Testing Antimicrobials

Modified from instructions developed by Susan Fleming for the Cell Lab at the Science Museum of Minnesota, 2001, a project supported by the National Science Foundation

Introduction

In recent years there has been a huge increase in production of household goods that have antibacterial properties. This includes not just soaps and other cleansers, but also such things as sponges, cutting boards, and toys. But are all these products really necessary to keep the average person healthy? Do they work? And do these products pose a risk by artificially selecting for stronger strains of bacteria? For most people, washing their hands with soap and water is good enough to remove most pathogens from the skin. In terms of selecting for "super bugs", there is some evidence of this happening, but not everyone agrees on the degree of danger that this presents to the public. It is important to keep in mind that we share our world (and our bodies) with a lot of bacteria that are not harmful and that are sometimes necessary for us to function well.

This experiment tests how well soaps, hand gels, and cleansers kill bacteria. The bacterium used in this experiment is Bacillus megaterium ('big beast'), one of the largest bacteria known. It is a rod-shaped bacterium found in dirt and soil. B. megaterium will not make you sick but can cause 'wet wood' disease in elm trees. It is able to survive harsh conditions by forming spores, which protect it from cold, heat and dryness. Other bacteria can be used for this experiment, but it works much better with B. megaterium.

The indicator solution contains fluorescein diacetate (FDA). FDA is sometimes used in cancer research to detect the presence of live cancer cells in a culture. In this activity FDA shows the presence of live bacteria. In cells that are alive, enzymes (called esterases) convert the colorless FDA into a green fluorescent compound called fluorescein. This makes the cells glow green under an ultraviolet (UV) light. When cells are dead and the enzymes are not active, the FDA doesn't get changed to fluorescein and the solution remains colorless.

Important concepts and links to other topics:

• Bacteria are an important part of our ecosystem.
• Not all bacteria are disease causing (pathogenic).
• The overuse of antibactieral products and antibiotics might help produce more deadly strains of bacteria.
• Some antibacterial products work better than others, but it may not be necessary to use these products all the time.

Supplies:

• Tubes
• Tube holder
• Plain LB broth
• Bacillus megaterium in liquid culture (LB broth)
• Indicator solution (FDA)
• Household cleansers, soap, hand gel, bleach
• UV light and viewing chamber

Instructions for making indicator solution:

The concentration of fluorescein diacetate used in this experiment is 1mg/ml in acetone. To make 100ml of indicator solution, dissolve 100mg of FDA in 100ml of acetone.

Procedure:

1. Place five sample tubes in the sample holder. Samples 1 and 2 are your control tubes. Sample 1 will not contain any bacteria and will be a negative (-) control. Sample 2 will contain bacteria, but will not have any antibacterial agents added and will serve as a positive (+) control for the experiment. Samples 3, 4, and 5 will be your experimental tubes and each will contain a different antibacterial agent.
2. Place twenty (20) drops of he liquid broth (which contains no bacteria) into sample tube 1.
3. Place twenty (20) drops of bacterial culture into sample tubes 2, 3, 4, and 5.
4. Select three different antibacterial agents. Place one drop of the first agent in tube 3. Place one drop of the second agent in tube 4. Place one drop of the third agent in tube 5.
5. Mix each tube by gently swirling the contents for 5-10 seconds.
6. Allow the antibacterial agents to exert their effects for one minute. While you are waiting record on a piece of paper which antibacterial agents you picked and whether or not you think they will kill the bacteria.
7. Add one drop of the indicator solution to each of the five sample tubes.
8. Wait 2-3 minutes for the reaction to proceed. You may see some color change in as little as 15 seconds.
9. Place the samples in the viewing chamber and turn on the UV light.
10. Look at the control tubes; sample tube 1, the negative control (-), should be colorless because it does not contain any bacteria. In the sample tube 2, the positive control (+), the bacteria are alive and convert the indicator solution to a bright green compound.
11. Compare the fluorescence of your test samples to the control samples. Were the antibacterial agents you selected able to eliminate the bacteria (colorless tubes), or were they ineffective (green tubes)?
12. Compare the results to your predictions. Were there any surprises?
13. Dispose of your samples (add bleach to all tubes, and then they can be poured down the sink).

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