254 PROCEEDINGS OF THE AMERICAN ACADEMY. 



strains which had been determined in the zygosporic culture A. In 

 other words, although the presumption was in favor of considering the 

 (+) and (— ) strains sexual in character, it might be found that such was 

 not the case, and that in other zygosporic cultures the opposite strains, 

 if present, were different in nature from those obtained in A. In order 

 to test the prevalence and nature of these strains in the formation of 

 zygospores, a detailed analysis has been made of the following seven 

 cultures, B to H, which were found producing zygospores in widely 

 separated parts of this country. But before further considering these 

 cultures, a brief account of the general procedure used in making them 

 may not be out of place. 



It has been found that a temperature of 26° to 28° C. accelerates the 

 growth and favors the production of zygospores in Rhizopus, and there- 

 fore in all cases where a different treatment has not been stated the 

 cultures have been run in the warm oven at the temperature above 

 mentioned. Excessive desiccation has been prevented by keeping a dish 

 of water on the same shelf with the cultures. Plain flour paste thickened 

 over a steamer and sterilized in the autoclave has been the nutrient 

 most frequently employed, and small stender dishes, 3.5 cm. in diameter, 

 and 2 cm. tall, have been constantly used, since the zygospores develop in 

 them in greater abundance than in the larger dishes, which are useful for 

 the culture of other forms. The advantage of the smaller dishes may be 

 due to their closely fitting ground-glass covers, which better prevent a loss 

 of moisture. In making contrasts, spore material from the cultures to be 

 tested is inoculated on opposite sides of stenders of flour-paste. Gener- 

 ally within forty-eight hours, when the cultures are run in the warm 

 oven, young zygospores can be readily seen with a hand lens forming a 

 more or less distinct and often yellowish line, clearly indicating the 

 demarcation between (+) and (— ) strains wherever they come in con- 

 tact. In all cases, however, whenever negative results have occurred, 

 the stenders have been re-examined on the fourth day or later. 



Culture B was obtained from nut shells from Mrs. J. W. Cushing, 

 Brookline, Mass., and after having been freed from other moulds and 

 bacteria was kept running on bread. A piece of this material which was 

 found to produce abundant zygospores was transferred to nutrient agar 

 December 15, and the day following terminal branches of the mycelium 

 were dissected out in a pure condition from different sides of the culture 

 and transferred to flour paste in stenders. Seven cultures were in this 

 way obtained, and their treatment is indicated in Table IV. 



Since when B^ or B^ were contrasted with Bo, zygospores resulted, as 



