502 MILK 



bacteria per cubic centimeter is found, daily counts shall be made, and if 

 normal counts are not restored within ten days the certificate shall be sus- 

 pended. 



59. Bacterial counts shall be made at least once a week. 



60. Collection of Samples. The samples to be examined shall be obtained 

 from milk as offered for sale and shall be taken by a representative of the milk 

 commission. The samples shall be received in the original packages, in 

 properly iced containers, and they shall be so kept until examined, so as to 

 limit as far as possible changes in their bacterial content. 



61. For the purpose of ascertaining the temperature, a separate original 

 package shall be used, and the temperature taken at the time of collecting the 

 sample, using for the purpose a standardized thermometer graduated in the 

 centigrade scale. 



62. Interval Between Milking and Plating. The examinations shall be 

 made as soon after collection of the samples as possible, and in no case shall 

 the interval between milking and plating the samples be longer than forty hours. 



63. Plating. The packages shall be opened with aseptic precautions 

 after the milk has been thoroughly mixed by vigorously reversing and shak- 

 ing the container twenty-five times. 



64. Two plates at least shall be made for each sample of milk, and there 

 shall also be made a control of each lot of medium and apparatus used at 

 each testing. The plates shall be grown at 37 C. for forty-eight hours. 



65. In making the plates there shall be used agar-agar media containing 

 1.5 per cent, agar and giving a reaction of 1.0 to phenolphthalein. 



The following is the method recommended by a committee of the Ameri- 

 can Public Health Association 1 for the making of the media, modified, however, 

 as to the agar content and reaction to conform to the requirements specified 

 in Section 65 : 



1. Boil 15 grams of thread agar in 500 c.c. of water for half an hour and 



make up weight to 500 grams, or digest for ten minutes in the 

 autoclave at 110 C. Let this cool to about 60 C. 



2. Infuse 500 grams finely chopped lean beef for twenty-four hours with 



its own weight of distilled water in the refrigerator. 



3. 'Make up any loss by evaporation. 



4. Strain infusion through cotton flannel, using pressure. 



5. Weigh filtered infusion. 



6. Add Witte's peptone, 2 per cent. 



7. Warm on water-bath, stirring until peptone is dissolved and not 



allowing temperature to rise above 60 C. 



8. To the 500 grams of meat infusion (with peptone) add 500 grams of 



the 2 per cent, agar, keeping the temperature below 60 C. 



9. Heat over boiling water (or steam) bath thirty minutes. 



10. Restore weight lost by evaporation. 



11. Titrate after boiling one minute to expel carbonic acid. 



12. Adjust reaction to final point desired +1 by adding normal sodium 



hydrate. 



13. Boil two minutes over free flame, constantly stirring. 



14. Restore weight lost by evaporation. 



15. Filter through absorbent cotton or coarse filter paper, passing the 



filtrate through the filter repeatedly until clear. 



16. Titrate and record the final reaction. 



17. Tube (10 c.c. to a tube) and sterilize in autoclave one hour at 15 



pounds pressure or in the streaming steam for twenty minutes on 

 three successive days. 



1 In a provisional report of the Committee on Standard Methods of Bacte- 

 riological Milk Analysis, published in the American Journal of Public Health, 

 1916, vol. 6, p. 1315, a change is proposed in the composition of standard 

 agar which, according to Sears and Case (Jour, of Bact., 1916, vol. 3, p. 351), 

 gives low and irregular counts. 



