EXAMINATION OF MILK 177 



Liquefy a number of tubes of plain 2 to 3 per cent, agar (+1.5 reaction) and 

 cool to 45C. 



In each of the first two tubes put i cc. of i : 1000 dilution and plate. 



In each of the second two tubes put 0.5 cc. of i : 1000 dilution and plate. 



In each of the third two tubes put 2 cc. of i : 10,000 dilution and plate. 



In each of the fourth two tubes put i cc. of i : 10,000 dilution and plate. 



In each of the fifth two tubes put 0.5 cc. of i : 100,000 dilution and plate. 



Incubate one set at room temperature, the other at 37C. for 3 days and 

 count colonies every 24 hours. 



Leucocytes. Fill a Stewart's tube (i cc.) with milk, insert rubber plug in 

 one end and push the rubber bulb down tight on the other. Place in centrifuge 

 with plugged end at periphery. Spin at 3000 revolutions per minute for 5 

 minutes. Hold the tube horizontal and remove plug without scraping against 

 edge*f tube. Smear the sediment collected on the plug over an area of i square 

 centimeter on a glass slide by rubbing the plug against it. 



Fix by heating over flame and stain with Loeffler's methylene blue for 5 

 minutes. Dry and examine under oil immersion lens. 



The leucocytes are counted in 10 fields. If a total of 230 or more leucocytes 

 is observed in unheated milk it is condemned; when less than this number are 

 observed associated with considerable numbers of streptococci it is condemned. 



When making this examination many fields will be observed in which there 

 are no bacteria, fields that contain them will only show a few if the milk is 

 clean less than 500,000 colonies per cubic centimeters when plated. If most 

 of the fields show a few bacteria plating will show more than 500,000 colonies 

 per cubic centimeter, and if numerous fields show large numbers or clumps of 

 bacteria, the milk is very dirty and will show more than 1,000,000 colonies per 

 cubic centimeter. 



ANIMAL INOCULATION TESTS 



Place 50 cc. of milk in centrifuge tube and spin at high speed, 3000 revolu- 

 tions per minute or more, for 10 minutes. Remove the cream, place it in a 

 centrifuge tube. Remove the skimmed milk without disturbing the sediment 

 in the bottom of the tube. Fill both the centrifuge tubes up to the 50-0;. mark 

 with sterile distilled water, shake thoroughly to evenly mix the contents of each 

 tube, then centrifugalize them as before. Remove the upper 48 cc. of fluid 

 from each tube without disturbing the sediment. Suck the remaining 2 cc. 

 of fluid and sediment from the cream into a syringe, and inject into the peri- 

 toneal cavity of a guinea-pig. In like manner inoculate a second pig with the 

 sediment from the skimmed milk. 



Healthy pigs, weighing between 200 and 300 Gm. should be used. 



Wholesome milk will not injure guinea-pigs. If the animals remain alive, 

 as they should if the milk does not contain large numbers of pathogenic organ- 

 isms other than tubercle bacilli, kill them 6 weeks after inoculation and examine 

 for tuberculosis. It requires a month or more for tubercle bacilli to produce 

 distinct, characteristic lesions in guinea-pigs. 



