Standards for Certified Milk. 263 



63. Plating. The packages shall be opened with aseptic pre- 

 cautions after the milk has been thoroughly mixed by vigorously 

 reversing and shaking the container 25 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 48 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 phe- 

 nolphthalein. 



The following is the method recommended by a committee 

 of the American Public Health Association 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 10 minutes in the autoclave at 110 C. Let this 

 cool to about 60 C. 



2. Infuse 500 grams finely chopped lean beef for 24 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 G. 



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 30 minutes. 



10. Eestore 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 20 minutes on three successive days. 



66. Samples of milk for plating shall be diluted in the pro- 

 portion of 1 part of milk to 99 parts of sterile water; shake 25 

 times and plate 1 c. c. of the dilution. 



The committee on bacterial milk analyses of the American Public Health 

 Association in Part IV of its report presented details with respect to plating apparatus 

 and technique in part as follows: 



Plating apparatus. For plating it is best to have a water bath in which to melt 

 the media and a water-jacketed water bath for keeping it at the required temperature; 

 a wire rack which should fit both the water baths for holding the media tubes; a ther- 

 mometer for recording the temperature of the water in the water-jacketed bath, sterile 1 

 c. c. pipettes, sterile Petri dishes, and sterile dilution water in measured quantities. 



Dilutions. Ordinary potable water, sterilized, may be used for dilutions. ^ Oc- 

 casionally spore forms are found in such water which resist ordinary autoclave steriliza- 

 tion ; in such cases distilled water may be used or the autoclave pressure increased. With 

 dilution water in 8-ounce bottles calibrated for 99 cubic centimeters all the 



necessary dilutions may be made. 



Short, wide-mouthed "blakes" or wide-mouthed French square bottles are more 

 easily handled and more economical of space than other forms of bottles or flasks. 



Eight-ounce bottles are the best, as the required amount of dilution water only 

 about half fills them, leaving room for shaking. Long-fiber nonabsorbent cotton should 



