414 CITY MILK SUPPLY 



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 (see 

 author's note at end of the methods) : 



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 min. in the autoclave at 1 10C. Let this cool 

 to about 60C. 



2. Infuse 500 grams finely chopped lean beef for 24 hr. with its own weight of dis- 

 tilled 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 tem- 

 perature to rise above 60C. 



8. To the 500 grams of meat infusion (with peptone) add 500 grams of the 2 per 

 cent, agar, keeping the temperature below 60C. 



9. Heat over boiling water (or steam) bath 30 min. 



10. Restore weight lost by evaporation. 



11. Titrate after boiling 1 min. to expel carbonic acid. 



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



13. Boil 2 min. 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 1 hr. at 15 Ib. pressure or in 

 the streaming steam for 20 min. 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 Asso- 

 ciation 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. Occa- 

 sionally spore forms are found in such water which resist ordinary autoclave sterili- 

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

 With dilution water in 8-oz. bottles calibrated for 99 c.c. * * * 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 non-absorbent cotton 

 should be used for plugs. It is well to use care in selecting cotton for this purpose 



