Exercise 4
ASEPTIC TECHNIQUE & STREAK PLATE PREPARATION
Introduction
In order to make laboratory studies with pure microbial cultures, microbiologists must start with sterile
culture media and must be able to prevent contamination of this media. At the same time, they must be
able to inoculate media with the desired pure cultures without introducing any other organisms. The
procedure utilized to accomplish this is referred to as aseptic technique and is essential to any
microbiology laboratory. Remember that all culture media must be sterile prior to inoculation. Media
samples that appear to be contaminated with unknown cultures (are already supporting growth) must be
discarded and not used for laboratory exercises.
Transfer Instruments
In order to inoculate a tube or plate with a desired microbial culture, students will use some type of
transfer instrument, usually a wire loop or a pipette. If a wire loop or needle is being used, it must be
sterilized (heated to redness by flaming) before and after making the microbial transfer. This heating
destroys any living forms on the surface of the needle or loop, thus preventing unwanted contamination.
To sterilize the loop or needle, hold it in the hottest part of the flame at an angle that will distribute heat
to the entire length of the wire. After the loop or needle is sterilized in this manner, it must be allowed
to cool briefly before being used to pick up the desired culture. Remember that contact with hot
metal will kill bacteria. When using a wire loop to transfer bacteria from cultures grown on solid
media it is recommended that students avoid filling the loop. Use a small volume of cells (just visible as
a patch of color on the wire surface) for routine inoculations. This practice will allow most bacteria to
be removed from the loop during inoculation and will prevent splattering when the loop is reheated.
Avoid filling your loop, i.e., practicing "scoop-shovel" microbiology, as this will hinder your ability to
obtain well-isolated colonies on streak plates.
In this laboratory students will occasionally use pipettes when making microbial transfers. Pipettes
provided in air-tight plastic containers (sleeves) have been pre-sterilized and if handled appropriately
will not introduce contaminants. Always open the plastic sleeve so that the pipette tip remains covered,
and if a pipette bulb will be used, attach the bulb before removing the pipette from the sleeve. Do not
allow the pipette tip to contact any foreign surface prior to making the transfer. When transfers are
made with digital pipettes, the tips that will contact microbial cultures have been sterilized prior to use.
Be careful to keep the pipette in a vertical position during use so the culture medium cannot contact the
pipette body. Keep the tip box closed to avoid the introduction of air contaminants.
Microbial Transfers Using Tubes
When transferring microbial cultures to or from glass culture tubes, students are encouraged to use the
following procedure. Hold the tube (or tubes) in your left hand and hold your transfer instrument with
your right. Remove the plastic cap (or cotton plug) with the fingers of your right hand not engaged in
holding the sterile loop or pipette. Never lay a cap or plug down on the surface of the table or bench.
Keep the tube horizontal as much as possible during the transfer, and do not leave it uncapped longer
than is necessary.
The mouths of tubes into which cultures are being transferred, as well as those from which cultures are
being taken should be heated (rolled through a hot flame) immediately before and after the transfer
instrument is introduced and withdrawn. In addition to destroying any organisms on the mouth of the
