434 PHYSIOLOGY OF THE FUNGI 



250-ml. flasks. Inoculate four flasks of each medium with one strain of 

 Phycomyces blakesleeanus or Ceratostomella Jlmbriata. 



B. Thiamine concentration and sporulation. Use media 51 to 53, 55, 

 and 57, but sohdify with 20 g. purified, vitamin-free agar (see Ex. 1) per 

 Hter. Pour into sterile Petri dishes (about 20 ml. each). Inoculate four 

 plates with Ceratostomella fimhriata (be sure to use ascospores or mycelium 

 producing perithecia), Phycomyces blakesleeanus (plus and minus strains 

 on opposite sides of plate), Choanephora ciicurhitarum, and Dendrophoma 

 obscurans. Incubate Phycomyces at 20 to 22°C., the others near 25°C. 

 Cultures of Choanephora must be adequately aerated and must receive 

 alternate light and darkness. Observe cultures of Ceratostomella for 

 production of perithecia, Phycomyces for zygospores, Choanephora for 

 conidial heads, and Dendrophoma for pycnidia. 



Allow sufficient time for the above cultures to grow; then add one or 

 two drops of sterile (autoclaved) solution of thiamine to some of the 

 thiamine-starved, nonsporulating cultures (leave controls). Observe the 

 effects. 



C. Effects of added thiamine on thiamine-starved mycelium 



Media 



60. Distilled water and purified agar 



61. Distilled water, 100 ^g thiamine per liter, purified agar 

 Note: The addition of agar in media 60 and 61 is not essential. 



From a thiamine-starved culture of C. fimbriata which has produced 

 no perithecia cut quarter-inch disks with a cork borer and place them in 

 tubes of media 60 and 61. If liquid media are used, make sure that the 

 disks of inoculum float on the surface. Observe the results after a few 

 days. 



D. Relation of required thiamine to sugar in medium. To show that the 

 concentration of thiamine required for the production of perithecia 

 depends upon the amount of sugar in the medium, this short experiment 

 may be performed. 



Media. Liquid glucose-asparagine 



62. Glucose 25 g., thiamine 1 ng per liter 



63. Glucose 2.5 g., thiamine 1 fig per liter 



64. Glucose 0.25 g., thiamine 1 ng. per liter 



Distribute the media in 250-ml. Erlenmeyer flasks, inoculate with C. 

 fimbriata, and incubate at 25°C. Observe the rate of growth, time of 

 perithecium formation, and relative number of perithecia formed in each 

 medium. Harvest and weigh the mycelium of each culture after peri- 

 thecia have formed. 



