November 26, 1915] 



SCIENCE 



769 



found in the lower and moister parts of the 

 culture below the sporangial growth but need 

 never be expected in the upper and dryer 

 regions. They frequently form in pure masses 

 free from sporangia in the folds of crumpled 

 paper directly below the substratum. The spe- 

 cies, however, is diecious and it may frequently 

 happen that the spores of only one of the two 

 sexes, which together are necessary for zygo- 

 spore production, have fallen upon the sub- 

 stratiun. The chances of finding zygospores 

 in the lower parts of a spontaneous culture 

 wiU be increased if bits of the fungus from 

 different sources in the field are laid on the 

 bread when the culture is started. The two 

 sexes are often found growing together in 

 nature although the moisture conditions may 

 not be such as to cause the appearance of 

 zygospores. Thus, out of seven mixed trans- 

 fers made this last month from as many indi- 

 vidual decayed squashes in the field, three 

 showed the presence of the two sexual races by 

 production of zygospores, and of the remainder 

 one was male and three were female. If even 

 a small mass of zygospores is found, their 

 production can be increased by laying on a 

 fresh culture of bread, a bit of such zygosporic 

 material freed as much as possible from 

 sporangial spores. "When an abundant pro- 

 duction of zygospores has been once secured, 

 the bread with the fungus growing on it can 

 be dried and kept six months or more to be 

 used later as " spawn " if broken up and laid 

 on fresh bread when a zygosporic culture is 

 again desired. Cultures have been kept over 

 a year in this way but the spores are rela- 

 tively short lived and fresh " spawn " should 

 be prepared at more frequent intervals. 



Zygospores of Sporodinia also may be 

 readily obtained from sowings of the spores on 

 bread in moist chambers. This mold is 

 hermaphroditic. If a large collection of differ- 

 ent fleshy fungi are left for a few days under 

 a beU jar, a sporangial growth of Sporodinia 

 win usually be found appearing on some of 

 the decaying fungi. 



In studying the habit of growth of the bread 

 mold, pieces of paper on which the mold has 

 spread will be found convenient or masses of 



the mold-infected bread (about half the size 

 of an English walnut) which is beginning to 

 show sporangia may be left for 24 to 48 hours 

 in Petri dishes. If the Petri dishes are kept 

 in too moist an atmosphere, stolons with but 

 scanty sporongia may result, if kept too dry 

 no stolons will be produced. If the Petri 

 dishes be wrapped in paper, the proper condi- 

 tions are generally secured. The presence of 

 the columella within the unopened sporangium 

 may be shown by drawing a solution of KOH 

 under the cover. 



To observe the swelling and germination of 

 the sporangial spores, they may be sown in 

 suitable fluid media or on nutrient agar. The 

 filtrate obtained after boiling a couple of 

 prunes for five or ten minutes in 100 c.c. of 

 water makes a convenient fluid for the pur- 

 pose without the necessity of further steriliza- 

 tion. Only enough spores should be added to 

 make the fluid slightly clouded or to give a 

 sufficient number of germinations in a drop 

 taken for observation. At room temperature 

 the formation of germ tubes may be expected 

 in five or six hours. The process may be 

 hastened or delayed by keeping the spores at 

 higher or lower temperatures. 



The methods suggested in the preceding 

 paragraphs have been purposely such as can be 

 adopted by any teacher without the facilities 

 for sterilization. It will not be necessary to 

 give detailed directions to those familiar with 

 cultural methods. It may be said, however, 

 that Bhizopus and especially its zygospores 

 develop best upon nutrients rich in carbo- 

 hydrates. If agar is used, 4 per cent, to 8 per 

 cent, dextrose will be found a desirable in- 

 gredient of any formula. Bread is an ideal 

 substratum but ordinary sterilization renders 

 it pasty and unfit for use. A short steriliza- 

 tion with steam, but not imder pressure, con- 

 tinued for less than five minutes wiU prob- 

 ably be found satisfactory. 



The two sexes may be isolated from a cul- 

 ture, producing zygospores by making trans- 

 fers from individual sporangia to well-sepa- 

 rated points in the outer margin of a large 

 dish of some suitable substratum. Zygo- 

 spores wUl appear between the growths of oppo- 



