FUNGI 253 



The Chicago formula is a good chromo-acetic-osmic solution for 

 Rhizopus, especially if iron-alum haematoxylin is to be used for mate- 

 rial to mounted whole or for sections; but for material to be mounted 

 whole, the osmic acid should be reduced to 2 or 3 c.c. to 100 c.c. of the 

 solution, because material is hard to bleach. A thin section is easier to 

 bleach. Wash for 24 hours and follow the Venetian turpentine method. 



To bring out the nuclei, use iron-alum haematoxylin. Rhizopus 

 stains very rapidly, so that an hour in iron-alum and an hour in the 

 haematoxylin may be long enough; it may be necessary to stain 

 longer. The time should be such that it will require about an hour 

 for differentiation in the second iron-alum. The sporangia stain more 

 readily than the mycelium; consequently to show the coenocytic char- 

 acter of the mycelium, the action of the second iron-alum must be 

 stopped earher than when staining for the sporangium. Extract the 

 stain until the nuclei of the mycelium show clearly, and then remove 

 part of the material to wash in water. For the rest of the material, con- 

 tinue to extract the stain until the sporangia are satisfactory. Mount 

 some of each lot on each slide. 



The finer details of the sporangium can be seen only in thin sections. 

 Rhizopus is the most easily obtained material for showing the pro- 

 gressive cleavage of protoplasm by vacuoles. 



The zygosporic stage in the life-history is rarely met in nature or in 

 cultures, but when once secured it may be propagated indefinitely. We 

 had a culture which furnished illustrative material for twenty years. 

 When a particularly good culture appears, lay aside some of it to start 

 the next culture. 



Dr. Blakeslee has shown why zygospores are so infrequent. The 

 conjugating filaments belong to different strains of mycelia which he 

 calls "plus and minus strains," and which, for convenience, may be 

 called "female and male strains." The more vigorous mycelium is + 

 and the less vigorous — . When the two strains come together, zygo- 

 spores are formed along the line of meeting. If + and — strains are 

 started at opposite sides of a dish, they will meet near the middle and 

 form a dark line of zygospores. Through Dr. Blakeslee's generous dis- 

 tribution of material, the + and — strains are now available in prac- 

 tically all of the great universities of the world. 



While Rhizopus may be grown on bread, it is better to use culture 

 media in Petri dishes. While it grows well on agar media, it is hard to 

 pick it off; and on liquid media, the growth is abnormal. Dr. Alice 



