MONOSPORIC ISOLATIONS 11 



poured immediately into a Petri dish. Similarly poured plates 

 should be made from the remainder of the series of suspensions. 

 After incubation some of the Petri dishes may bear a few widely 

 separated colonies that are suitable for transfer to other media. 



With either of these methods difficulties may be encountered 

 in separating the desired fungus from bacterial contaminants. 

 Several techniques have been developed to overcome this diffi- 

 culty. The fungus may be of a t\^pe that will leave the bacteria 

 behind as it grows awav from the original inoculum. Advantage 

 may be taken of this fact by cutting off portions of the hyphal 

 tips when making transfers to new substrata. In other studies 

 the medium upon which the inoculum was first planted or the 

 medium in subcultures may be acidulated with a drop of 25 or 

 50% lactic acid. By this means media may be so acidified as to in- 

 hibit bacterial growth and at the same time to permit the fungus 

 to grow at the usual rate. 



Brown (1924) described a procedure which appHes generally 

 in freeing fungal cultures from bacteria. Its basis is the fact that 

 hyphae tend to penetrate the medium. After the colonies have 

 grovv^n for 1 to 5 days, the medium is cut through wdth a sterile 

 knife in advance of the grrowiuCT colony. The colony can then 

 be inverted, and bits of material from the undersurface can be 

 carefully cut away in making transfers. The same principle 

 underlies a method recently successfully employed by Raper 

 (1937) in isolating species of Achlya. He fused glass beads % 

 to % mm in diameter on one rim of a van Tieghem ring and 

 placed the beaded rim downward in a Petri dish. Agar was 

 poured into the dish to bring the medium well up on the ring. 

 The mixed inoculum was placed inside the ring, and as growth 

 proceeded the hyphae extended under the ring and into the agar 

 lyinsr beyond. The bacteria, however, remained confined within 

 the ring. The hyphae outside the ring therefore provided inocu- 

 lum for pure cultures. 



AIoxospoRic ISOLATIONS. Numerous methods are available for 

 single-spore isolations of fungi. The techniques involved have 

 been assembled in a recent report by Hildebrand (1938). Among 

 these methods may be mentioned those of Barber (1914), Edger- 

 ton (1914), Keitt (1915), LaRue (1920), Dunn (1924), Brown 

 (1924), Hanna (1924, 1928), and Ezekiel (1930). With each 

 technique, practice greatly facihtates successful operation. That 



