J. F. SHIRLAW AND P. R.
KRISHNA
IYER
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negative pressure of 50 mm. mercury. Filtration was slow, taking 90 minutes for the passage of
250 c.c. fluid. Three cattle were subcutaneously inoculated with 5, 10 and 15 c.c. filtrate respectively
and no reaction was observed over a period of 25 days. The test was repeated in two cattle,
each being given subcutaneously 225 c.c. Berkefeld N filtrate of a different sample of fresh lymph
diluted 1/50 saline; the result was again negative. All five animals were later tested for immunity,
the first batch 25 days and the second batch 110 days after inoculation. The three animals tested
at the 25th day reacted to a test dose of 2 c.c. fresh lymph virus given subcutaneously and died,
whereas the two animals similarly tested at the 110th day showed a strong immunity, both controls
to the test reacting fatally. Later work showed that the organism cannot be isolated in culture
from Berkefeld N filtrates of lymph virus and the presumptive evidence from this small-scale ex-
periment is; (a) some degree of immunity is obtained in susceptible cattle following the subcutaneous
inoculation of lymph virus filtrates proved free of the causal organism; the immunity so conferred
being possibly antitoxic; (b) immunity is of slow development, a fact which has been commented
on by several workers while discussing immunity in general. Further filtration experiments showed
that the organism in lymph virus passed and L3 and occasionally an L5 but not an L7 filter, whilst
results with a Seitz E. K. filter were erratic and seemed to depend on the degree of positive pressure.
The Assam 1 virus was used in these experiments.
(B) Culture
We have consistently failed to isolate the organism in culture from filtrates of lymph virus stored
for more than a week at 5°C. The Assam organism is much more exacting in its cultural require-
ments than the Australian 264 strain, which has been studied in equal detail. The most direct
method of obtaining primary culture of the Assam organism is to incubate at 37°C. a few c.c. Berkefeld
V filtrate of fresh lymph virus. In 2-3 days the clear filtrate has acquired a faint opalescence and
a few drops of this material inoculated in Bennett's broth yields visible growth within 48 hours.
This procedure is recommended for primary isolation of the organism from filtrates as cultures from
direct filtrates of lymph virus are feeble in comparison, more slow growing, and sometimes fail.
Further, the growth phases of the organism are better studied by dark-ground illumination in in-
cubated filtrates than in culture media rich in serum; the manner in which artefacts liable to be
confused with these growth phases tend to appear in serum media has been commented on by many
workers [Titze and Seelemann, 1921; Freiberger, 1912; Merling-Eisenberg, 1935; and others].
The Assam organism will grow feebly in Bennett's broth with 1 per cent added horse serum when
it has adapted itself to a medium with 10 per cent added serum. Cultures cannot, however, be
passaged or maintained in 1 per cent serum medium, nor will growth occur in this medium if the seed
material is a Berkefeld V filtrate of lymph virus. At the other range, the Assam organism will not
grow in Bennett's broth with 20 per cent added serum. It seems that a 10 per cent serum concentra-
tion is optimal. Maximum growth occurs at pH. 7.4-7.8; a pH below 7.2 or above 8 will not permit
growth of the organism. The inoculation of three drops of Berkefeld V incubated filtrate of Assam
lymph virus in Bennett's broth results in visible growth within three days, this showing as a faint
opalescence which is best seen in contrast with incubated controls. The opalescence daily increases
and growth seems maximal in ten to fourteen days. On gently shaking the culture, a delicate sedi-
ment of growth ascends as a spiral veil. This cultural character is noteworthy. Cultures older
than three weeks undergo some degree of clearing and the sediment becomes decidedly granular.
Surface growth on Bennett's serum agar seems conditioned to no little extent by the hygroscopic
qualities of the medium, and the Assam organism is exacting in this respect. The correct texture
of the medium can only be judged by experience. Dewdrop-like, glistening, smooth colonies are
first visible on the third day after inoculation and incubation at 37.5 C. The colony gradually en-
larges to about ½ mm. and buds off daughter colonies (Plate IX, fig. 3). Viewed at a magnification
of x 40, the umbonate centre is granular and somewhat opaque, and the outer part of the colony
transparent (Plate IX, fig. 4).The colony is adherent to the medium in about a week. This phase
does not seem, as stated by some workers, confined to older cultures and is not conditioned by gradual
desiccation of the medium. Following heavy inoculation with filtrate an irregular film of metallic
sheep slowly develops, a cultural character which we have not seen previously described, and which