Sterile method and sterile technique are very important concepts when working with microbes. We want to be confident that what we see growing on our plates and in our tubes and flasks are the organisms we placed there. Contamination costs time and money and may sometimes result in the devastating loss of irreplaceable material. Cutting too many corners can compound errors and increase the likelihood for contamination. Implementing sterile technique requires conscious thought about the process of contamination and the potential points at which contamination could possibly be introduced. Always Be Aware.
There are several steps that we take to ensure the media and media components that we prepare or handle remain contaminant free.
Good sterile technique is the first and most important step in ensuring consistent results when working with microbes. Sterile technique refers to procedures by which cultures may be manipulated without infecting the worker or contaminating the cultures or the laboratory environment.
Because contaminating microbes are ubiquitous and are found on fingertips, bench tops, etc., it is important to minimize contact with these contaminating surfaces. When students are working with the inoculation loops and agar plates, you should stress that the round circle at the end of the loop, the tip of the pipetter, and the surface of the agar plate should not be touched or placed onto contaminating surfaces.
The flaming of lips of tubes and flasks must ALWAYS be done whenever culture liquid is to be poured from a container (e.g., pouring plates). Flaming should be routinely done when caps are removed from tubes during transfer of cultures. The purpose of flaming is not to sterilize, but to warm the tube and create warm air convection currents up and away from the opening. This "umbrella" of warm, rising air will help to prevent the entrance of dust particles upon which contaminating bacteria reside.
Petri dish lids prevent dust from falling directly onto plates but allow diffusion of air around the edges. There are no direct air currents into the plate, and to enter, dust particles would have to rise vertically more than a centimeter. This does not often occur because of the density of the particles. Whenever the lid is removed, it should be held over the plate as a shield. Do not place the lid on the bench top. Do not leave plates uncovered. Do not walk around the room with an open plate.
When working with cultures in test tubes, work rapidly while maintaining sterile technique. Keep the tubes open a minimum amount of time. While the tubes are open, hold them at a 45 degree angle so that dust cannot fall into the open tube. Hold the tubes away from your face while transferring.
Test tubes are handled in the following manner:
Always clean all work areas (your bench, balance area, sink area, gel area, etc.) thoroughly before leaving the laboratory! The last step before leaving the lab is to wash your hands thoroughly.
These are guidelines. You may find a set of techniques that best suite your working style. This is fine as long as you adhere to the basic concepts of good sterile technique.
Adapted from Chazen Lab Sterile Technique at Vanderbbilt University