12-mm discs of Whatman no. 1 filter paper which were
then dried at 37 C for 30 min. The quantity of antibiotic
on each disc was that required to give the desired
agar concentration, assuming complete distribution
throughout the agar after the period of diffusion. Experimental
work had shown that diffusion of the antibiotic
from the paper into the agar was almost complete,
and, since the volume of agar in the wells was
known, the appropriate quantity of antibiotic on the
disc could readily be calculated. After the period of
diffusion, the discs of paper were removed and the agar
surface was inoculated by use of a swab.
In the serial dilution test, the minimal inhibitory
concentrations were determined by adding 2-ml volumes
of the appropriate concentrations of antibiotic
to 18-ml volumes of cooled molten agar which were
then poured into petri dishes. The plates were dried at
37 C and inoculated with the test organisms by use of
an automatic replicator delivering an inoculum of approximately
0.003 ml. In both the serial dilution test
and the diffusion test, Blood Agar Base (Oxoid) was
used, except in the case of sulfamethoxazole, for which
D.S.T. agar (Oxoid) containing 5% lysed horse blood
was employed. In both tests, overnight nutrient broth
cultures of the test organisms were used as inoculum,
diluted as indicated in Table 2.