490 NUMBER AND SIGNIFICANCE OF LEUKOCYTES IN MILK 



be condemned as improper for use, repulsive, and probably unwhole- 

 some. The leukocyte contents of wholesome milk from healthy cows, 

 however, varies so much as shown by more recent investigations based 

 upon accurate methods, that older standards resting upon inaccurate 

 methods and upon insufficient data must be given up. 



Methods of Estimating Leukocytes in Milk. STOKES' METHOD. 

 Place 10 c.c. of milk into a centrifuge tube and centrifuge in an 

 electric or other good centrifuge for ten minutes. Draw off the fat 

 and the clear fluid by the aid of a pipette and leave only the sediment 

 in the tube. Spread a platinum loopful of the sediment over 1 square 

 centimeter on a glass slide or cover-glass. Air dry, fix and stain with 

 methylene blue; wash in water, dry between filter paper and mount. 

 Count ten fields of a one-twelfth oil-immersion lens and calculate the 

 average per field. It has been customary on the basis of this method 

 to declare milk unfit for use when the number of leukocytes exceeded 

 ten leukocytes per field. 



STEWART'S METHOD. Small glass tubes are used, closed at one 

 end with a rubber stopper. Place 1 c.c. of milk into one of the tubes, 

 centrifuge for ten minutes. Then hold the tube horizontally and 

 draw out the rubber stopper, to which the sediment adheres. Spread 

 the sediment over one square centimeter area of a glass slide or 

 cover-glass. Air dry, fix and stain with methylene blue, and count 

 the leukocytes in ten fields of a one- twelfth oil-immersion lens. Milk 

 has been considered unfit for use if the average per microscopic one- 

 twelfth oil-immersion field is above twenty- three. 



TROMMSDORF'S METHOD. In this method specially constructed 

 centrifuge tubes are used holding 10 c.c. and drawn out at the bottom 

 into a fine caliber. The narrow part of the tube has twenty gradua- 

 tions, each one representing 0.01 percent, of 10 c.c. If the sediment, 

 therefore, reaches to the tenth mark it indicates that the 10 c.c. of 

 milk contain 0.1 per cent, sediment or one volume of the latter to 1000 

 of milk. According to Trommsdorf this is the maximum amount 

 admissible, and anything above represents an excess in leukocytes, 

 i. e., pus. While this method simply indicates the percentage of sedi- 

 ment and gives no information as to its composition, it is said to be a 

 fairly accurate and good practical method. 



DOANE-BUCKLEY METHOD. This is the most accurate, reliable 

 procedure, because actual count of the leukocytes is made with a 

 Thoma-Zeiss counting chamber (see below). The method, as modified 

 by Campbell and described in Bulletin 117 of the Bureau of Animal 

 Industry, includes the heating of the milk to be tested, as this separates 

 the leukocytes from the fat globules, and gives higher and more 

 accurate values. The effect of heating on the leukocyte count had 

 .been previously described by Russell and Hoffmann. The steps in 

 the Doane-Buckley method are as follows: 



1. In this as in any other method the sample examined should be 

 as fresh as possible, because the formation of lactic acid favors the 

 precipitation of casein, and this interferes with obtaining a clear 



