful not to tear the agar.

8. Incubate as designated by the instructor (~72 hours).

9. At the next lab observe the plate for the pattern of bacterial growth and then place it back into the incubator. At the next observation draw a picture of it. Circle any isolated colonies growing in a region of higher antibiotic concentration and show them to the instructor.

UV MUTATION

1. Before proceeding read pg. 91-92 in A Photographic Atlas for the Microbiology Laboratory and make sure you understand what is going on there.
2. Label nutrient agar plates as indicated by the instructor.
3. Swab Serratia marcesens across the surface of the plates as before, covering the complete surface, including the strip around the edge.
4. Turn the room lights down.
5. Remove the lid and cover ½ of the plate with an opaque material (paper towel). Put on your SAFETY GLASSES. Expose the plates as indicated by the instructor to the UV lamp Record the LENGTH of the UV exposure.
   • UV light is dangerous; follow the instructor's directions and avoid exposing your eyes or any skin to the UV.
   • Each bench will do a different exposure time; write down the time.
6. Place the exposed plate at room temperature in a sandwich bag in your drawer IN THE DARK until the next lab.
7. At the next lab period examine the plates carefully for: (1) the number of survivors vs. exposure time to UV; and (2) the number of WHITE colonies vs. time of exposure to UV light. Compile and write up your observations, putting any quantitative data in table form.

TABULATIONS OF RESULTS

SPONTANEOUS MUTATION: Label the plate as to orientation of the antibiotic gradient.

UV MUTATION

DISCUSSION OF THE RESULTS:

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• Explain the relationship between UV and skin cancer. On which part of the body do the most skin cancers develop?
• What is the role of ozone in protecting the earth from harmful doses of UV?
• Why is the "Ozone Hole" considered so dangerous? What country in the world is most affected by the "Ozone Hole"?
• Define "random nature of spontaneous mutations".
• Explain the statement "the cold winds of natural selection".
• How can DNA replication lead to mutations?
• What is a "neutral mutation"?
• Develop a scenario where a mutation that increased the body hair on a person might have a "beneficial" survival effect if certain environmental conditions changed? How about a scenario where this same mutation might be a "detriment" to survival?
• If an evil alien were to infect every human on earth with the HIV (AIDS) virus, do you think that would eliminate the human race. Why or why not?

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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