ESTIMATION OF THE BACTERIAL CONTENT OF MILK ()85 
agar. The number of colonies which develop after forty-eight hours' 
incubation at .37° C. multiplied by the dilution is taken as the bacterial 
count of the milk. It is customary in some laboratories to make 
a parallel count at 20° C, after four days' incubation. The numbers 
of colonies developing on agar at the lower temperature may be nuich 
greater than those incubated at body temperature. The difference 
between the counts is usually more marked in samples of milk which 
have been maintained for some time at a relatively low temperature, 
and in ice-cream. In such cases bacteria whose minimal temperature 
of growth is relatively low— 4° to 12° C— may multiply with con- 
siderable rapiditv. These organisms frequenth' fail to develop at 
37° C. 
The cultural count possesses advantages and disadvantages. The 
principal advantages are: the simplicity of the method, comparative 
accuracy of results provided uniform conditions are maintained, and 
some differentiation of the types of organisms present in the milk. 
The disadvantages are: the time required to obtain results— milk 
is perishable and cannot be held pending examination by this proce- 
dure. Furthermore, by no means all the bacteria which may theoreti- 
cally gain access to the milk will grow upon plain agar; this is par- 
ticularly true of pathogenic microorganisms. Bacteria which remain 
adherent in groups or chains are frequently not separated during the 
shaking of the sample and a single colony may originate from such 
a clump or chain. This naturally introduces an error which may be 
very considerable if, for example, a long chain of streptococci develops 
as a single colony. 
(b) Direct microscopic count. Milk hygienists have long recognized 
the advantages of a direct estimation of the bacterial count of milk 
and numerous methods have been proposed, from time to time, to 
accomplish this object. The most practical method thus far prescribed 
appears to be that of Prescott and Breed.' The theory involved is to 
spread a definite volume of milk upon a definite area on a glass slide, 
evaporate the fluid, fix the sediment (w^hich contains all the bacteria 
in the sample), anfl stain it in such a manner that the microorganisms 
are distinctly colored. The organisms of a definite area are counted 
under the microscope. The number in the original sample are readily 
computed, knowing the volume of milk examined, the area o\er which 
it is spread and the size of the microscopic field. 
In practice 0.01 cc. of a well-mixed sam])le of milk is spread uniformly 
over an area of 1 square centimeter on a glass slide. (This area is 
readily outlined with a wax pencil, using a pattern previously- ruled on 
a piece of paper as a guide and following the outline on the glass slide; 
the wax pencil mark tends to limit the spread of the milk beyond the 
limits of the square.) The film of milk is then air-dried or dried at 40° 
C, immersed in absolute methyl alcohol for a few minutes to fix the 
sediment to the slide and to remove some of the milk lipoids and fats 
Jour. Infec. Dis., 1910, 7, 632. 
