248 RESEARCHES ON FUNGI 



No. 2 by the spore-fall method already described. However, it is 

 best to leave sub-culture No. 1 undisturbed for at least a week before 

 using it as a source of inoculum. By this time, the sub-culture is 

 well established and is producing basidiospores in great numbers. 



Sub-culture No. 1, or any similar sub-culture, may be used for 

 making not merely one new sub-culture but many sub-cultures, as 

 it produces and violently discharges basidiospores continuously for 

 several weeks. 



By taking the usual bacteriological precautions, one may make 

 numerous new sub-cultures by the spore-fall method without the 

 culture medium becoming contaminated by extraneous organisms 

 such as moulds and bacteria. 



It is to be emphasised that the sterigmata (Brefeld's primary 

 conidia) produced on a basidium-body (promycelium) are never 

 violently discharged. If an agar culture which was sown with 

 chlamydospores and in which there are developing numerous sterig- 

 mata and basidiospores is inverted over a glass slide for some hours 

 without being disturbed, and if the spore-deposit is then carefully 

 examined, one observes that the deposit consists entirely of basi- 

 diospores {cf. Fig. 124, p. 251) among which one seeks for fallen 

 sterigmata in vain. Hence one may safely conclude that sub-culture 

 No. 1, which was made from a chlamydospore culture by the spore - 

 fall method, originated solely from fallen basidiospores. 



By employing the spore-fall method of inoculation, and by using a 

 sub-culture as soon as it is established as a source of inoculum for a 

 succeeding sub-culture, Tilletia tritici may be kept in culture in an 

 active state indefinitely. Twelve successive sub-cultures were made 

 by the spore-fall method and, although the twelfth sub-culture was not 

 obtained until about three months after the first, yet sub-culture 

 No. 12, when from one to two weeks old, was producing and dis- 

 charging its basidiospores just as freely as sub-culture No. 1 when 

 this was of the same age. 



A vigorous test-tube culture of Tilletia tritici, such as that shown 

 in Fig. 121, can be obtained within two weeks as follows. The 

 upper end of an agar slope is inoculated with a mycelium -bearing 

 piece of agar about 1 cm. square cut from a Petri-dish culture. One 

 now sets the test-tube in such a position that the surface of the agar 



