20 



ISOLATION AXD CULTIVATION OF FUNGI 



taminants. Bv means of this simple procedure they essentially 

 isolate themselves in pure culture. If an attempt is made to 

 isolate these same species occurring in decaying leaves and twigs 

 by using macerated tissues as inoculum, there is difficulrv^ in 

 separating the organism sought from protozoa, bacteria, and 

 other fungi present. 



In isolating Hymenomycetes advantage may be taken of their 



ability to discharsre their ba- 

 sidiospores. In such studies 

 the spores to be used as inocu- 

 lum can best be collected on 

 a sterile microscopic slide. By 

 methods previously described 

 they can then be transferred 

 directly to a^ar tubes or 

 plates. Instead of this pro- 

 cedure the sporophores may 

 be placed above the surface 

 of agar plates, so that the ba- 

 sidiospores can fall upon the 

 surface of the agar. 



One of the difficulties that 

 attend efforts to isolate plant- 

 pathogenic fungi is early oc- 

 cupation of lesions by second- 

 ary invaders, both bacteria and 

 fungi. Under these conditions special techniques may be re- 

 quired to isolate the primary organism. This situation is exem- 

 plified by a study of the citrus melanose pathogen, Diaporthe 

 citri. \\^hen infected leaves, fruits, or t\vigs were dipped into 

 alcohol and flamed, and lesions were excised and planted on agar, 

 the pathogen soon became overgro\^n by Colletotrichinn gloeo- 

 sporioides unless subcultures were made as soon as hyphae 

 appeared [Bach and Wolf (1928)]. Similarly other species of 

 fungi may act as "weeds" and stifle the growth of the organism 

 to be isolated. 



Observation of pure cultures. Two essentially different pro- 

 cedures are available for the study of pure cultures: (a) cultiva- 

 tion in test tubes or in Petri dishes, or (b) cultivation in hanging 



T^nr r^ T-j II I 



Fig. 6. Diagrammatic arrangement of 

 Van Tieghem cells attached within 

 Petri dish, for repeated observations 

 on germination of spores and subse- 

 quent development. A. Surface view. 

 B. Side view. 



