1. ASEPTIC TECHNIQUES IN THE MICROBIOLOGY LAB PETER FORSON

2. Introduction Aseptic technique is a set of routine measures that are taken to prevent cultures, sterile media stocks, and other solutions from being contaminated by unwanted microorganisms. While such actions are sometimes called “sterile technique,” that terminology is appropriate only in reference to preventing introduction of any organisms to laboratory or medical equipment and reagents. Microbiologists use aseptic technique for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests.

3. Aseptic technique is also essential for isolation of a single species of microorganism from a mixed culture to obtain a pure culture. Proper aseptic technique prevents microbes used in the laboratory from accidentally being released into the environment and/ or infecting people working in the laboratory. One should always remember that a completely sterile working environment does not exist.

4. Examples of aseptic technique are: -Cleaning and disinfecting lab surfaces prior to use. - Limiting the duration that cultures or media are uncapped and exposed to the air. -Keeping petri dishes closed whenever possible. -Effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media. -Avoiding breathing on cultures or sterile instruments.

5. There are some general rules to follow for any aseptic technique: Close windows and doors to reduce and prevent sudden movements which might disturb the air. Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its rapid action. If the bench surface is difficult to clean, cover the bench with a sheet of tough material which is more easily disinfected. Start the operations only when all apparatus and materials are within immediate reach. Complete all operations as quickly as possible, but without any hurry.

6. There are some general rules to follow for any aseptic technique: Vessels must be open for the minimum amount of time possible. While vessels are open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards. On opening a test tube or bottle, the neck must be immediately warmed by flaming with the vessel held as near to horizontal as possible and so that any movement of air is outwards from the vessel.

7. General rules to follow for any aseptic technique During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to contamination from the air. The parts of sterile pipettes which will be put into cultures or sterile vessels must not be touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the surface of the working area, or the outside of bottles/ test tubes. All items which come into contact with microorganisms must be sterilised before and after each such exposure (for example, in the case of glassware to be used), or by the worker during the course of the practical (for example, in flaming a wire loop).

8. Using an Inoculating Loop Before each use, an inoculating loop must be sterilized using a burner, as follows: 1. Place the junction between the loop wire and the handle just above the inner blue cone (hottest point) until the wire turns red. 2. Slowly draw the wire through the blue flame, making sure that every part of the wire is heated to glowing red. The loop tip is heated last. 3. Cool the loop by making contact with another sterile surface, e.g., an unused section of an agar plate. Do not blow on the loop or wave it in the air to cool it. Such actions will contaminate the loop.

9. Using an Inoculating Loop 4. The loop is now ready for immediate use. Do not put the loop down or touch it to a non-sterile surface before using. 5. Flame the loop again immediately after use before setting it down. As an alternative, pre-sterilized inoculating loops may be purchased. These are particularly handy for use within a laminar flow hood, where a Bunsen burner should never be used. Plastic, disposable loops are available either individually wrapped or packaged in bulk.

10. 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.

11. The loop will automatically cool once it contacts the broth culture but wait a one or two second 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. These 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).

12. 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.

13. 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 vv 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.