Aseptic Technique

The technique is all measures taken to limit exposure of the worker and the environment to the bacteria; in addition, it includes the prevention of contamination of the bacterial culture by other unwanted organisms.

The handling of materials and equipment is an important aspect of safety. The inoculating loop that is used to transfer bacteria must be sterilized and cooled before and after each use. After use it must be replaced into the rack for loops and never laid on the countertop. The lids of Petri dishes containing media should be opened just enough to perform the necessary procedure.

Small Heading

Pure Culture and Transfer Techniques

Step One:

Culture Techniques In the bacteriology laboratory, transferring a population of microorganism to a growth medium is called inoculation. The group of microorganisms is called the inoculum. Successful acquisition and identification of bacteria depends on proper collection and transfer of the inoculum, using aseptic technique.

Step Two: Inoculation of Media

A - The first step in inoculating a plate of medium is labelling. The Petri dish is always labelled on the bottom, not the lid, since the lid could get switched from one dish to another. Also, agar plates are always incubated turned upside down. This is to prevent condensate which would form on the lid from dripping onto the agar. The inoculation of medium is accomplished by a gently rolling the specimen swab onto one quadrant of the agar.

B - A bacterial inoculating loop is used to spread the inoculating material over the agar plate in a manner to produce isolated colonies. This is accomplished by transferring less culture material into each successive quadrant. Gentle pressure should be used to avoid tearing the surface of the agar.

C - The loop is sterilized by flaming, cooled, and then used to spread the material from the first quadrant into the second quadrant (see figure three). After the loop is flamed again it is used to spread material from the second quadrant into the third quadrant, and then from the third into the fourth (if there is a fourth is used). In the fourth quadrant the procedure is done carefully in order to produce isolated colonies of the bacteria. The lid of the Petri dish should never be removed completely, exposing the medium to contamination. In addition, the lid should never be laid on the countertop.