IV] LOUIS A. KLEIN, DAVID W. CRISMAN, AND JOHN W. MOOR 271
TABLE II
Results of examination of milk samples collected immediately before the first injection of penicillin
Cow
91
10
10
1
27
216
10
Quarter
LH
LF
RH
RH
RF
RF
RF
Staphylococci per c.c.
80
125
5400
470
175
510
5100
Pigmentation
Medium
Orange
Light
Light
Medium
Medium
Light
Hemolysis
Hemolytic
Hemolytic
Hemolytic
Nonhemolytic
Hemolytic
Hemolytic
Hemolytic
Coagulation
—
+
+
+
+
—
+
Leucocytes
per c.c.
1,200,000
60,000
900,000
1,200,000
120,000
90,000
300,000
Plating. Blood-agar plate were prepared by adding 5.0 per cent of defibrinated cow's blood to
melted standard nutrient agar containing 0.5 per cent of sodium chloride and inoculating two plates
from each milk samples, one with 1.0 c.c. and one with 0.1 c.c. After 48 hours incubation
at 37° C., the plates were examined for hemolytic colonies and for nonhemolytic colonies showing
pigmentation. Two plates of standard nutrient agar were inoculated with the same quantities of
each sample and examined after 48 hours incubation, since pigmentation could be more
readily detected on this medium. Hemolytic colonies of staphylococci and those showing any degree
of pigmentation were picked off and streaked on standard nutrient agar. A film was also prepared
from one of these colonies and gram-stained for microscopic examination. After the ox-blood agar
and standard nutrient-agar plates were prepared, each milk sample was incubated for 24
hours and then streaked on ox-blood agar. These plates were examined after 48 hours
incubation at 37 C.
Coagulase test. The technique used by Chapman, Berens, Peters, and Curcio, 9 as modified by
Plastridge and associates, 8 was used to determine ability to coagulate human-blood plasma. (Rabbit-
blood plasma is equally satisfactory.) A 4 m.m. loopful of a 24-hour-old culture on nutrient agar
was mixed with 0.5 c.c. of fresh plasma contained in a ⅜ in. by 3 in. test tube, held at room tempera
ture, and examined after one, two, three, and twenty-four hours, for evidence of clot formation.
Evidence of clotting during the period of observation is a positive result. The human-blood plasma
was obtained by drawing 75 c.c. of blood in a flask containing 7.5 c.c. of a 10 per cent solution of
sodium citrate, and centrifuging to separate the plasma from the blood cells. The plasma should
be used within forty-eight hours.
Leucocyte Count. The slides for the leucocyte count were prepared and the count determined
as specified for the direct microscopic method of counting bacteria in milk in Standard Methods for
the Examination of Dairy Products, 8th ed ition, pp. 44 to 54.
On the information obtained by the methods of examination described previously, seven quarters
were selected for treatment with penicillin—three in 1 cow (10) and one each in 4 others (91, 1, 27,
and 216). None of the cows showed any clinical symptoms of mastitis. According to the history
obtainable, all had previously had attacks of catarrhal mastitis due to streptococci but none recently.
On palpation of the selected quarters, indurated area were found in four, viz. to a 2 in. by 4 in. area in
the left front quarter of cow 10 and a similar area in the right hind quarter; a nodular area 2½ in. in
diameter in the right hind quarter of cow, 1 with thickening of a part of the mucous membrane of
the milk cistern, and an area 2½ in. in diameter in the right front quarter of cow 27.
Schedule of dosage and sampling
Penicillin was used in the form of the sodium salt. This material) at the time of its preparation,
assayed 320 Oxford units per milligram. It was contained in sealed glass vials, 120 mg in each vial,
and the vials were closed with rubber stoppers. Difficulty was encountered in making other
Q 2