ISOLATION METHODS IS 



erlv to appreciate and evaluate the variation that exists within the 

 given species of fungus. 



ISOLATION METHODS 



In all the methods that are described in the following account 

 the ul^iquitous presence of contaminants is presupposed. For this 

 reason culture media, glassware, scalpels, needles, forceps, and 

 other laboratory materials and apparatus must be sterilized be- 

 fore being used. The atmosphere of laboratories always contains 

 bacteria and spores of fungi, and they may contaminate plates 

 of media during pouring or planting, or during the transfer of 

 cultures. In order to avoid contamination from this source, isola- 

 tions and transfers are sometimes made by use of special transfer 

 hoods or culture chambers. Their use, however, is unnecessar\^ 

 and constitutes a form of "self-inflicted inquisition" or "purpose- 

 less purgatory," since contamination mav be avoided in the open 

 laboratory if reasonable care is exercised. 



Surface disinfection. Isolation from diseased tissues or from 

 the interior of fruit bodies of the larger fungi is usually accom- 

 pHshed by planting bits of tissues directly on the surface of agar- 

 poured plates or of tubes of slanted agar. Generally it is advan- 

 tageous to disinfect the surface of such tissues before using frag- 

 ments for planting, for the reason that contaminants occur at the 

 surface. With care the action of the disinfectant may be limited 

 to destruction of those organisms at the surface. 



Surface disinfection may be accomplished by the use of:. 

 (a) 95% alcohol applied for a few seconds and removed by 

 flaming or by washing; (b) 1 : 1000 solution of HgCU applied for 

 15 to 45 seconds and removed by washing; (c) a solution of 

 calcium hypochlorite applied for about 1 minute and removed 

 by washing; (d) a 50% solution of H2O2 appHed for 15 seconds 

 to 5 minutes and removed by washing. In all cases washing con- 

 sists in placing the tissues for about 5 minutes in several changes 

 of sterile water, thus removing the disinfectant. It is entirely 

 possible with thick or massive tissues to obviate surface disin- 

 fection, for instead the outer tissues may be removed \\ith a 

 sterilized scalpel. The underlying tissues can then be transferred 

 directly to the surface of appropriate media. After incubation, 

 transplants can be made from the marginal growth of colonies. 



