Isolation of Pure Cultures and Their Maintenance


Principles:

Once discrete, well-separated colonies develop on the surface of the streak plate, selected ones may be picked up with an inoculating needle and transferred to separate culture tubes such as tryptic soy agar slants (the type of agar will depend on the microorganism). Where possible, bacteria from the center of a colony are transferred, because the center is less likely to be contaminated than the edges. Each slant now represents the growth of a single species of microorganism and is called a pure or stock culture.
One of the more important problems in a microbiology laboratory is the maintenance of pure stock cultures over a long period. Ideally, one should employ a technique that minimizes the need for subculturing the microorganism. This is achieved by storing the microorganism in a state of dormancy either by refrigeration or desiccation.
Short-term maintenance (generally between one to three months) of aerobic bacteria can often be achieved by storing slant cultures in the refrigerator at 4 to 10. The use of screw cap tubes for these slants will minimize desiccation during storage.
One way in which many cultures may be maintained for relatively long periods is by sealing them with sterile mineral oil in order to prevent moisture loss. The white mineral oil used can be sterilized by heating at 110 for one hour in a drying oven. After an agar slant culture has grown, the slant surface is aseptically covered with the sterile oil. The mineral oil surface should be about 1/4 inch above the top of the slant. The oil-covered slant is then stored at the normal storage temperature. A number of genera may be stored under oil (e.g., Bacillus, most Enterohacteriaceae,some species of Miclwcoccus, Proteus, Pseudomonas,and Streptococcus). There are genera that may not be stored successfully under oil (e.g., Azotohacter andLeuconostoc). Table 19.1 summarizes maintenance conditions for a few representative bacteria.
In many cases, long-term maintenance of cultures does not even require mineral oil. E. coli and many other members of the family Enterobacteriaceae,Pseudomonas aeruginosa, staphylococci, and enterococci can often be successfully stored for years at room temperature with the following procedure. Stab inoculate screw cap deeps containing either half-strength nutrient agar or 0.7% agar in distilled water. Incubate overnight at optimal temperature. Finally, screw down the caps tightly and seal the tubes with tape or paraffin. Store the cultures in a safe place at room temperature.
The best way to preserve many stock cultures for long periods is through lyophilization (freeze-drying). This eliminates the need for periodic transfers and reduces the chance of mutations occurring in the stock culture. In !yophilization, the bacterial culture is suspended in a sterile solution of some protective medium such as milk, serum, or 3% lactose. Small amounts of the thick suspension are transferred to vials and then quickly frozen in a dry-ice/alcohol mixture. The frozen suspension is finally dried under vacuum while still frozen, and the vial sealed. These sealed, desiccated cultures may often be stored for years. Strict anaerobes and some facultative anaerobes will be injured by exposure to 02. They can often be maintained as agar stab cultures. In this procedure, one allows a tube of the desired agar to solidify in an upright position and then inoculates it by thrusting an inoculation needle coated with bacteria into the center of the agar. The anaerobes will grow deep within the agar in the anaerobic environment it provides. After suitable growth, the stab may be refrigerated. Anaerobes can also be maintained on thioglycollate broth or cooked meat medium as described in exercise 20.
Commercial sources of cultures and more information on stock culture maintenance are given inAppendix K.

Procedure:

1. With a wax pencil, label the tryptic soy agar slants with the names of the respective bacteria. Do the same for the broth tubes. Add your name and date.
2. Using aseptic technique, select a well-isolated colony for each of the three bacteria and pick off as much of the center of the colony as possible with an inoculating loop. It may be necessary to obtain material from more than one colony.Prepare a slant culture and a tryptic soy broth tube for each of the bacteria. If screw cap tubes are used, they must be loosened slightly before incubation to keep the slant aerobic.
3. After incubation 24 to 48 hours, you should have three pure slant and three pure broth stock cultures.
4. Observe the broth cultures while taking care not to agitate them. Record your observations in the report.
5. Place the pure cultures in the refrigerator for later use.