42 BACTERIOLOGICAL METHODS 



1. Substances in which the organisms are not too numerous to 

 permit the use of the ^5 sq. mm. areas without making dilutions. 

 That is, substances in which the number of organisms does not 

 exceed 10,000,000 per cc., hence the number of yeast cells, spores, 

 bacteria, etc., may not exceed forty in one of the Kso c.mm. areas 

 of the hemacytometer. The limit for the spore and yeast counter 

 would be 5,000,000, before making the dilution is necessary. 



2. Substances in which the number of organisms and spores 

 are too numerous to permit the use of the H 5 sq. mm. areas of the 

 hemacytometer, but permitting the use of the J^QO sc l- mm - areas 

 without making dilutions. The total number of spores, bacteria 

 and other organisms may range from 10,000,000 to 100,000,000 

 per cc., numbers derived from finding on an average from 2.5 to 

 25 organisms in one of the Kooo c.mm. areas of the hemacytometer. 



The counter is used as follows: A bit of the thoroughly mixed substance, as 

 jam, jelly, tomato paste, catsup, etc., is placed on the slide in the ruled areas and 

 covered with a rectangular cover glass (No. 2). Slight pressure may be necessary 

 to make the cover glass rest evenly on the two slips. The counting is done in areas 

 entirely filled (from slide to cover glass) by the substance mounted. The larger 

 areas may prove useful in estimating the amount of sand particles, dirt, etc., present. 

 The 2^125 c.mm. areas will be used in counting spores, yeast cells and mold hyphae 

 and similar contaminations. It is possible to make counts without dilutions as 

 long as the number of organisms in the areas does not exceed forty. If more 

 organisms are present in one area dilution becomes necessary, as already ex- 

 plained. Making dilutions of i-io, i-ioo and i-iooo makes the counting limits 

 50,000,000, 500,000,000 and 5,000,000,000 per cc. The 3^25 c.mm. areas are also 

 used in estimating the quantity of mold hyphae present. Finding clusters of mold 

 hyphae in 25 per cent, of these smallest areas is presumptive proof that the substance 

 is unfit for human consumption. Naturally the more finely divided the substance 

 is the more numerous are the mold fragments. For making mold counts the material 

 to be examined should be reduced to uniform fineness. This could be accomplished 

 by rubbing a thoroughly mixed sample through a sieve of standard mesh, say Y mm. 



B, a simplified modification of the counting apparatus just described, is made as 

 follows: The two slips 0.2 mm. thick are placed in position, but the ruling is omitted 

 and in place thereof an eye-piece scale C is used, the measuring value of which has 

 been carefully determined by means of the stage micrometer. The rulings on the 

 eye-piece must be delicate and the analyst must be careful not to move the eye or 

 change the direction of his vision while making counts. 



The ruled slide (Z>) will be found useful for making quantitative estimates of 

 seeds, sand particles, dirt, larger parasites such as vinegar eels, ova of intestinal 

 parasites, etc., in catsups, crushed berries (strawberries, raspberries, loganberries, 

 etc.), jams and in other vegetable substances. Definite quantities of the substance 

 to be examined are placed upon the ruled area of the slide by means of a small 

 measuring spoon (0.25 gram, 0.5 gram, i gram), spread and covered with a suitable 

 cover glass and the counts made in the entire amount placed on the slide, using the 

 low power (80 diam.) of the compound microscope. 



