CULTIVATION AND EXAMINATION 43 



progeny which represent essentially the outside limits of such variation. 

 This is worth while since it offers one means of securing strains which may 

 prove more useful than the parent culture for some particular purpose, 

 industrial or otherwise. The single spore method can be of real value in 

 purifying a culture. It can be of real value as a means of "dissecting" a 

 culture. But once a promising strain has been discovered and its purity is 

 established, perpetuation by the transfer of masses of conidia is the best 

 safe-guard for preserving, in a constant condition, its morphological and 

 physiological characteristics. 



Some of the techniques by which such single spore isolations can be made 

 follow : 



(1) Serial Dilation: Spores are thoroughly suspended in water and the 

 resulting suspension is subsequently diluted in sterile water blanks in steps 

 of 1:5, or 1 :10. One cubic centimeter aliquots from two or three selected 

 dilutions, depending upon the density of the original suspension, are added 

 to sterile petri dishes. Melted agar at approximately 45° C. is then added 

 to these plates, which are rotated to secure uniform mixing of the still liquid 

 agar and the diluted spore suspension. The plates are then incubated at 

 room temperature and isolates are made from plates showing a limited num- 

 ber of colonies which are uniformly separated. Using this technique, one 

 cannot be certain that any particular colony results from a single spore, but 

 if the original suspension was properly prepared, one can feel sure that more 

 then 95 percent of the colonies resulted from single spores. More uniform 

 suspensions of spores can be obtained by adding to the suspending medium 

 some suitable detergent or aerosol. For this purpose we have successfully 

 used sodium lauryl sulfonate in concentrations of 1 : 10,000 or 1 : 100,000 

 without apparent harmful effect upon the molds under study. The dilu- 

 tion method of securing "single spore" isolations is more rapid than any 

 other, and for many purposes it is quite satisfactory. 



(2) Selection and Removal of Individual Spores: Where the investigator 

 wishes to be positive that every colony results from a single spore, it is 

 necessary to employ some technique combining actual microscopic examina- 

 tion with some device for the mechanical removal of selected spores. The 

 various types of micro-manipulators are well-suited for this work and, with 

 sufficient practice, single spore isolations can be made quite satisfactorily 

 with any of these. Dilute spore suspensions in water are mounted on the 

 undersurface of a cover slip supported by a glass chamber open at either 

 one or both ends. With the aid of mechanical controls and a micro-pipette, 

 a single spore is withdrawn from the suspension and ejected upon a suitable 

 substratum where the spore develops into a mature colony. 



Single spores can likewise be removed by mechanical cutting devices 

 such as those described by LaRue (1920), Keitt (1915), and Lambert (1939). 



