VOLATILE ANTISEPTICS AND SOIL ORGANISMS. 161 
EFFECT ON PROTOZOA. 
The casual observation of the abundance of certain types 
of protozoa in cultures of Azotobacter from soils previously 
‘partially sterilized,” led the writer to undertake a series 
of experiments to determine whether subjecting local soils 
to the treatment described by Russell and Hutchinson 
actually freed them of protozoa. This series of experiments 
has brought forth some very startling and interesting results, 
compared with the results of the above mentioned writers. 
The same soils as mentioned under the bacteriological 
part of this paper were also investigated as to protozoa. The 
methods used were as follows, and while they were by no 
means satisfactory, they are the best the writer has been 
able to devise, and do reveal certain facts that seem worthy 
of mention :— 
Samples of treated soil were mixed with twice their 
weight of sterile water or culture medium in an Erlenmeyer 
flask, thoroughly shaken, and set aside at room temperature 
to incubate. At first 20 grams were used, later 50 grams. 
In the first work, only water cultures were made, but it was 
later found that if a culture medium, composed of 1% hay 
decoction (1% hay infusion heated one-half hour in Arnold 
sterilizer, filtered and sterilized) and 5% white of egg, was 
used, certain types of protozoa, notably Amoeba, Colpoda, 
and certain flagellates that escape observation in water 
cultures, would develop in large numbers. No doubt if 
other media were employed other types would be observed. 
Now every sample is cultured both in water and in the 
medium given above. 
After incubating the water cultures for twenty-four hours, 
5 or 10 ce. of the supernatant liquid were removed (without 
stirring) with a sterile pipette to a sterile centrifuge tube. 
The centrifuge was then run until experience had shown 
practically all protozoa to be thrown down; 1 to 5 minutes 
usually sufficed. This can be determined by examining 
the upper portions of the liquid. A sterile pipette was 
inserted to bottom of tube and sediment removed and 
