112 TRANSFORMATION OF THE INTESTINAL FLORA 



of fifteen pounds for twenty to thirty minutes, or longer when more 

 than 1000 cubic centimeters are sterilized in a container. For this 

 purpose Pyrex Florence flasks have been employed in the present in- 

 vestigation. The cooled milk is inoculated with a pure culture, prefer- 

 ably mixed strains, of B. acidopJtilus, and incubated at 35 to 37° C. 

 for twelve to twenty-four hours. The inoculum should be a milk culture 

 not more than seventy-two hours old. 



It is at this point that special effort is required to obtain a product 

 that is well soured and viable. Recently isolated strains of B. acidoph- 

 ilus are slow and ineffective. Therefore, it is necessary to employ 

 strains which have been grown in milk for at least two or three weeks 

 and which have been transplanted from milk to milk at frequent inter- 

 vals, preferably every day. The amount of inoculum for each trans- 

 plantation should be large, as compared with the usual procedure. 

 For every 1000 cubic centimeters five to ten cubic centimeters of the 

 inoculum are required to obtain maximum development within twenty- 

 four hours' time. The transfers are made with sterile pipettes made 

 from sections of glass tubing. These pipettes are drawn out at one end, 

 but the opening must be large enough to admit coagulated milk. Plati- 

 num loops are useless for making the transfers. 



As soon as the milk has undergone coagulation it is removed and 

 placed in the refrigerator. It is important that the refrigerator com- 

 partment in which the milk is kept be clean and free from odors which 

 may be absorbed. When kept under these conditions the milk changes 

 very little in the course of a few days, but should be used very soon. 



If it is desired to reenforce the milk with lactose, the sugar may be 

 added at the time the milk is taken. It will be expedient to shake the 

 lactose and milk well and allow the mixture to stand for thirty minutes 

 or more in a cool place. This will allow a goodly portion at least of 

 the sugar to dissolve and to reduce the gritty character of the lactose- 

 enriched milk. For those who crave sugar the addition of the lactose 

 greatly adds to the richness of the milk. 



In the present investigation four strains of B. acidophilus have been 

 employed for the production of acidophilus milk. These strains were 

 isolated from human feces in October, 1919, and were grown on whey 

 agar for five months, transfers being made three times a month. For the 

 inoculation of the first milk tubes the four strains were combined. 

 Transfers were made almost daily. At first it required five or six 

 days, and at times even more than a week for the milk to show indica- 

 tions of acidity and coagulation. The coagulation time was gradually 

 shortened tintil three weeks after the first milk culture was prepared, 

 when coagulation was obtained within twenty- four hours. At the end 

 of the fourth week of almost daily transfers the coagulation time was 

 reduced to twelve to fifteen hours, where it stands at the present time, 

 many months after the first milk inoculation. 



