398 MILK AND MILK PRODUCTS 
unfiltered stains are to be avoided, as they may contain troublesome 
precipitates. The slides remain in this solution for five seconds 
to one minute or longer, depending upon the effect desired, and are 
then rinsed in water to remove the surplus stain, and decolorized in 
alcohol. The decolorization takes several seconds to a minute, during 
which time the slide must be under observation in order that the decol- 
orization may not proceed too far before they are removed from the 
alcohol. When properly decolorized the general background of the 
film should show a faint blue tint. Poorly stained slides may be decol- 
orized and restrained as many times as necessary, without aay apparent 
injury. After drying the slides may be examined at once, or they may 
be filed away and preserved for further reference. 
Standardization of the Microscope. The microscope to be used 
must be adjusted in such a way that each field of the microscope covers a 
certain known fraction of the total square centimeter’s area. This 
procedure is simple, with the proper materials at hand. The micro- 
scope should have a 1.9 mm. (1/12 in.) oil immersion objective, and an 
ocular giving approximately the field desired, and should preferably 
be fitted with a mechanical stage. To standardize the microscope, place 
upon the stage a stage micrometer, and by the selection of oculars or 
adjusting the draw tube, or both, bring the diameter of the whole 
microscopic field to .205 mm. When so adjusted, the microscope field 
will cover almost exactly 1/300,000 of a cubic centimeter of the milk 
(actually 1/302,840). This means that if the bacteria in one field only 
are counted, the number should be multiplied by 30,000 to give the 
total number for a cubic centimeter. If the bacteria in a hundred 
fields are to be counted, the total should, of course, be multiplied 
by 3000. 
Inasmuch as it is difficult to count bacteria lying near the margin 
of the microscopic field, it is much better to have an eyepiece micrometer, 
with a circular ruling, 8 mm. in diameter, and divided into quadrants. 
This will give, in the microscopic field, a smaller area within which the 
bacteria may be seen most sharply, and which may be more easily 
counted. Such eyepiece micrometers are now manufactured by labora- 
tory supply houses, and may be casily obtained. In the use of this eye- 
piece micrometer the inner circle, by the adjustment of the draw tube, 
should be made to cover a circle with a diameter of .146 mm. In this 
case this inner circle will cover 1/600,000 of a cubic centimeter of milk, 
meaning, of course, that the number of bacteria in a single field should 
be multiplied by 600,000, or, if a hundred fields are counted, by 6000, 
to obtain the number per cubic centimeter. 
