NUMERICAL DETERMINATION OF BACTERIA. 147 



Plaling technique. The agar after melting should be kept in the 

 water-jacketerl water bath between 40° C and 45° C. for at least fifteen 

 minutes before using, to make sure that the agar itself has reached the 

 temperature of the surrounding water. If used too warm the heat may 

 destroy some of the bacteria or retard their growth. 



For routine work in cities in order to bring down the actual number 

 of colonies in a plate around the standard of two hundred, it is well to 

 use a dilution of 1-10,000. To make this dilution use two bottles of 

 sterile water each containing 99 cc. 



Shake the milk sample twenty-five times, then with a sterile pipette 

 remove 1 cc, put into the first dilution water and rinse the pipette b\ 

 drawing dilution water to the niark and expelling ; this gives a dilution 

 of 1 to 100. 



Shake the first dilution twenty-five times, then with a fresh sterile 

 pipette remove 1 cc, put into the second dilution water, rin.sing the 

 pipette to the mark as before ; this gives a dilution of 1 to 10. (Mid. Shake 

 the second dilutipn -twenty-five times, then with a sterile pipette remove 

 1 cc, and pvit it into the petri dish, using care to raise the cover only .so 

 far as necessary to insert the end of the pipette. 



Taking a tube of agar from the water bath, wipe the water from out- 

 side of tube with a piece of cloth, remove the plug, pass the moutli of 

 the tube through the flame, and pour the agar into the plate, using the 

 .same care as before to avoid exposure of the plate contents to the air. 



Carefully and thoroughly mix the agar and diluted milk in the petri 

 dish by a rotary motion, avoiding the formation of air bubbles or slop- 

 ping the agar, and after allowing the agar to harden for at least fifteen 

 minutes at room temperature place the dish bottom <loun in the incuba- 

 tor. The practice of mixing the diluted milk with the agar /// the liihr, 

 leaving a certain portion of the bacteria unplated, is not recommended 

 by the Committee. 



Controls. Plating should ahva\s be checked by controls. A blank 

 plate should be made with each set of milk plates for control of the water, 

 petri dishes, pipettes, etc. 



For control on technique of plating it is recommendeil that for work 

 on "market milk," duplicates be made each day on several plates. 



"Certified milk" should always be plated in duplicate, and where pos- 

 sible it is well to have one man's work occasionally checked by another. 



Unless duplicate plates show as -a rule approximately the same count, 

 the worker should see if there is error in his technique. 



Racks are very useful for stacking the plates and to prevent breakage. 



Plating sliould be done always in a place free from dust or currents 

 of air. 



In order that the colonies may have sufficient food for proper develop- 

 ment, 10 cc. of agar shall be used for each plate. In plating a large 

 number of samples at one time the dilution and transfer of diluted milk 



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