220 PRINCIPLES OF SOIL MICROBIOLOGY 



34.5° to 35°C, without solidification. Six to eight cubic centimeters of agar are 

 placed in a Petri dish 10 cc. in diameter. If the alga is filamentous, it is first 

 washed in sterilized nutrient solution; if it is a unicellular form, it is diluted, 

 the extent of dilution depending on the abundance of organisms. The material 

 is added to the tube of liquid agar, which is then vigorously shaken so as to 

 separate the adhering bacteria, and the contents are poured into a Petri dish. The 

 plates are allowed to cool, then they are turned upside down, so as to prevent the 

 moisture from spreading bacteria over the surface, and are placed in the light of a 

 north window, preferably in a glass case. The plates are examined frequently 

 and, if rapidly spreading bacteria and fungi are found, they are dissected out. 

 The algal colonies usually appear in from three to four weeks. If the inverted 

 plates are examined from time to time with the compound microscope (12 m. 

 objective), the algal colonies may be located in the very early stages of develop- 

 ment. The colonies are marked with a glass pencil and are transferred, by means 

 of a platinum loop, to sterile agar slants. The purity of the culture ma}' be further 

 tested by transferring it to media suitable for bacterial growth. 



This method may have to be modified for particular forms of algae : 

 in some instances the method of Barber is used; 15 in other cases the fact 

 is utilized that certain species readily produce zoospores or other 

 free endogenous spores; in some species the vegetative cells are either 

 free from bacteria or can be rendered so by mechanical means. Pure 

 cultures of various algae, particularly of Chlorophyceae, were thus iso- 

 lated. The Cyanophyceae presented more difficult problems of isola- 

 tion, since the gelatinous investments are all impregnated with bac- 

 teria, which cannot be removed even by most vigorous washing. By 

 the use of silicic acid gel, one species of Oscillatoria and one Microcoleus 

 were isolated. However, as soon as these two organisms are completely 

 separated from bacteria, the media, otherwise favorable, seem to become 

 unfavorable and the organisms eventually die. 



By repeated transfer to sterile silicic acid gel plates, a species of 

 Nostoc was isolated 10 in pure culture. Another method 17 consists in 

 growing the organisms in a dilute mineral salt solution (Detmer's), 

 either placed in flasks or impregnated in silica gel. Subcultures are 

 made for enrichment purposes. Dilute suspensions of the algae, well 

 shaken for the separation of the cells, are then inoculated into flasks 



15 Barber, 1907 (p. 55). 



16 Pringsheim, E. G. Kulturversuche mit Chlorophyllfi'ihrenden Mikroor- 

 ganismen. I. Die Kultur von Algen auf Agar. III. Zur Physiologie der Schizo- 

 phyceen. Reitr. Biol Pflanz. 11. 1912; 12: 49-108. 1913. 



17 Chodat, R. fitude critique et experimentale zur le polymorphisme des 

 algues. Geneve. 1909; Monographic d'algues en culture pure. Mat6riaux pour 

 la flore cryptogamique Suisse. Vol. IV, Fasc. 2, 1913. Berne. 



