INTRODUCTION 31 



4. Corn meal agar (use 2-4 heaping teaspoons full to 1 litre water, de- 

 pending upon strength desired. Heat gently in water bath, temperature 

 about 60°, 1 hour. Filter. Add water to make 1 litre. Agar 2 %). 



The spores seem to germinate better on plain agar or agar with very small 

 amounts of nutrient material. Before taking time to spread the spores care- 

 fully on agar it is worth while to drop a few on the medium used to deter- 

 mine whether or not the spores go to pieces or settle down and encyst. 

 Naturally, if the spores are plasmolized by the nutrient agar it is a waste of 

 time to go further with that particular medium. Spores to be isolated should 

 be as free from bacteria as possible. Hence, it is well to isolate one or a 

 few sporangia about to discharge spores in a drop of water on a sterile 

 slide. However, where only a few spores are available it is possible to pick 

 them up with a platinum loop or a small pipette directly from the original 

 dish. The essential part of this technic is to spread the spores so well on the 

 surface of the agar that some of the spores will be completely separated from 

 the bacteria and other organisms. A successful spreading requires a firm 

 agar (2-3 % agar) and a steady hand. The spores are picked up with a plat- 

 inum loop and the loop dragged along the surface of the plate in a straight 

 line. Several east-west lines may be made and then another group of north- 

 south ones. It is unnecessary to mark these lines for the bacterial colonies 

 will make the lines quite evident. If the laboratory is clean and free from 

 spores of Penicillium, etc., it is possible to do this spreading with the un- 

 covered dish on the table. However, if the air is dusty, the dish should be 

 held at an angle with the agar surface down. If the spores germinate at all 

 they will germinate within 12 to 24 hours or in even less time. After 12 hours 

 the plate should be examined under the binocular dissecting microscope 

 (x 40 — x 100). If the spores have been properly spread, one may now cut 

 out a tiny block of agar with a single sporangium descended from a single 

 spore. The cutting out operation requires a very fine tool. For this we use 

 a tiny chisel made by sharpening down a steel needle under the microscope. 

 Individual sporangia may be transferred to a drop of water in a sterile Petri 

 dish on a piece of leaf. Such a culture descended from a single spore may be 

 kept free from bacteria for a few generations in water cultures. In some 

 chytrids the sporangia mature and discharge their spores on agar. It is 

 possible, though exceedingly tedious, to keep such cultures pure, growing on 

 agar and free from bacteria. The labor involved, however, is excessive 

 and we have carried such cultures on agar for only a few generations. 



In some of the polycentric chytrids as Cladochytrium replication the 

 spores germinated on agar to produce a distinct mycelium. It is therefore 

 possible to isolate this species by cutting out a single thread or several threads. 

 This method (6), however, is useless with the monocentric chytrids. 



Stanier (1942) developed methods for isolating and culturing Rhi- 

 zophlyctis rosea. He outlined them as follows : 



