218 PRINCIPLES OF SOrL MICROBIOLOGY 



The composition of the undiluted solution is as follows: 



NH4NO3 0.5gram CaCl 2 0.1 gram 



KH2PO4 0.2gram FeS0 4 trace 



MgS0 4 -7H 2 0.2 gram Distilled water 1000 cc. 



The bottles are inoculated with about 10 grams of soil taken at the desired 

 depth. To lessen the amount of evaporation, waxed paper covers are placed 

 over the cotton plugs. The sand is slanted in the bottle so as not to be wholly 

 submerged, giving various moisture conditions. The bottles are placed so as to 

 get good light for at least part of the day. The water lost by evaporation is 

 replaced from time to time with sterile water. 



The following two media are also recommended for the isolation of algae : 



Bristol's solution 7 Detmer's solution 



NaN0 3 0.5 gram Ca(N0 3 ) 2 1.0 gram 



KH 2 P0 4 0.5 gram KH 2 PO, 0.25 gram 



MgS0 4 -7H 2 0.15 gram KC1 0.25gram 



CaCl 2 0.05 gram MgS0 4 -7H 2 0.25 gram 



NaCl 0.05 gram Tap water 1000 cc. 



FeCl 3 0.005 gram 



Distilled water 1000 cc. 



Detmer's medium is diluted to one-third of its strength and 0.01 per cent FeCl 3 

 is added. Distilled water is always prepared in a silver or glass still. Sand is 

 placed into wide-mouth culture bottles to a depth of about 1.5 inches and moist- 

 ened with one of the above media; the bottles are plugged and sterilized, then 

 inoculated with a suspension of the soil in a sterile mineral salt solution. 



The soil may also be packed in a Petri dish to a depth of about 1 cm., well 

 moistened with sterile distilled water, and the surface covered with a piece of 

 pure filter paper. The cultures are kept in diffuse light, preferably at a tem- 

 perature of 20° to 25°C The paper is moistened from time to time with sterile 

 distilled water. After 2 to 60 days, various blue-green algae are found to grow 

 through the pores of the paper to the light. 8 The mixed cultures are transferred 

 to sterile culture solutions or proper agar media for the isolation of the individual 

 species. 



Isolation of pure cultures. The separation of various species of algae 

 can be done either mechanically by the use of a loop and the micro- 

 scope, or culturally by the use of solid media. 9 The Barber pipette can 

 also be employed as a mechanical means of separation. 



7 Bristol, B. M. On the algal-flora of some desiccated English soils: an im- 

 portant factor in soil biology. Ann. Bot 34: 35-79. 1920. 



8 Esmarch, F. Untersuchungen liber die Verbreitung der Cyanophyceen auf 

 und in verschiedenen Boden. Hedwigia, 55: 224-273. 1914; Diss. Kiel. 1914. 



9 Pringsheim, E. G. Algenkultur. Abderhald. Handb. biol. Arbeitsmeth. 

 Abt. XI, T. 2, 377-406. 1924. 



