324 PRINCIPLES OF SOIL MICROBIOLOGY 



b. Delafield's haemotoxylin: 4 grams of haemotoxylin crystals are dissolved 

 in 20 cc. of 95 per cent alcohol and added to 400 cc. of a saturated solution of 

 ammonia alum. The mixture is exposed for a few days, filtered, and 100 cc. of 

 glycerol and 100 cc. of methyl alcohol are added. Solution is allowed to stand 

 until dark, then filtered. The preparation is placed in a dilute solution of the 

 stain in water for 30 minutes or more. When overstained, acid alcohol may be 

 used to remove excess. 



c. Safranin light green. The preparation is placed for 1 to 24 hours in a con- 

 centrated aqueous safranin solution, then washed in alcohol and placed in an 

 alcoholic light green solution. 



Among the other stains, the Giemsa stain, Mann's methylene blue-eosin 

 stain, gentian-violet and safranin may also be mentioned, while for ciliates 

 simple staining in borax carmine is usually sufficient. 



After staining, the preparation is dehydrated by passing through increasing 

 strengths of alcohol (after washing with water, a low concentration of alcohol 

 is used at first) to 90 per cent, then in a mixture of 1 part of absolute alcohol + 

 1 part xylol, then in pure xylol. The preparation is then mounted in Canada 

 balsam. 



Martin and Lewin 46 examined the active soil fauna in fresh films by using 

 picric alcohol (50 per cent saturated solution picric acid in water+50 per cent 

 pure alcohol), or corrosive alcohol (50 per cent saturated solution of corrosive 

 sublimate in water + 50 per cent pure alcohol). The soil is placed in a porce- 

 lain dish and enough of the fixative is poured through a funnel to the bottom 

 of the soil layer until the soil is just covered: the dish is then slightly shaken. 

 A film is formed which contains protozoa in a fixed and stained condition. By 

 floating coverslips on the surface of the liquid, the protozoa are removed and 

 can be examined microscopically. 



Life history of protozoa. The life history of a protozoan in the soil 

 consists of a period of activity, when the animal moves, feeds and repro- 

 duces, and a period of rest, when a thick wall is secreted around the body 

 and the cell (or cyst) becomes capable of resisting adverse conditions; 

 the animal is distributed from place to place in the cyst state. 

 When conditions become favorable, the wall is ruptured and the animal 

 again becomes active. Sometimes, actual reproduction takes place 

 within the cyst, as in Colpoda steinii. More seldom the cyst results 

 from conjugation of two similar animals forming a large body known 

 as the zygote. 46 The physiology of encystment and excystation are 

 obscure points concerning which a considerable literature is growing up. 

 Earlier writers regarded cyst formation as a direct response to unfavor- 

 able external conditions and excystation as occurring whenever a cyst 

 found itself again in an environment suitable for active life. Experi- 



45 Martin and Lewin, 1914-15 (p. 318). 



46 Martin, C. H. A note on the protozoa from sick soils, with some account of 

 the life h : story of a flagellate monad. Proc. Roy. Soc. B, 85: 393-400. 1912. 



