294
J. C. KAKAWAS
poured on and thoroughly mixed with the culture. A
sterile marble was placed in the centre of the agar.
When the agar hardened, the marble was removed,
and the cup formed by the marble was filled with
approximately 1 c.c. of 37.8 per cent homogenized
suspension of sulfanilamide in liquid petrolatum.
The preparation of the homogenized sulfanilamide
has been described in a previous publication. 1 The
Petri dishes were then placed in the incubator at
37. 5° C. and at intervals of twenty-four hours, sub-
cultures were made from the plates by cutting small
pieces of agar at approximately 10 mm. and 30 mm.
distance from the edge of the cup and inoculating
into tryptose broth. The subcultures were then
incubated at 37.5° C. and the presence or absence of
growth was recorded at the end of seventy-two
hours. The Petri dishes were incubated for 120
hours, and at the end of this period, the zone of
inhibition was measured from the edge of the cup.
TABLE 2
Effect of inoculum on the size of inhibition zone by
sulfanilamide on Streptococcus agalactiae (F-12a-4)
—cup-plate test
Hrs. incubation
before sub-
culturing
11 . . . .
26 . . . .
46 . ..
70 . . .
94 . . .
125 . . .
142 . . .
Inhibition Zone
No. bacteria per Petri dish
348
a
—
—
—
—
—
—
—
b
+
+
+
+
+
+
+
25 mm.
3480
a
—
—
—
—
—
—
—
b
+
+
+
+
+
+
+
16 mm.
34800
a
+
—
—
—
—
—
—
b
+
+
+
+
+
+
+
12 mm.
348000
a
+
+
+
+
+
+
+
b
+
+
+
+
+
+
+
8 mm.
*a=Results of subcultures made from cup-plate at 10 mm. from
edge of cup.
**b=Results of subcultures made from cup-plate at 30 mm. from
edge of cup.
+ = Viable organisms present.
— =No viable organisms.
The amount of sulfanilamide which diffused through
the agar was determined at the end of the 120-hour
period at two points ; namely, approximately 10 mm.
and 30 mm. from the edge of the cup. The sulfa-
nilamide determinations were made by cutting small
pieces of agar and placing them in test tubes which
were immersed in boiling water to melt the agar.
This was then diluted with distilled hot water
sufficiently high so that when the test was completed,
a reading could be made with the standard sulfanila-
mide tubes. The test for sulfanilamide was made
according to the method of Marshall and Litchfield,
The sulfanilamide concentration in the agar at the
end of the test period was, on the average, about
730 mg. per cent at 10 mm. distance and 400 mg.
per cent at 30 mm. distance.
TABLE 3
The inhibitory effect of sulfanilamide by streptococcus
agalactiae (F-12a-4) metabolites determined by the
cup-plate test
Petri dish
No.
1
2
3
4
5
6
Hours
incubated
before
adding
sulfanilamide
0
3
8
19
33
Control
Subculture
growth
before
adding
sulfanilamide
+
+
+
+
+
+
Subculture
growth
after
adding
sulfanilamide
—
—
+
+
+
+
Size of
inhibition
zone
15 mm.
8 mm.
0
0
0
0
Number Bacteria per plate = 1620.
The results of this study are summarized in
Table 1. It will be noted that sulfanilamide was
bactericidal against all the strains studied, as shown
by the inhibition zone around each cup. It should
be noted, too, however, that the size of the zones
varies with the different strains. The smallest
inhibition zone was that of strain 1B with a 6 mm.
radius, and the largest inhibition zone was that of
strain 090 R with a 25 mm. radius. It should be
further noted that the Petri dishes containing the
smaller inoculum developed larger zones of inhibition
compared to those with larger inoculum. This is
particularly well demonstrated in Table 2.
In an attempt to find an explanation for this
variation in inhibition as a result of the difference in
the size of the inhibition zones, the following cup-
plate experiment was conducted. A series of Petri
dishes were prepared in the same manner described
previously, using strain F-12a-4 as the test organism.
The plates were incubated at 37.5° C. and the sul-
fanilamide-oil preparation was added at different
intervals in the agar cup. In Table 3, the result of
this test shows that when the organisms had an
opportunity to grow for a period of approximately
eight hours before sulfanilamide was added in the
cup, no inhibition zone developed around the cup.
It would appear, therefore, that when the organisms
are allowed to multiply without the interference of
sulfanilamide during the early growth stages, bacterial
metabolites accumulate in the medium which nullify
the bactericidal effect of sulfanilamide.
The
difference in susceptibility of the various strains to
sulfanilamide may be explained on the basis of the
above findings ; namely, that group B streptococci
produce a sulfanilamide inhibiting substance or
substances and that some strains produce more active
or more abundant inhibiting metabolites than others.
P-aminobenzoic acid and sulfanilamide activity
for group B streptococci.—The problem of the nature