1911.] Knowledge of the Protozoa of the Soil. 167 



Peck* found protozoa in the mannite medium used for the cultivation of 

 Azotobacter from soil. 



However, no comprehensive account of the various species of protozoa 

 found in the soil has as yet been published, and, in view of the fact that 

 such highly important functions have been attributed to them, it is very 

 desirable that our knowledge of them should be extended. It is with this 

 object in view that the present paper is put forward as a preliminary 

 communication. 



Part I. — Protozoa Found. 

 (a) Methods. — The method of cultivation which has been chiefly used is as 

 follows : — To a quantity of sterile 1-per-cent. hay infusionf contained in a 

 small Erlenmayer flask or test tube are added a few grammes of the soil, the 

 protozoa of which it is desired to investigate. The mixture is shaken up, 

 a plug of cotton wool inserted in the mouth of the vessel, and the culture 

 placed in the incubator either at 20° or 30° C. After allowing it to stand 

 for a day or two, numerous protozoa can be taken from the surface of 

 the liquid. 



As an intra-vitam stain, neutral red diluted 1/100,000 has been found 

 very useful for showing up food vacuoles. For instantaneously killing the 

 organisms with their flagella or cilia fully displayed, the preparation is 

 exposed to the action of osmic vapour for a few seconds. A saturated 

 solution of methyl green in 1-per-cent. acetic acid has proved very 

 serviceable for quickly staining and showing up nuclei. It has been used 

 in the following manner : — A small drop of the culture solution is taken 

 on the sterilised platinum loop, and spread out on a cover-slip. This is 

 then exposed to the action of osmic vapour for a short time and a small 

 drop of methyl-green solution is added. The cover-slip is then inverted 

 over a cavity slide or placed on an ordinary slide and examined. It can 

 be waxed down by painting round its edge with the hot wick of a candle, 

 the flame of which has been just previously blown out, and thus kept from 

 drying up for a long time. 



* Peck, S. S., ' Bull. 34, Hawaiian Sugar Planters' Expt. Stat.,' September, 1910. 



t The hay infusion used throughout has been slightly alkaline in reaction ; sufficient 

 N/1 NaOH solution being added to the boiled and filtered liquid to render it just alkaline 

 to litmus. Other media found useful, especially for film preparations, are 1-per-cent. hay 

 infusion + egg albumen, 075 per cent. NaCl solution + egg albumen ; to each 100 c.c. of 

 liquid is added in each case about 15 c.c. of fresh white of egg. Cultures of amoeba and 

 other protozoa can be made on the agar medium made up according to the recipe given 

 by Berliner (' Arch, fiir Protistenk.,' 1909, vol. 15). 



A filtered soil extract has also been used as a liquid medium. 



