INTRODUCTION 29 



Berdan (1939), Haskins (1939), Ward (1939), and others. Whether these 

 reports refer to bacteria-free unifungal cultures is not always made 

 clear. 



Couch (1939a) cites several methods which have been particularly 

 effective in securing unifungal (but not bacteria-free) cultures from 

 materials such as decaying grass leaves infested with various chytrids. 

 He describes these in (1) — (4) below: 



(1) Isolation in water of a single sporangium or (2) isolation on agar of a 

 single sporangium; (3) isolation of spores from a single sporangium on slide; 

 (4) isolation of single spore in a capillary tube or (5) isolation of single spore 

 on agar or (6) isolation of a single thread or several threads on agar. 



With method ( 1 ) the procedure may be as follows : Place leaf with chytrids 

 in a drop of water in a Petri dish and in another drop of water about one 

 cm. away in the same dish put a new piece of sterile leaf. Then under 

 a wide field binocular dissecting microscope (x40— 100) with sharp, 

 smooth, steel needles dissect out a small piece of leaf to which only one 

 sporangium is attached. Transfer this to a small drop of water in the same 

 dish and examine under compound microscope to be sure only one sporan- 

 gium and no spores are present. The fragment of leaf with the sporangium 

 may now be transferred to the fresh piece of leaf. All transfers up to this 

 stage are made from drop to drop in the same dish, because if the delicate 

 chytrid were transferred from one dish to another, it might dry up in the 

 operation. After the chytrid sporangium is on the large, moist leaf, the 

 latter is transferred to a drop of charcoal water in a fresh sterile dish and 

 other drops of water are added to the floor and ceiling of the dish to prevent 

 desiccation. In this operation many of the single sporangia are injured in 

 transfer. Hence it is advisable to make large numbers of transfers. 



If one is lucky, each isolated sporangium will form zoospores which will 

 infect the new leaf, thus establishing the chytrid in pure culture save for the 

 presence of bacteria. 



The above technic is useful only if the sporangia are large. A more use- 

 ful technic (2), particularly with small sporangia, and where several species 

 are mixed and are discharging spores simultaneously, is as follows. An 

 infected leaf is transferred after washing to the surface of a 3 % agar plate 



The desired chytrid sporangium is now dissected out from the leaf tissue 



and dragged along on the surface of the agar to free it from spores, bacteria, 

 etc. After examining under the compound microscope to make sure that 

 only one chytrid sporangium is now present, it is cut out with a little cube 

 of agar and transferred to a fresh Petri dish in a drop of water with a fresh 

 piece of leaf, other drops of water being added to the bottom of the dish 

 to prevent desiccation. This is a very useful technic because it enables one 



