234 STATE BOARD OF AGRICULTURE. 



INVESTIGATIONAL WORK. 



This work was carried out under commercial conditions and the data 

 sliould represent the numerical numbers of bacteria and include the 

 most prevalent types of organisms present at different stages in the 

 manufacture of butter. 



METHODS. 



f^ourcc of Cream. Cream was obtained from dairj'men in the vicinity 

 of the College. 



Taking fiamples. Samples (about 50 cc. each) of cream, pasteurized 

 cream, starter, ripened cream and buttermilk, were taken with sterile 

 pipettes and introduced into sterile 100 cc. Erlenmeyer flasks. Analyses 

 of these samples were made immediately after collection. Four sam- 

 ples of butter (about 100 grams each), one before washing and salting 

 and three when the butter was ready to tub, were taken from the churn 

 under aseptic conditions and placed in sterile deep culture dishes. The 

 sample taken before salting and one of the samples taken after were 

 analvzed at once, the other two were stored at 40° to 45° F. in a dark 

 room and examined after seven days and one month respectively. 



Bacterial Content and Isolation. In all steps except those concerning 

 butter, 1 cc. of the medium was added to 99 cc. of sterile physiological 

 salt solution. The mixture then was shaken and other dilutions made 

 by introducing 1 cc. of this into another 99 cc. of sterile salt solution. 

 Dilutions of 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000 were then plated 

 in litmus lactose agar and duplicate dilutions in casein agar. But- 

 ter was introduced into a small Erlenmeyer flask and placed in a water 

 bath at 35° C. until the butter had softened, then the flask was shaken 

 to insure uniformitv. One gram (1.15 cc.) was measured by means of 

 a sterile pipette and introduced into 99 cc. of warm (35° to 40° C.) 

 sterile salt solution. This was shaken to a milky emulsion and higher 

 dilutions were made as above. Samples were plated in litmus lactose 

 agar and in casein agar, the same dilutions were used as for cream. 

 All plates were incubated at 20° to 22° C. After bacterial counts were 

 recorded many of the organisms were isolated and transferred from the 

 agar plates to sterile nutrient bouillon. 



Litmus lactose agar used for plating was made according to the 

 rules adopted for nutrient agar by the Committee on Standard methods 

 (Jour. Inf. Dis. Suppl. No. 1, 190o) to which 1 percent lactose and 0.003 

 percent azolitmin was added. 



Casein agar Avas made up according to the formula given by S. H. 

 Ayers in the 28th Annual Eeport of the Bureau of Animal Industry. 



Peroxidase. The presence of peroxidase was determined by the gum 

 guaiac test. A tincture of guaiac was made by dissolving a little of the 

 powdered resin in alcohol. About 10 cc of the sample was placed in a 

 test tube shaken with a few drops of hydrogen peroxide, then two drops 

 of guaiac tincture were allowed to run down the sides of the tube coming 

 in contact with the sample but not being mixed with it. The appear- 

 ance of a blue ring within a few minutes is considered positive for 

 peroxidase. 



Reductase. The presence of reductase was determined by adding 1 



