Dec. 15,1924 Differentiation of Bad. melitensis from Bad. abortus 589 
On February 14, when the remain¬ 
ing 15 guinea pigs, 14 of which had 
been inoculated with blood from the 
four goats, were destroyed, no lesions 
indicative of Bacterium melitensis infec¬ 
tion were detected. Negative aggluti¬ 
nation and negative cultural results for 
Bact. melitensis were obtained. 
Since the isolation of the Texas 
strains of Bacterium melitensis, the writer 
has endeavored to determine with 
what frequency Bact. abortus may be 
isolated from the spleens of infected 
guinea pigs, following their inocula¬ 
tions with Bact. abortus infected milk, 
when sown on serum-agar slants in 
cotton-plugged tubes and placed in an 
incubator kept moist by the introduc¬ 
tion of a receptacle containing water. 
Success has been attained only when 
the guinea pigs were inoculated with 
milk from cows which had been arti¬ 
ficially infected with Bact. abortus 
strains accustomed to propagation in 
a normal atmosphere, although such 
tests have numbered between 75 and 
100. Sealing the tubes after heating 
their upper portions caused isolation 
of the organism in some cases, but 
growth was slow as compared with 
the carbon-dioxid method. 
THE WORK OF SOME PREVIOUS 
INVESTIGATORS 
Bruce (2 ), the discoverer of Bac¬ 
terium melitensis , in announcing his 
discovery makes no mention of using 
special medium or of incubating his 
cultures in other than the usual man¬ 
ner. Agar-agar nutrient jelly is re¬ 
corded as having been used for medium. 
Some of his tubes sown with spleen 
tissue of a fatal human case were left 
at room temperature, and some sub¬ 
jected to 37° C. incubation. Under 
37° incubation, growth was visible in 68 
hours. At room temperature 168 hours 
elapsed before growth was observed. 
Horrocks (13) describes the isolation 
of Bacterium melitensis from the milk 
of a goat by spreading milk sediment 
on litmus-nutrose-agar plates. After 
four days incubation at 37° C. colonies 
of the organism appeared on all the 
plates sown. 
Shaw (23) describes investigational 
studies of 91 goats. The milk sediment 
of 30, which gave positive agglutination 
reactions, was sown on nutrose-litmus- 
agar plates. Bacterium melitensis was 
isolated from 7 of the number following 
incubation at 37° C. He further de¬ 
scribes serological and cultural work in 
connection with 33 cows on the island 
of Malta. Ten of the number gave 
agglutination reactions varying from 
1 to 30 to 1 to 1,000. From the milk 
sediment of 2 of this number Bact. 
melitensis is mentioned as having been 
repeatedly isolated, 3 plates yielding 
in some cases more than 200 Bact. 
melitensis colonies. The method de¬ 
scribed in the isolation of the organism 
consisted in centrifugalizatipn of the 
milk samples and the sowing of the 
sediment on nutrose-litmus-agar in 
Petri dishes. Examination of the cul¬ 
tures was made after five days’ incu¬ 
bation at 37°. 
Keefer (17), in referring to the re¬ 
sults of Shaw, suggested that Shaw 
was probably dealing with Bacterium 
abortus. The writer’s experience has 
been that in the isolation of the abor¬ 
tion organism from the milk of natur¬ 
ally infected cows considerable diffi¬ 
culty is encountered in obtaining the 
original cultures, even when seemingly 
ideal conditions were provided. These 
observations and the results reported 
by different workers in this connection 
lead to the assumption that Shaw’s 
conclusions may have been wholly 
correct, regardless of the fact that at 
the time the Malta fever investigations 
were made the close similarity in mor¬ 
phology, biochemical, and serological 
characteristics of the Malta fever and 
abortion organism was little suspected. 
Huddleson (15), who deserves credit 
for greatly simplifying the method of iso¬ 
lation of Bacterium abortus from infected 
tissues and fluids, and the discovery 
that CO 2 gas in combination with air in 
the proper proportion is the essential 
factor rather than the degree of oxy¬ 
gen tension, states that to simplify the 
isolation of the organism from milk the 
following factors must be considered: 
The medium and its proper preparation; the 
proper H-ion concentration of the medium; the 
employment of an agent which will eliminate fast¬ 
growing organisms and the method of incubation. 
Cooledge (3) in discussing the isola¬ 
tion of Bacterium abortus from milk of 
infected bovines calls attention to the 
defects of the Nowak method, in which 
cultures of Bacillus subtilis are grown 
in closed jars with those sown with 
suspected Bact. abortus infected ma¬ 
terials. While he mentions having 
succeeded in isolating Bact. abortus 
through the use of this method he 
refers to it as— 
too tedious a process for application to a considerable 
number of samples, requiring weeks to isolate and 
identify the cultures, 
a feature which prompted him to inves¬ 
tigate the value of the agglutination 
and complement-fixation tests in con¬ 
nection with milk whey as a means of 
detecting Bact. abortus udder-infected 
cows. 
