C. H. Martin and K. R. Lbwin 113 



The over-staining in eosin will be found of great assistance in searching 

 rather poor films for active forms, especially in the case of flagellates. 



These methods have been found to give very fair results as regards 

 small flagellates, small amoebae, and thecamoebae. Up to the present 

 we have only very rarely found large flagellates and cihates, but to this 

 question we return in a later part of the paper. 



(3) Cultural Methods. It would we feel be premature at present 

 to attempt a formal list of the culture media on which soil protozoa 

 flourish. In all cases of cultures of soil protozoa, so far as we are aware, 

 as Vahlkampf clearly insisted in his paper on the biology, etc., of 

 Amoeba Urn ax, the protozoa feed upon the bacteria of the culture, 

 and hence almost any culture media on which soil bacteria flourish 

 will probably support a large number of protozoa. 



Therefore in those cases in which the expression "pure animal 

 culture" is used we only wish to indicate that the culture contained 

 only one form of protozoon, though of course it contained large 

 numbers of bacteria. It may of course be possible in the future to 

 obtain cultures of some saprozoic protozoa free from bacteria, and in 

 certain cases we have foxmd indications that certain amoebae show a 

 distinct preference for certain culture media, though here, again, this 

 eileet may be a secondary one due to the encouragement of a certain 

 type of bacteria. 



Up to the present we have mainly used sohd media for our cultures, 

 as we find that they are far more convenient for isolating any given 

 form. We used two types of culture media, one an ordinary agar made 

 up of 1000 c.c. meat extract and 15 grm. of agar; but we have found 

 a culture medium of Friedberger and Reiter described in Kolle and 

 Wassermann's Handhuch der pathogenen Mikroorganismen, vol. i, gives 

 very good results for most soil protozoa ; it consists of a horse-dung 

 agar made up of three lumps of horse dung and 500 c.c. of water, this 

 mixture is boiled for one and a half hours, then filtered through cloth, 

 and finally about 8 grm. of agar is added. In many cases where it is 

 used to get a very strong growth of protozoa it is advisable to add a 

 small amount of water or dilute albumen to the culture plates to 

 about a depth of 2 mm. This addition of water seems to obviate the 

 vacuolated appearance which some workers have noted as characteristic 

 of culture amoebae on plates. 



The stock cultures are made up by adding a httle soil directly to 

 the plates. If these stock cultures are examined from time to time it 

 will be found that in any given culture there is a more or less definite 



Journ. of Agrio. Sci. vil 8 



