Advanced Higher Biology
Lab Exercise
Aseptic Technique
Aseptic techniques will be used in this lab exercise as is usual in the clinical lab.
On a more personal note, adherence to aseptic technique assures that infectious agents are not spread to you, fellow students, or the laboratory surfaces.
The following general rules should be adhered to when working in the microbiology laboratory.
1. The inoculating loop is usually used for making transfers of bacterial cultures. Instructions for the proper technique used to flame a loop with a Bunsen burner are provided on the following page. Allow the loop to cool sufficiently so that any organisms to be tested will not be killed by the hot wire, but do not allow the loop to contact anything during the cooling period or contamination will result.
2. Learn to remove and replace the closures (usually caps) of the test tubes with the same hand that holds the loop. The caps must be held during the entire procedure and never placed on the desktop or contamination will result.
3. After the transfer is completed the loop must be sterilized again. Follow the procedure outlined on the following page in Figs. 1-3 to prevent splattering of infectious materials.
4. Always work sitting down.
5. Attention to details and practice will allow you to work both rapidly and accurately.
FLAMING
A
Heat from the base of the wire first (Fig. 1) and slowly move towards
the loop (tip) (Fig. 2). Heat the wire until it is red-hot (Fig. 3). 
Remove caps from liquid specimens and replace the caps of the test tubes
with the same hand that holds the loop. The caps must be held during the entire
procedure, as shown below in Figures 4-6, and not placed on the desktop or
contamination may result. 
Flame the neck of the tube (as in Fig. 4). If using a plastic tube, flame briefly to avoid melting the opening of the tube. Remove a loop full of the culture with the cooled loop (Fig. 5) and briefly flame the opening of the tube again (Fig. 6).
Return the cap to the specimen tube (as in Fig. 7) and transfer the inoculum from the loop to the surface of an agar petri dish. Then flame the loop as in Figures 1-3 above before placing on a counter or in a rack.
LABORATORY CULTIVATION AND ISOLATION OF BACTERIA
Since accurate studies of the biochemical and the antigenic properties of a bacterial species are possible only through the use of pure cultures, it is necessary to have a reliable and rapid method that will permit the isolation of possible pathogenic organisms. An inoculum from the specimen is streaked on solid agar in a manner, which physically separates most of the bacterial types, permitting them to form discrete colonies. This procedure is facilitated whenever possible by the use of either a selective medium that inhibits the growth of species not sought or by the use of a differential medium, which imparts a recognizable appearance to the colonies of the type sought. It is possible that colonies of the bacterial type selected for by the selective medium will be contaminated with bacteria of a different type that are inhibited from growing but not killed by the selective medium. Upon transfer of this mixed colony to a medium without the inhibitors, both types of bacteria may grow, and a pure culture will not be obtained. Consequently, it is often necessary to streak a second plate of the same selective medium with a colony from the first selective plate in order to obtain a pure culture of the bacterial species that you are attempting to isolate.
The method used most often for colony isolation from a clinical specimen or mixed culture utilizes the four-phase streaking pattern described on the following page.
COLONY ISOLATION
Streak Plate Colony Isolation
Step 1 Using a sterile loop, streak cultures (liquid broth) over one-fourth of the surface of an agar plate. Then flame the loop as described on the preceding page.
Step 2 Flame the loop and allow it to air cool.
Step 3 Pass the cooled loop three or four times over the initial streaked portion of the plate. Streak it, without overlap, to the next quadrant.
Step 4 Flame the loop and allow it to cool as described above in Step 2.
Step 5 Pass the loop over the streaked portion of the second quadrant two or three times and then streak the material without overlapping over the third quadrant of the plate.
Step 6 Pass the loop over the streaked portion of the third quadrant and then streak in a continual zigzag pattern.
Most bacteria do not move appreciably from the sites of inoculation but give rise there to clones of bacteria called colonies. Isolated colonies should arise in the third and fourth quadrants depending on the concentration of bacteria in the initial inoculum.