374 STATE BOARD OF AGRICULTURE. 



METHODS. 



Obtaining Samples. — Samples of butter were taken from tubs with sterile 

 triers and placed in sterile one pint Mason jars. A separate sterile butter 

 trier and short cheese trier were used for each sample, and about two inches 

 from top and bottom discarded. These samples (about ^ pint each) were 

 melted in a water bath at 35° C, partially cooled, \agorously shaken to 

 insure uniformity, and the samples required for bacterial and chemical 

 determinations poured into the appropriate dishes. 



Bacterial Content and Isolation. — A small amount of butter, about 25 cc, 

 having been poured from the Mason jar into a 60 cc. Erlenmeyer flask, this, 

 together with the dilution flasks containing 99 cc. each of sterile 0.6% salt 

 solution, was kept in a water bath at 35° C. Pipettes sterilized in plugged 

 test tubes were kept in a water bath at 50° C. After shaking the butter 

 sample well, 1.15 cc. (= 1 gram) were measured from a warmed sterile 

 pipette into 99 cc. of salt solution. This was shaken well to a milky emulsion 

 and other dilutions similarly made by introducing 1 cc. of the dilution already 

 prepared into 99 cc. of sterile salt solution. Dilutions of 1-1000, 1-10,000, 

 and 1-100,000 were then plated in ordinary agar, whey agar, whey gelatin 

 and litmus glucose agar. In the November and February examinations, 

 dilutions of 1-100, 1-1,000 and 1-10,000 were employed. 



Ordinary agar was made accorcUng to the rules adopted by the Com- 

 mittee on Standard Methods (Jour. Inf. Dis., Suppl. No. 1, 1905.) It was 

 adjusted to 15°. 



Whey agar was made as follows: Fresh skimmed milk at 41° C, was lop- 

 pered by adding sufficient rennet (about i% liquid rennet in 20 cc. of cUstilled 

 water). After the curd was firm it was cut in fine pieces and placed in steam 

 for 40 minutes. It was then strained through muslin to remove curd. The 

 reaction of the whey was adjusted to 15° acid with standard NaOH, and 

 1% of dry peptone and 1.5% finely -shredded agar added. It was then 

 placed in the steam for one hour. The reaction was readjusted to 15° acid, 

 cooled to 60° C, clarified with egg albumen, counterpoised, boiled over free 

 flame for five minutes, loss made up with distilled water, filtered through 

 a hot washed plaited paper filter, tubed and sterilized 15 minutes in steam 

 for three consecutive days. 



The ordinary and whey agar plates were kept at 20-22° C, and counted 

 at the end of seven days, descriptions and numbers of each type of colony 

 being noted. Typical colonies from each plate were isolated into bouillon, 

 incubated two days at 20°-22° C, replated in gelatin and fished as pure 

 cultures to slant agar tubes. Each culture was then sub-cultured to bouillon, 

 gelatin, milk (20 and 37° C), potato, Dunham's broth for indol, and the 

 three sugar broths in fermentation tubes. 



Whey gelatin was prepared according to the method of Conn (Bacteria 

 in Milk and its Products, 1903, p. 270). Plates were kept in cold storage 

 at about 4° C. for 30 days undisturbed, when they were counted and cul- 

 tures isolated as in the case of agar plates. 



Litmus glucose agar plates were placed in Novy jars and kept in hydrogen 

 gas at 20-22° C. for the development of anaerobes. After 7-10 days the 

 jars were opened, the chffereht typical colonies from each plate described 



