By David M (Ed) Prescott
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Extra resources for Methods in cell physiology. / Volume IV
Sample text
Thus, any diploids arising by an endomitosis-like mechanism would be spontaneously eliminated. C. Contamination The problem of microbial contamination in primary cultures of amphibian cells is a severe one, since many frogs are heavily parasitized. In addition to bacterial growth, which may also result from inadequate aseptic technique, infestations of yeast, mold, and even ciliates have been experienced. In selecting primary cultures for further use, careful examination of the cultures should be made to detect microorganisms.
The cells are resuspended in 5 or 10 ml of modified Leibovitz medium and agitated with a Pasteur pipette to loosen them. , the volume is noted, and a few drops are removed for a hemocytometer count. After centrifugation for 10 minutes at 500 g, the medium is decanted and enough 10%dimethylsulfoxide (DMSO) in modified Leibovitz medium is added to give loGcells/ml. 2 ml), with black ceramic ink serial numbers already baked on with an oxygen torch. The ampoules are sealed with the oxygen torch and tested for leaks by immersing for 10 minutes in 1:20 HC1:ethyl alcohol ( a t about 4°C).
A platinum hook is the simplest means for handling and selecting larger cells, which can be hooked by the rhizoids or caps. It is made from platinum wire (25 gauge) and is sterilized by flaming. Very small cells are counted or manipulated using finer wire or a hair loop. 54 DAVID C. SHEPHMUI D. Antibiotics and Bacteriocidal Agents Table I presents much of our experience in using chemical agents to obtain sterility in Acetabularia cultures. Many agents are more toxic to Acetabularia than to contaminants.