30 BULLETIN 1, U. S. DEPARTMENT OF AGRICULTURE. 
erly 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 betiveen 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 40 hours. 
63. Plating. — The packages shall be opened with aseptic precautions after the 
milk has been thoroughly mixed by vigorously reversing and shaking the con- 
tainer 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 phenolphthalein. 
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° 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 30 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 20 minutes on three successive days. 
66. Samples of milk for plating shall be diluted in the proportion 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 thermometer for recording the temperature of the water in the 
water-jacketed bath, sterile 1 c. c. pipettes, sterile Petri dishes, and sterile dilu- 
tion water in measured quantities. 
Dilutions. — Ordinary potable water, sterilized, may be used for dilutions. 
Occasionally spore forms are found in such water which resist ordinary auto- 
