BLAKESLEE. — SEXUAL REPRODUCTION IN THE MUCORINEAE. 247 



saying. That lu-retofore, however, the (jiiestion has baflled all inquirers 

 is due to the ditficulties inherent in tlie manipulation of this species, and 

 it was not until some time after the real thallic condition was strongly 

 suspected that the writer was able to separate out the two sexual strains. 



Preliminary Tests of " Zygosporic Strains." 



After the running out of the original Harvard strain the first appear- 

 ance of more than isolated cases of zygospores in the laboratory was in 

 a paste culture of nut shells which will be called culture " A" to distin- 

 guish it from later zygosporic cultures. The zygospores were rather 

 abundant, mixed with other moulds and with bacteria, but by transfer- 

 ring a mass of the zygospores to sterilized bread, and from this again to 

 a third culture, it was possible to obtain eventually a culture containing 

 only Rhizopus. On the supposition that a new zygosporic strain had 

 appeared, pure transfers were made from separate sporangia, but only 

 sporangial growth resulted. As many as thirty pure transfers were 

 made on such nutrients as experience had shown to be productive of 

 zygospores whenever a mass of the zygospores themselves were used 

 for the inoculation, and in only one case — a bread culture — did 

 zygospores appear. It seemed therefore probable that the ordinary 

 Rhizopus, which was not an uncommon weed in the laboratory, had 

 become mixed with the culture, and perhaps from its greater luxuri- 

 ance had crowded out the zygosporic strain. 



To avoid the contamination of these "weed" spores, small masses of the 

 young zygospores with the mycelium adherent were washed and teased 

 out in sterilized water till a microscopic examination showed that they 

 were free from spores, and a few of these zygosporic hyphae were then 

 used in the inoculation of a number of steuder dish cultures. In one of 

 these, zygospores were almost exclusively present in the middle of the 

 dish with sporangia only toward the sides, and while the growth was still 

 young the process was repeated, but the cultures were covered with raps 

 of filter paper to insure increased moisture and placed in a chamber kept 

 damp by a bottom layer of wet filter paper. All produced a great abun- 

 dance of zygospores, which grew up into the paper caps, and before the 

 sporangia had matured, zygospores with attached hyphae were washed, 

 and having been found free from spores were transferred to van Tieghem 

 cell cultures* Although they were finally infected with bacteria and no 

 new zygospores formed, a miscroscopic examination showed that the few 

 small sporangia which developed came from the hyphae that had been 

 transferred and not from the spores. It seemed thus unquestionable that 



