CULTIVATION AND PRESERVATION OF PENICILLIA 73i 



lum be extended into a second plate. This method has special merit in 

 separating molds from contaminating bacteria. 



Streak cultures have another important application in the study of 

 natural variation within an unstable strain of Penicillium since variant 

 growth types have an opportunity to develop as separate colonies if the 

 inoculum is representative of the parent culture. The same type of cul- 

 ture can be very useful when it is desired to obtain conidial structures 

 suitable for microscopic examination in situ within two or three days. 



Single Spore Cultures: Dilution of spores in sterile water, followed by 

 plating in agar as described above, constitutes a satisfactory means of 

 isolating "single" spore cultures where it is sufficient if 90 or 95 percent of 

 the resulting colonies develop from individual conidia. That such a 

 percentage actually develops from single cells can be determined by com- 

 paring the number of developing colonies with the haemocytometer count 

 of conidia present in the original suspension. Using such cultures, how- 

 ever, it is impossible to know whether any particular colony developed 

 from a single conidium, or from two or more adherent cells. 



Where the investigator needs to know with certainty that every colony 

 has developed from a single spore, it is necessary to employ some tech- 

 nique combining microscopic examination with a device or means for the 

 mechanical removal of selected spores. This can be accomplished with 

 the aid of some type of micro-manipulator with which individual cells are 

 removed from a water suspension and subsequently transferred to a 

 suitable culture plate or tube. It may also be accomplished by means of 

 some type of cutting disk attached to a holder or frame which can be 

 mounted in the nose-piece of the compound microscope and alternately 

 s\vung into position and out again as described by Lambert (1939) and 

 others. A somewhat simpler and more rapid method can be used when 

 one is dealing with cells 3/i or more in diameter. Such a procedure has 

 been developed and used extensively at this Laboratory. Briefly de- 

 scribed, the method is as follows: Conidia of a selected culture are thor- 

 oughly dispersed in sterile water containing a detergent, sodium lauryl 

 sulfonate, in a concentration of 1:10,000. Appropriate dilutions of this 

 suspension are then spread evenly over the surface of a carefully filtered 

 nutrient agar. The plates are incubated at a favorable temperature 

 over night and the conidia allowed to germinate. The plates are examined 

 on the following day with a wide field binocular microscope and the po- 

 sitions of isolated germinating cells are marked. These are then carefully 

 checked with a compound microscope using an 8 mm. objective to insure 

 that no ungerminated spores are present in the immediate area of the 

 cells selected for isolation/j^By'means of a micro-scalpel fashioned from 

 thin platinum-iridium wire, a minute block of agar surrounding the se- 



