Objectives

The major objective in this experiment is to determine if compounds in tobacco can cause DNA mutations in a strain of bacteria (Serratia marcescens), in as little as 2 days. Mutations in bacterial DNA cause the bacteria to change from red to white. After completing the exercise, the student should be able to:

• Discuss the effect of tobacco on DNA
• Describe how a DNA mutation is manifested in S. marcescens
• Explain how fast a DNA mutation can occur
• Explain how one could determine if nicotine can cause mutations in the same system

HYPOTHESIS AND EXPERIMENTAL DESIGN

To address the objective of this exercise the following hypothesis can guide the experiment:

Compounds in tobacco will cause mutations in the DNA of the bacteria, Serratia marcescens, within 2 days.

To test the hypothesis, one needs to design how to set up the experiment, including conditions that will serve as “controls”. In this experiment, there are be 3 experimental conditions to measure DNA mutations in S. marcescens: 1) extract of tobacco (in water), 2) UV light exposure, and 3) water alone. The water controls for possible mutations produced by something in the water that is used to make the extract. Second, the UV light causes mutations in many organisms, including humans. It is used to show that the experiment actually works… the UV light should cause DNA mutations regardless of what happens with the tobacco condition. OK, now let’s take a look at the actual procedure.

MATERIALS

• Online access to the 10 minute “virtual lab”, performed by peer students.
• Color printouts of the 3 agar plates containing bacterial colonies.

METHODS

Serratia Marcescens is a strain of bacteria commonly found in the environment. S. Marcescens produces a red pigment (i.e. colonies are red) when grown at 24-30°C. Mutations in the bacterial DNA for the red pigment will cause the colonies to turn white (they don’t make the red pigment) or pink (they make less of the red pigment).

UV light is used as a control condition to show that the experiment works. UV radiation causes mutations in the bacterial DNA and the colonies should turn white. (UV light will also kill some of the colonies).

What’s not in the video: The preparation

Prior to the actual experiment shown in the video, the teacher carried out some preparation. The preparation included the following:

• Make stock plates of bacteria by inoculating agar (nutrients in gelatin) petri plates with the bacteria purchased from a biological company
• Store the plates for 2 days at room temperature (24-25°C)
• Transfer a single colony of bacteria to a tube containing liquid nutrients (nutrient broth)
• Store the liquid bacteria culture for 1 day before starting the experiment
• Before starting the experiment, dilute the bacteria (1 drop) in a new tube containing 10 milliliters of fresh nutrient broth. Then repeat. This dilutes the bacteria to be used for the experiment.

What’s in the video: Experiment day

Step 1: Students make the tobacco extracts

• Heat water, cut open a cigarette and put tobacco in the water, stirring for 5 minutes
• Heat another beaker of water for the control
• Filter the extract through cheesecloth into a bottle

Step 2: Students add tobacco extracts to plates

• Label the underside of 3 agar petri plates as follows:

• Place 1 drop of the following solutions in the middle of each agar plateWater plate: add previously heated waterUV plate: add previously heated waterCigarette plate: add cigarette extract (in water)

  You might ask why water is added to the UV plate. The reason is that with this design all 3 plates contain the same water, providing a fair comparison.

• Use glass beads to roll the solution all over the plate, let solution dry for ~20 minutes

Step 3: Add bacteria to the plates

• Place 1 drop of bacterial solution into the middle of each plate
• Spread the bacteria on the plate using fresh glass beads; let dry for ~10 minutes
• For the UV plate, place the UV lamp over a box with a hole in the top and shine the UV light through the hole onto the plate for 15–20 seconds. This should cause mutations in the bacterial DNA. It will also kill many bacterial cells.
• Store the bacterial plates for 2 days at room temperature
• Take pictures of the plates and send to a computer

Collect Data: Done in class, this is no longer virtual

Data collection is done 2 days after the bacteria were treated; students will count the number of mutated bacterial colonies in each plate that was prepared by the students in the video to determine the number of mutations.

• The teacher should hand out color copies of the 3 plates, one set for each group (see below). Images of the plates can be downloaded here.
• The plates are already coded as follows:

Plate A = UV; B = cigarette; C = water control

• Students should indicate on their lab data sheet what they expect to find BEFORE looking at the plates. Ask the students to circle the level of mutations they expect for each plate:

• Teacher hands out pictures of Plates A, B, and C, to each group; explain that these are the actual results from the experiment that the students did in the video
• Explain to the class that the plates are coded so that no bias will be introduced when counting colonies. The students should not know the treatment groups of the plates until after the data are collected.
• Students count the number of white spots on the printout of each plate.

red = normal DNA         white = mutated DNA

To count most efficiently, have students draw a line through each white spot as they count so that spots are not counted twice.

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• Students should record their bacteria counts in the following table that is found in the student lab data sheet (download here ). They should get similar data to that shown in the table:

Plate Treatment	# of white colonies on plate
Plate A	~100
Plate B	30
Plate C	1

 

Remember:

red = normal DNA

white = mutated DNA

• After the students have recorded their data in the table, “break the code”–tell them which plate is which. Have them write the actual treatment in the table under the code letters (A = UV exposed; B = cigarette; C = water)
• Have students answer the questions on their data sheets so that a discussion about the experiment can be conducted. The discussion questions can be seen below as well.

Based on your results, what do you think was added to each plate (water, UV light, or cigarette extract)?

Plate A: ________UV__________Plate B: _________cigarette__________Plate C: ________water control________

Explain in 2-3 sentences why you chose your answers.

Now, ask your teacher to identify the conditions for each plate. Write them into the table in the first column. Did you guess correctly which plate was which? If not, what are some reasons why your answers were wrong?

Some possible answers:

Water had more mutations in it than expected

UV light did not mutate all of the bacterial colonies

Tobacco extract did not mutate all of the bacterial colonies

Go back and look at your predictions (before you counted colonies) for the level of DNA mutations produced in each condition. Which of your predictions were not supported by the results of the experiment?

What are some reasons why the experimental results did not support your predictions?

Possible answers:

Perhaps some background mutations in bacterial DNA are normal

Colonies were difficult to count giving errors

Didn’t think that 2 days was long enough for tobacco to mutate DNA