ASEPTIC TECHNIQUE GUIDE

1. Sterilization by Flame

   The flame from a gas burner effectively sterilizes small glass or metal objects, such as inoculating loops, but one must avoid "frying" the yeast by contact with objects heated in a flame. Dip glass spreaders in alcohol and then use the flame to ignite the alcohol. Place metal tools such as loops in the flame until they are red-hot. Cool hot tools by touching them to the agar or inside a sterile glass tube. However, since flames are dangerous this method can and should be avoided whenever possible. We have found that flaming is really only necessary to sterilize inoculating loops and small instruments. We have not been able to demonstrate any value in flaming the openings of tubes, bottles, or flasks in these experiments.

2. Sterilizing with Alcohol

   In many experimental procedures, the most effective way to sterilize objects is with ethanol. Either 95% or 70% will work. The latter is actually more effective, but the former is often more convenient. Of course the alcohol must be allowed to evaporate or be burned off before the object is used in contact with microbes. Use a flame to burn off the alcohol, a candle is generally less expensive and safer than a gas burner. The alcohol, more than the heat, does the sterilizing, so just "light" the alcohol to minimize heating and speed up the process. Also, wipe the bench with alcohol before starting an experiment to remove mold-laden dust, the most common source of contamination. If your skin is not particularly sensitive, wipe your hands with a small amount of alcohol, too.

3. Keeping Sterile Things Sterile

   The most common sources of contamination during an experiment are from dust, air and people. This dictates several obvious principles:

   1. Keep things covered as much as possible.
   2. Don't touch anything that will come in contact with the culture and if you do touch it sterilize it again before using it.
   3. Wipe down the surface around the experiment with alcohol and minimize air turbulence.
   4. Avoid talking, singing, whistling, coughing or sneezing in the direction of things that should be sterile. Long hair, if not tied back, may be a source of contamination.
   5. Maintain a suitable area for preparing, storing and using sterile media. Unfortunately, house plants, animals and other materials such as Drosophila media that are commonly found in biology classrooms are abundant sources of mold and must be kept far away from the area used for sterile procedures.

SPREAD PLATE TECHNIQUE: