50 Stinli<:s nil Soil I 'rot ()':<)(( 



submitted to microscopic examination. As a result of his work he 

 found that drying the soil caused a reduction in the numbers of protozoa 

 and that this reduction was first noticeable in the case of the amoebae. 

 Killer^ also used the dilution method, with a number of the solutions 

 employed for cultivation of soil bacteria as media. 



The method employed in these experiments is in principle the same 

 as that generally apphed in bacteriological work. Four parallels in 

 each dilution are used. The medium is soil extract (prepared as 

 described in Ijohnis' Pndlikum, p. 118, but undiluted) -1 ",j K^HI'O^ 

 in 1 c.c. (juantities in small test-tubes. To each tube 1 c.c. of the 

 dilution-water is added, so that the medium, so far as the protozoa are 

 concerned, is ordinary soil extract f •()") % K^HPOj. But. if the 

 dilutions are put up simply as above described, it has been found that 

 the multiplication of the protozoa after excystation is rather slow and 

 the microscopic work as a consequence is very tedious. It has been 

 observed that inoculation of the soil extract with a protozoa-free 

 culture of bacteria, prepared from a bloodmeal culture as described in 

 the previous paper (p. 604), hastens the multiplication of the protozoa. 

 The microscopic work is thus considerably facilitated. The procedure 

 is to inoculate the soil extract with the protozoa-free cidture and 

 incubate for two days before inoculation from the dilutions. 



Subsequent work on the effects of moisture, etc. on the protozoal 

 content of soils has shown that the dilutions l(»i, looo, Ki.ikmi. ico.dOO, 

 etc., are not close enough to bring out differences due to the treatment 

 of the soils. It has, therefore, been found necessary to employ closer 

 dilutions. Those used are, for example, 100, KOO, 500, 750, 1000. 

 3000, 5000. etc. 



When now the method is applied to the enumeration of protozoa 

 in soils, it is found that the results are rather irregular. Up to a certain 

 dilution all four parallels in each case give positive results. Then in 

 the next three or four dilutions 1-3 of the parallels in each are positive, 

 the remainder negative. Table 1 shows a typical case. The figures 

 given in the columns indicate the nnniher of parallels showing positive 

 results. 



It will be observed that after five days' incubation the development 

 is regular up to and including 5000 with single positive results in each 

 of the next four dilutions. On incubation for a further period of 25 

 days the regular development stage is pushed forwaid to the 10.000 

 dilution. But beyond this irregularities still remain. From the jinint 

 1 Centmlbl.f. Bakt. .M.t n. B-l. 37, ji. r.2l. 



