August 27, 1915] 



SCIENCE 



285 



2. It is more accurate than any other 

 method in use, because it is a standard instru- 

 ment and no mechanical variation is possible. 



3. It is rapid and saves considerable time 

 in contradistinction to other methods, and the 

 technique is simple. For example, triplicate 

 counts on any medium were recorded in ten 

 minutes. 



4. The counts check more closely than those 

 of any other method. 



5. It can be used to advantage whether the 

 number of protozoa present be large or small. 



6. It can be used for living, killed or 

 stained organisms and permits of a thorough 

 observation of the individual organisms. 



Its disadvantages are that the initial cost is 

 greater than that of other methods, and the 

 sample is too small to be representative. The 

 error of count is considerable where the pro- 

 tozoa are very few or many in number. And a 

 number of fields must be counted because of 

 the uneven distribution, if an accurate coun* 

 IS required. 



Despite the logical thoroughness of Eussell 

 and Hutchinson's work, there appears to be 

 one point which they seem to have neglected. 

 N^amely, the production of ammonia, etc., is 

 used as a criterion for measuring the effect of 

 soil protozoa on bacterial activity, while the 

 fungi in the soil, which are known to be ca- 

 pable of producing ammonia,^ were not taken 

 into account. Thus there is an added unrec- 

 ognized factor operating in their experiments 

 as well as those of others, i. e., soil fungi. 



Taking cognizance of this factor, a method 

 was devised for its elimination, based upon the 

 principle of dilution, in such a way as to re- 

 duce the possibilities for the occurrence of 

 fungi. The method of procedure was to pour 

 plates of ten different fungi media in dupli- 

 cate. These agars were: potato, oat, cornmeal, 

 rice, bean, raisin, apple, synthetic, soil extract 

 and Cook and Taubenhaus's No. 2J' 



Upon cooling, a block of each medium about 



sMiintz and Coudon, Compt. Bend., 116 (1893), 

 395. 



7 Oook and Taubenhaus, Dela. Bull. No. 91 

 (1911), 11. 



2 cm. square was cut out with a sterile knife, 

 and 1 c.c. of sterile soil extract was introduced 

 by means of a sterile pipette into the cavity 

 formed. A platinum loopful of a three-day- 

 old culture of soil organisms in soil extract, 

 known to contain numerous bacteria, protozoa 

 and fungi, was then carefully rinsed off in the 

 medium occupying the cavity. 



At the same time poured plate cultures of 

 one loopful of the three-day-old culture of or- 

 ganisms were made on the ten different agars 

 mentioned above. Likewise after one week 

 poured plate cultures were made on the ten 

 different media by inoculation with one loop- 

 ful of the solution present in the cavity of the 

 agar plate. 



The results show that on the plates where a 

 portion of the agar was removed and 1 c.c. of 

 soil extract substituted, the bacteria and pro- 

 tozoa developed in large numbers, which 

 might in all probability be due to the fact that 

 a large surface is exposed for such a small 

 quantity of media. The important point, how- 

 ever, which is to be noted from this experi- 

 ment is that despite the fact that suitable 

 media were furnished for the grovrth of fungi, 

 none was evident, even after thirty days' incu- 

 bation. 



From the observation of the poured plate 

 cultures made from the original three-day-old 

 culture we note that fungi appear after four 

 days upon three out of ten plates ; namely, ITo. 

 2, synthetic,^ and raisin agars. The fungi pre- 

 dominating were species of Penicillium, 

 Alter n aria and Fusarium. 



On the poured plate cultures made from the 

 solution in the cavity of the agar plates, no 

 fungi developed. This experiment was re- 

 peated and corroborated the previous results. 



Thus it is certain that whereas fungi were 

 present in the original culture the process of 

 high dilution was responsible for their elimi- 

 nation from the specially prepared cavity on 

 the agar plates. 



Thus the dilution method followed by the 

 peculiar manner of plating, as outlined, makes 

 it possible to separate fungi from bacteria and 



s Lipman and Brown, N. J. Ann. Ept. (1908), 

 132. 



