872 
Journal of Agricultural Research 
Vo!. V, No. 19 
In studying the presence of Bact . abortus in milk it was found neces¬ 
sary to develop new technic in order to study a large number of samples. 
Knowing that this organism is sometimes present in considerable num¬ 
bers in milk as it comes from the cow's udder, it was thought that this 
might indicate an infection of the udder and a consequent local produc¬ 
tion of antibodies. With this in mind, agglutination and complement- 
fixation tests were made, using milk and milk serum, instead of the usual 
method of using blood serum. Bact, abortus was used as antigen. The 
object of this paper is to report upon this method. 
TECHNIC EMPLOYED 
Complement-fixation test. —The complement-fixation test as used 
by Surface 1 and others, was employed in this work. Rennet milk serum 
was used in the following quantities: o. 1, 0.04, 0.02, and 0.005 c. c. Milk 
was considered positive only when the tube containing 0.04 c. c. of 
serum was positive. Preliminary tests run upon samples of milk show 
that the agglutination and complement-fixation tests correspond closely. 
For this reason only the results of agglutination tests will be given in 
this paper. 
Agglutination test. —Antigen was prepared for the agglutination 
test by growing a culture of Bact. abortus upon ordinary agar for 48 hours. 
The growth was then washed off with a solution containing 0.9 per cent 
sodium chlorid and 0.5 per cent phenol. The suspension was then 
filtered through a coarse filter paper and standardized so that the tur¬ 
bidity compared with tube 1.5 of McFarland's nephelometer. 2 Four c. c. 
of this bacterial suspension are placed in each of the small test tubes 
used and the following quantities of milk added: 0.1, 0.05, 0.025, 0.01, 
and 0.005 c. c. In this way approximate dilutions of 1 to 50, 1 to 100, 
1 to 200, 1 to 500, and 1 to 1,000 were obtained. It was found that 
turbidity due to the whole milk added did not interfere with the reading 
of the reaction. When a dilution lower than 1 to 50 was made, rennet 
milk serum was used. 
For the experiment given in Table I, a cow was selected whose milk 
had given a negative agglutination reaction since first tested, October 
10, 1914, using Bact . abortus as antigen. Thirty-five c. c. of a 48-hour 
broth culture of Bact . abortus was introduced into the right rear quarter 
after it had been milked dry. As shown in the table, the agglutinins 
had appeared in the right rear quarter the following day and soon spread 
to the other quarters. This spreading was probably brought about by 
the organism being carried from quarter to quarter upon the hands during 
milking. After the cow freshened the reaction was seen to gradually 
die out. 
1 Surface, F. M. The diagnosis of infectious abortion in cattle. Xy. Agr. Exp. Sta. Bui. 166, p. 301-365, 
5 fig. 1912. 
2 McFarland, Joseph. The nephelometer. . . In Jour. Amer. Med. Assoc., v. 49.no. 14, p. 1176-1178, 
2 fig. 1907. 
