Objectives:
- To acquire the skill of aseptic technique in the field of Microbiology.
- To prevent contamination of cultures and media from microbes in the environment.
- To transfer cultures from one medium by inoculating another medium. This is called subculturing.
- To isolate a microorganism from a mixed culture to obtain a pure culture.
- To prevent lab microorganisms from being spread in the environment and/or infecting the investigator.
Principle:
Aseptic technique is a fundamental and important laboratory skill in the field of microbiology. Microbiologists use aseptic technique for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests. Proper aseptic technique prevents contamination of cultures from foreign bacteria inherent in the environment. For example, airborne microorganisms (including fungi), microbes picked up from the researcher’s body, the lab bench-top or other surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and equipment, etc. may potentially contaminate cultures, thus interfering with the lab results. Using proper aseptic technique can greatly minimize or even eliminate the risk of contamination. In addition, aseptic technique is of utmost importance to maintain pure stock cultures while transferring cultures to new media. Aseptic technique is also essential for isolation of a single species of microorganism from a mixed culture to obtain a pure culture. Furthermore, proper aseptic technique prevents microbes used in the laboratory from accidentally being released into the environment and/ or infecting people working in the laboratory. This is especially relevant when pathogens are being handled.
Significance of Flaming:
Flaming the loop : Holding the loop in the flame of the Bunsen burner kills all contaminating organisms, thus sterilizing the loop. The loop should glow red-hot for a few seconds. After flaming, make sure to slightly cool the loop before picking up organisms from the inoculum culture (the culture that is to be transferred.) When transferring a culture from a plate, cool the loop by touching on the very edge of agar. When transferring from a broth, the red-hot loop will make a sizzling noise as soon as you insert it into the culture. The loop will automatically cool once it makes contact with the broth culture, but wait a one or two seconds before removing the loopful of inoculum from the tube. (The hot loop may create aerosols when it touches the media containing microorganisms. It will cause some of the broth and bacteria to boil briefly, creating a bacteria-containing aerosol. This airborne bacteria have the chances of entering into the respiratory tract or into the body parts. If you hear a hissing sound when you place the heat sterilized loop into the broth culture indicates that the loop is not cooled sufficiently).
Flaming the Mouth of the Test Tube: Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube. This prevents airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures.
Agar Slants: Cultures are often transferred to agar slants, in addition to broth tubes and agar plates. An agar slant is a test tube containing agar, in which the solid agar forms a slant in the test tube. When inoculating an agar slant, draw the loop containing the inoculum very lightly over the surface in a zigzag formation while being careful not to break the surface. A needle can be used instead of a loop to inoculate an agar slant by stabbing the needle containing the inoculum into the agar ( Fig 1).
Fig 1: Inoculation of culture into agar slant
Note:
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