30 AQUATIC PHYCOMYCETES 



to transfer nothing but the sporangium and its rhizoids. We have used a 

 slight modification of this method by tearing the leaf tissue apart on the 

 surface of the agar and spreading it out. If water is present, some of the 

 sporangia may discharge their spores on the surface of the agar. The spores 

 may then germinate in contact with the leaf, sending their rhizoids into the 

 agar. It is possible then to remove the leaf, wash the surface of the agar 

 with water from the wash bottle and then to dissect out one sporangium 

 from the agar surface. 



If the rhizoidal system is very complex and the spores of other fungi 

 abundant, the method just described may be unsatisfactory, in which case 

 the following method (3) is useful. A single sporangium about ready to 

 discharge spores is isolated by method 1 or 2 and put on a sterile slide in a 

 drop of water and kept under observation so that one may determine just 

 when the spores emerge. The moment this happens some of the spores are 

 drawn up in a capillary tube and blown out in a drop of water with a piece 

 of sterile leaf in a fresh Petri dish. If ordinary care is used one may secure 

 cultures by this technic descended from a few spores. It is possible so to 

 perfect this method that one can, with a little practice and skill, make single 

 spore cultures. This may be done as follows: (4) By using a very fine capil- 

 lary tube and picking up only a few spores, then diluting the spores by 

 mixing in another drop of water and so on by further dilutions, it is pos- 

 sible finally to get only one or two spores in the capillary tube. If the tube 

 were clean to begin with, it may be examined on the surface of the Petri 

 dish to determine how many spores it contains. If only one spore is present, 

 the tube is then transferred to a drop of water containing a piece of leaf. 

 If it contains two or even several spores, it is possible to break the tube in 

 such a way that one can separate a single spore from the others. 



The securing of bacteria-free cultures of chytrids on artificial media 

 is difficult, owing primarily to the slow rate of growth of the rhizoidal 

 system and, in many species, to the monocentric nature of the thallus. 

 Couch's methods 5 and 6 (below) refer to the preparation of bacteria- 

 free cultures: 



So far we have developed two methods for doing this. The easier, if the 

 spores will germinate on agar, is by the isolation of a single spore on agar 

 (5). After much experimenting with spore germination on nutrient agars, 

 we have found several which are useful in this work. 



1 . Plain agar 3 % (the agar shreds should be washed over night in several 

 changes of water to free from trash). 



2. Agar No. 12 (Leitner's agar) 2 % agar and 0.004 % peptone (meat). 



3. Agar No. 13 (Foust's agar) 2 % agar and 0.15 % maltose and 

 0.004 % peptone (meat). 



