512 Journal of Agricultural Research voi. iv. No. 6 



IMPROVED LOOP METHOD 



Because of the inadequacy of the methods heretofore used, the loop 

 method which was employed by Miiller (14) for counting bacteria was 

 improved upon. The improved method proved much more accurate 

 and required but a very small amount of manipulation. 



A platinum wire was bent into a permanent loop. The quantity of 

 solution that could be transferred by means of this loop was then deter- 

 mined by carefully weighing films of the culture solution on a sensitive 

 analytical balance. The average of several weights was taken and the 

 quantity of liquid that could be transferred by the loop was calculated 

 into cubic centimeters. To facilitate the counting of the organisms on the 

 slide, a quarter of an inch square in the center of an ordinary glass slide 

 was carefully ruled into 60 to 80 small squares by means of a sharp quartz 

 crystal. A film of the culture solution containing the living protozoa 

 was then transferred to the ruled area on the clean glass slide. In this 

 manner the living protozoa were counted with the low powef of the 

 microscope, and from the number of organisms transferred in the loop 

 the numbers per cubic centimeter were calculated. 



The platinum loop was slightly bent, making an angle of 30° to 35°, 

 so as to facilitate touching the slide in the same manner at each transfer 

 and to prevent the draining of the solution which would adhere to the 

 support of the loop. 



In making the counts the platinum loop was first sterilized in a flame, 

 and every precaution ordinarily obser\'ed in bacteriological work was 

 taken. The protozoa of not less than three loops of culture solution 

 were counted, and the average of the several counts was recorded. It 

 was found by experience that not more than 300 to 400 protozoa could 

 be counted in a film which had been transferred by a loop of 0.0020 to 

 0.0025 gm. transference capacity, as the culture solution on the slide 

 would be evaporated before all the organisms could be counted. There- 

 fore, two platinum loops, one of 0.0020 to 0.0025 &ni- transference capacity 

 and the other of o.ooi gm., were employed. When the number of 

 organisms became so great that they could not all be counted in the large 

 film, the loop of smaller capacity was employed. Where the organisms 

 numbered more than 300 for the small loop, the film of culture solution 

 containing the protozoa was transferred to a specially prepared slide. 

 This glass slide has a square cell in the center (3.7 by 2)-7 mm.), the 

 capacity of which is about 0.002 gm. of water at 22° C. (when the readings 

 are made the cell is almost completely filled with culture solution, thus 

 reducing the possibility of error due to capillarity). The surface within 

 the cell is accurately ruled into 25 large divisions, and one of the large 

 divisions again ruled into 25 small fields. The film of solution con- 

 taining the organisms was carefully spread over the entire surface and a 

 cover slip laid over the cell, thus preventing evaporation. The organisms 



