TIIJ; DILUTION METHOD 97 



disc (c) o.i inm. thinner. On the upper side of this latter are etched two 

 systems (crossing each other at right angles) of twenty-one parallel lines at 

 regular intervals of 0.05 mm., and therefore enclosing compartments each of 

 which has an area of 0.0025 S( 3- mm ' 



If now a sufficiently large droplet of the sample to be counted be laid on the 

 centre of c and covered with a cover-glass (b) about 0.5 mm. thick, then various 

 portions of the liquid, which has a uniform thickness of o.i mm., can be 

 examined under the microscope for the number of germs present therein. 



A number (ten to fifty) of the square divisions are counted and the mean 

 of the resulting figures is taken. This being denoted as M, the germ content 



of the liquid will then be OO02 ^ = 4000 M per cubic millimetre. 



In order to arrest the movement of motile forms or prevent the multiplica- 

 tion of rapid-growing cells (e.g. yeast), a portion of the sample, well shaken up, 

 is previously mixed with an equal volume of 10 per cent, sulphuric acid, which 



A 



e c t 



FIG. 32. Counting Chamber. Nat. size. Description in text. 



will kill the organisms. This dilution must be taken into account in calculating 

 the germ content from the numbers found in the counting ; so that M must be 

 multipled by 8000 instead of 4000. 



Assuming the germ content to have been ascertained in the way described 

 above in the bacterial mixture, the different species in which are being isolated ; 

 then, the number of cells present per unit of space being known, a portion of 

 the sample- but not that diluted with acid must be thinned down with 

 sterilised water to such an extent that only one cell is present in two to five 

 drops. One drop of this diluted liquid is then placed in each of a series of flasks 

 containing a sterile nutrient medium, which flasks are subsequently kept at a 

 suitable temperature, whereupon some of them will, after a while, exhibit 

 signs of development ; these will constitute the wished-for pure cultures. They 

 are, however, not unconditionally reliable, since it not infrequently afterwards 

 becomes evident that, in despite of calculation, some of the flasks contained more 

 than one germ. By this method, generally known as the dilution method, 

 Lister in 1878 prepared a pure culture of Bacterium lactis, which was (chrono- 

 logically) the first bacterial pure culture, and FRITZ (VII.) also employed the 

 same method in his studies on fermentation by fission fungi. The first six species 

 of the Saccharomycetes studied by E. Ch. Hansen, and which stand out so 

 prominently in the literature of fermentation physiology, were also isolated by 



I Q 



