Antibacterial Properties of Plants — Bushnell, et ah 
169 
was about to be performed, 0.5 ml. of the 
broth culture of the test organism was inocu- 
lated into 100 ml. of melted nutrient agar 
cooled to 41° C. This heavily seeded agar 
medium was then poured into sterile petri 
dishes, about 10 ml. to a dish, and the me- 
dium was allowed to solidify at room tem- 
perature. After the medium had solidified, 
one or two sterile porcelain penicups were 
placed upon the surface of the agar. 
The plant extract being tested was then 
placed in the appropriate penicup, 0.2 ml. of 
extract in each cylinder. At first extracts 
were tested in duplicate until, with time and 
the perfection of our techniques, we found 
that duplicate platings were unnecessary. The 
plates were incubated in the upright position 
at 37.5 ° C. for 24 hours, at which time they 
were examined for the degree of inhibition 
achieved by the plant extracts as they diffused 
into the medium from the bases of the peni- 
cups. In most of the instances in which inhi- 
bition was achieved, it was denoted by a clear 
halo-like zone in the medium around the 
penicup, with the heavy growth of uninhib- 
ited bacteria making the medium opaque 
around the periphery of the zone of inhibi- 
tion. A few of the plant extracts produced 
a considerable discoloration or opacity in the 
medium around the penicups, but in only a 
very few instances did this discoloration inter- 
fere with determinations of the extracts’ ef- 
fects upon the test organisms. 
The degree of antibacterial effect was re- 
flected directly, of course, in the size of the 
zone of inhibition: the greater the zone, the 
more potent the extract. The diameter of the 
zone could be measured quite easily in most 
instances and was recorded, in millimeters, 
for each extract tested. 
Several other techniques for determining 
the efficacy of the plant extracts were also 
tried — such as using filter paper discs of vary- 
ing sizes soaked in the plant extract before 
they were applied to the inoculated agar, or 
actually incorporating 1 ml. of the plant ex- 
tract or of varying dilutions of it into the 
inoculated medium before it had solidified — 
but the penicup method seemed to be the 
one which gave the most consistent and the 
most clear-cut results, and it was adopted for 
continued use. 
Just before each extract was tested for its 
effect upon the bacteria, its pH was deter- 
mined by means of a Macbeth line-operated 
pH meter. A series of tests was also per- 
formed to determine the inhibitory effect of 
H-ions and OH-ions in buffer solutions of 
assorted pH in order to ascertain whether or 
not the inhibitory effects of the different plant 
extracts might be merely a reflection of their 
pH values. The buffer solutions were pre- 
pared from tablets each of which, upon being 
dissolved in 100 ml. of distilled water, gave 
a solution of specified pH value. 2 
Information concerning the plants reported 
in this paper is presented in Table 1, which 
shows ( 1 ) the major plant group and the 
family to which each plant belongs; (2) the 
scientific name of the plant; (3) the com- 
mon names of the plant, in both English and 
Hawaiian; and (4) epitomes of the usual 
purposes for which the plant was employed, 
according to the references consulted in pre- 
paring the list (the specific references are 
cited in the table). 
We can make no claim to being taxono- 
mists and have based our presentation of the 
systematic relationships of these plants upon 
the manner in which Neal has set them forth 
in her recent book In Gardens of Hawaii 
(1948). In her introduction to th : s compen- 
dium, Miss Neal states that she has followed 
the system of Engler and Prantl for the flow- 
ering plants, and the arrangement of A. J. 
Eames for the ferns and fern allies. 
The single specimen of alga collected, 
Gracilaria furcellata, was identified by Dr. 
Marion L. Lehman, associate professor of bo- 
tany at the University of Hawaii. Most of the 
2 The tablets are produced by tie Coleman Elec- 
tric Company, Maywood, 111. 
