SOIL PROTOZOA IN SOUTH AFRICA. 375' 



tains, and various other sites. Cultivated, uncultivated and virgin 

 soils were examined and compared, and their acidity tested in each 

 case by the litmus compression method. 



Samples of soil were taken from three inches to twelve inches 

 deep and the soil collected in some cases from the bottom of the 

 hole, in other cases from the sides, the latter case yielding "mixed" 

 samples containing all the layers down to the required depth. 

 Metallic diggers and sterilised containers were used, thus avoiding 

 contamination from outside. The season and prevailing weather 

 conditions were noted. A few samples of waterlogged soils have 

 also been examined by direct observation. Some of these water- 

 logged soils formed the basins of shallow pools or vleis, while others 

 were taken from the banks of running streams. 



Various culture media for soil Protozoa have been tried else- 

 where, but none has been found perfectly satisfactory. In South 

 Africa various media have also been tried, but the best results were 

 obtained by using boiled, neutral tap water to ensure sterility, add- 

 ing it to soils in definite proportions (for example, in some cases 1 

 gram of soil to 12 cc. of water, in other cases 1 gram of soil to 5 c.c. 

 of water), and allowing it to stand for some time in test-tubes 

 plugged with cotton wool, using ordinary bacteriological precau- 

 tions to prevent contamination both at the time of incubation and 

 during the periodical examinations. Samples of the water cultures 

 were examined fresh by means of cover-slip or hanging drop pre- 

 parations. The cultures were kept at room temperature in most 

 cases. Some, however, were placed in incubators kept at 37°C. and 

 45°C. Some cultures were kept in the light, while others were 

 placed in the dark. All the results accruing were compared. We 

 know that water cultures may not give an accurate impression of 

 the constitution of the active fauna of the soil, as excystation is 

 brought about. However, this may be partly corrected by the 

 comparative examination of specimens of naturally water-logged 

 soil. 



Determination of the approximate number of protozoan cysts 

 in soils was attempted in some cases, and the intimate attachment 

 of the cysts to the soil particles was demonstrated by successive 

 triturations of the same sample of soil in boiled water and 

 counting the number of cysts recognisable under the microscope 

 after each trituration. 



In every case the soil was examined previous to culture for 

 Protozoa by microscopic examination of a small portion of soil in 

 a drop of cold boiled water. Trophic Protozoa were rarely found, 

 but encysted forms were fairly often recognised. 



The use of fixatives and stains has been found difficult by most 

 workers as distortion is easily caused in the Protozoa. Picric 

 alcohol was a useful fixative. Methyl green proved a good stain 

 for observing the nuclei of ciliates. Acid carmine and haematoxy- 

 lin were useful general stains. As before stated, examination has 

 so far been chiefly limited to fresh material. 



Daily observations on the culture tubes were essential, as each 

 organism appears to increase in numbers to a maximum, then 



