210 
METHODS IN PLANT HISTOLOGY 
the Venetian turpentine method. Eosin gives good results for general 
topography. Stain over night or 24 hours, treat with 2 per cent acetic 
acid in water for 5 or 10 minutes, changing several times, and theri 
put the material into 10 per cent glycerin, merely pouring off the acid 
but not washing it out with water. Wash out the glycerin with 
alcohol containing about 0.5 c.c. acetic acid to 100 c.c. of the 95 and 
100 per cent alcohol. Leave about 1 c.c. of the slightly acid alcohol 
on the material as you add the 10 per cent Venetian turpentine. 
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. The sporangia stain more readily 
than the mycelium; consequently to show the ‘coenocytic character 
of the mycelium, the action of the second iron-alum must be stopped 
earlier 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, 
continue 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 sec¬ 
tions. Rhizopus is the most easily obtained material for showing the 
progressive 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 indefi¬ 
nitely. We have a culture which has been furnishing illustrative 
material for nearly twenty years. Once in a while, when a particu¬ 
larly good culture appears, lay aside some of it to start the next 
culture. The best series of stages generally appears between the 
fourth and seventh days. Dr. Blakeslee shows 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, zygospores are formed along the line of meeting. 
If -f- 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 distribution of material, the + and — strains 
are now available in practically 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 
