Antimicrobial Agents and Experiments

Introduction to Experiments for Assaying Antimicrobial Agents

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PURPOSE This page is designed for students who want to test the power of detergents, bleach, alcohols and antiseptics for their true power in killing bacteria and other microbes. (Psst! Don't be afraid to use your dictionary to look up words you don't understand here. "Big" words are used to expand your own vocabulary, and for your use in writing your own project reports.)

INTRODUCTION You are probably interested in this page because you have a notion that killing bacteria and other microbes like fungi is a very good thing to do. However, this is far from being true because many of the microbes around us are helpful - indeed, we cannot live in the absence of many of them. You must have about a million of certain kinds per square inch on your skin because those certain kinds act like guard dogs and kill undesirable types of germs that you encounter during your normal lives. In fact, the National Centers for Disease Control and Prevention in Atlanta strongly recommends against using antibacterial soaps because they kill the good bacteria. The CDC has noticed a sharp rise in skin infections in this country since the advent of sales of antibacterial body soaps. The scientific field that deals with the good, guard-dog bacteria is a very new one, and it is called "probiotics." This website supports a number of probiotic research, and has been instrumental in seeing several students publish their work as well as winning prizes.

Yet we are still interested in knowing something about how to measure the efficacies of various antimicrobial methods and agents. Two general ones will be mentioned here - heat (a method), and detergents (agents). Of course, there are other methods (ultraviolet light, sunlight, and so on); and there are many other agents (bleach, alcohols, sugar, salt, heavy metals and on and on). Just so that this web-page does not become too lengthy, it will be confined to one method and one agent. You must use your own creativity to expand beyond these. Afterall, if all you do is blindly follow the steps given here - well, that is called plagiarism. You should try doing the steps here to make sure that you can make things work the way they are supposed to work, and once you get them to do that, then you can, with confidence, move on to create your own projects.

HEAT: a method for killing microbes

This protocol will deal with two new activities: (1) a method by which you "add" some physics rather than a chemical agent, and (2) the use of a general population of microbes.

You will take a large collection of bacteria from ordinary garden soil and subject it to ever increasing temperatures. You will take samples as the temperature rises and test them for what survives. The results can be quite startling and very informative in a general biological sort of way. This is a very good first lab in general biology for studying microbiology.

PROTOCOL

To start you will need the following items
1. Ten sterile petri plates containing nutrient agar (how to make these)
2. A pan of cold water and a thermometer that goes up to 100c (or 212F)
3. Ten sterilized swabs individually wrapped
4. A marking pen that will write on glass or plastic (the petri plates).
5. A spoonful of garden soil.
Stir the soil into the water in the pan, and measure its temperature.
Dip a swab into that water and then smear it all over the surface of one of the petri plates. Label that plate with the starting temperature.
Raise the water's temperature 10 degrees C, dip in a new swab and smear another plate. Label that plate with the temperature.
Keep raising the temperature by 10 degree increments and swabbing plates.
As soon as the water boils, swab a plate and label it boiling.
Now let the water boil for 5 minutes, and swab a sample onto a plate
The final sample will be taken after the water has boiled for 15 minutes.
Your plates must now be "dried" so that there are no moist puddles on them
Set the plates aside to incubate. Check the plates every 12 hrs and record what you see on them at the various times. The following is all dependent on temperature. For example, on the first plate you should see nothing growing at the beginning (too small). Perhaps at 12 hrs you might imagine that you are seeing some new specks on the surface of the agar. By 24 hours you should see a number of small bacterial colonies, which at 36 hours may have grown to be different colors and some being gooey. By 48 hours you might start seeing something new - small stars (fungi or mold), and those soon grow to be fuzzy and scuzzy.
Once plates become really scuzzy with molds, take them outside and dispose of the plates (if plastic) or scoop out the "corruption" from glass plates into a plastic garbage bag. Do not inhale the spores from the molds. (While you normally breathe in a few of those same spores everyday, your body cannot handle that a large number at once, and often allergic reactions occur.)

In your final report, pay special attention to which groups of organisms can survive the temperature increases. Remember that there are bacteria in one realm of life (the prokaryotes), and the fungi are in another realm (the eukaryotes). Do you see a trend?

CETAVLON - a type of chemical agent

This protocol will deal with two new activities: (1) the use of a chemical agent to kill microbes, and (2) the use of specific microbes as your test organisms.

You will subject a specific organism to various concentrations of the chemical "cetavlon", which is a common ingredient in shampoos. Another trade name is cetrimide, and its chemical name is NNNN-trimethyl-hexadecyl-ammonium bromide. Shampoos consist mainly of cationic ("+") detergents, while bar soap, laundry and dishwashing detergents are anionic ("-") detergents. Your hair is "+" so that if you wash it with bar soap, the "-" soap molecules stick to your hair and make it feel terrible. Shampoo detergents don't stick to hair, but do stick very strongly to the "-" dirt that does stick to hair, and thus the dirt is lifted and rinsed away. And remember that while the shampoo might be mild to you, it could be extremely lethal to bacteria - all depending on which brand you test.

In general there are two ways to do this exercise: (1) to add various concentrations of your "agent" to the nutrient agar prior to sterilization, and then after plating bacteria on those plates seeing which concentrations, if any, were strong enough to inhibit the growth of bacteria, and (2) to use ordinary nutrient agar plates that have been smeared with a dilute suspension of your known microbe, and then placing small circles of agent-soaked blotting paper on the plates.

The agent diffuses out of the circles into the agar, and if potent prevent growth of the bacterium in a halo around the circle. The value of the first method is that it allows you to determine the minimum concentration of a potent agent that is effective; and the value of the second is that with only a few plates you can test many different agents and get some idea of just how potent they are by the size of the halo around them - the more potent, the wider the "zone of inhibition." Here we shall consider this second method only, and use cetavlon as the agent. Of course, most of you will not be able to obtain pure cetavlon, but you can still use various shampoos - just read and compare the various labels for their lists of ingredients. (As a hint to cetavlon's potency, it was found to be about 70-times more potent than the classic antibiotic streptomycin on many different bacteria. This has great implications for use as a sterilizing agent, especially in very out-of-the-way places.)

PROTOCOL

To start you will need the following items
1. Two or three sterile petri plates containing nutrient agar (how to make these)
2. A bottle of shampoo that contains cetavlon or cetrimide
3. Several other candidate "agents"
4. A marking pen that will write on glass or plastic (the petri plates).
5. Some "holes" punched from a paper towel or filter paper (it would be nice to have these sterilized, but for this "starter" project we will dispense with sterility)
6. About a square foot of aluminum foil
7. A tweezers, rubbing alcohol and a small teacup
8. A liquid suspension of some known nonpathogenic bacterial species (see elsewhere on how to make these)
9. A few sterile, individually wrapped, cotton swabs.
Start by placing single drops of the shampoo and other candidate agents on the aluminum foil.
Using a pencil, write on both sides of disks some code letter or number indicating the various agents. For example, one disk might have a "C" for cetavlon.
Into each appropriately place one of the punched circles and allow the disk to soak up and be saturated with the liquid. While this soaking is happening go on to the next step.
Dip the cotton swab into the suspension of bacteria, and then rub the swab all over the agar surfaces of the plates. One dipping is all that is needed. The major mistake made here is insufficient smearing of the bacteria onto the agar surfaces. Smear, rotate the plate a few degrees and smear it again, and rotate and smear, and so on.
If the plates so signs of any moist puddles on them (seen in reflections), the surfaces should be dried.
Plan on where you will place the various disks on the plates. You can place up to seven different disks on a single plate (one in the center and six others in a circle around it - about a cm or half inch from the side of the plate).
Pour about an inch or 2.5 cm of rubbing alcohol into the teacup and place the tweezers into the alcohol.
Shake the excess alcohol from the tweezers and pick up one of the disks. Touch the disk to a paper towel to dab off the excess liquid. Set the disk on its reserved spot on the agar surface. Tap the disk lightly to make sure that it is in full contact with the agar surface.
Rinse the tweezers free of the previous agent under running water. Replace the tweezers in the alcohol. Go back to the previous step and do the same with a disk containing a different agent.
Cover and set the plates in a warm and dark location.
The next day, the bacteria should have grown up as a confluent "lawn" on the plates, but around some of the circles you should see "zones of inhibition." Those indicate that the agent was either bactericidal or bacteristatic.
Record your results, take photos, if desired, and then dispose of the plates. Because you did not use aseptic techniques (sterile disks, etc), lots of molds and other things will soon appear. So get rid of the stuff while it doesn't look too "gross."

There are other ways to do this besides using small circles. One of the newer ways is to use a piece of porous paper that is cut into the shape of a sharp wedge. When this is placed on the surface of a freshly inoculated (notice: only one "N") plate, there is a lot of agent in the thick end of the wedge to diffuse out, and only a small amount at the pointy end. Thus if the agent is very powerful, you will see a halo that goes completely around the wedge, but if the agent is weak (but not "zero") there will be a halo around the wide end, but no inhibition around the point.

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