158
White's Method for Collection of Infective Larvae
culture are so little and within the provisions of
any veterinary institution, that it can be adopted
as a routine for the gathering of infective larvae,
for the specific diagnosis of verminous infesta-
tions. In the main, it consists in replacing the
crystallizing dish and the watch-glass cover with
two petridishes. The materials required are two
petridishes of the same diameter (about 5 inches)
and a petridish of half that diameter with a cir-
cular blotting paper to cover the bottom and
distilled water or water boiled and cooled to avoid
contamination with the larvae of free-living forms.
These form a set sufficient for each culture.
The blotting paper is moistened with a little
water. Material for culture preferably fresh
faeces, prepared according to the method described
later, is loosely spread on the blotting paper to
fill half to three-quarters of the petridish. The
petridish is then transferred to the centre of one
of the larger petridishes, so that a trench of
uniform width forms between the two. Water
is run into the trench to a height quarter inch
below the brim of the petridish. A belt of water
is thus formed to trap any larvae migrating from
the culture. The third petridish is placed
inverted over the culture, the opposing brims of
the two large petridishes being adjusted to flush
against each other. The apparatus after labelling,
is placed for incubation, preferably, where a high
humidity may be maintained.
The cover serves to prevent undue evaporation
of the culture ; the water belt delays it still more.
It is unnecessary during ordinary weather condi-
tions to attend to the moisture content of the
culture under such arrangement, as the water
evaporating from the trench condenses on the
inside of the cover and, so keeps the culture moist ;
in addition it creates and maintains throughout a
high humidity within which is so essential for
migration. However, in very dry weather, when
the surrounding atmospheric conditions maintain
only a low humidity, sprinkling of the culture
with a little water everyday is desirable.
Under optimum conditions, the infective larvae
of Strongyloides papillosus migrate into the water
by the end of the second day after culture while
all the rest of the infective larvae do so from the
fourth to the seventh day.
After use, the apparatus with the culture, may
then be dismantled and steamed.
THE CULTURE—SOME ESSENTIAL REQUISITES
(i) Consistency
The method described, in this paper, depends,
for successful results, upon the preparation of a
culture of a correct consistency.
Nematode larvae that migrate upwards from
cultures do so only under certain conditions of
temperature, moisture and consistency of the
culture medium. Variations within limits of the
temperature and moisture content, permit
development within the egg and through the
free-living larval stages; but changes from the
optimum consistency of the culture medium
cause failure of the migratory tendencies of the
infective larvae. In preparing the culture, watery
or even semi-solid consistency should be avoided,
as the surface tension then prevents the larvae
from leaving the medium. A culture prepared
according to the instructions given by Monnig
[1930] to resemble 'in consistence and moisture
content, that of normal, fresh sheep pellets
broken down into a loose mass' is the ideal.
While maximum conditions for migration are
then ensured the percentage of infective larvae
developing out of the culture is also raised to a
maximum.
Faeces passed by verminous animals may not
always be fit for immediate culture. When very
hard, it will have to be broken up and a little water
sprinkled. Soft or watery evacuations have to
be adjusted to' the proper consistency by the
addition of the necessary amount of sterilized
earth, fairly dry sterilized sheep dung or a good
grade of animal charcoal.
The author has obtained very satisfactory
results with sterilized sheep faeces used to moderate
horse and cattle dung and faeces of the cat, dog,
man and the pig; but with sterilized animal
charcoal the infective larvae not infrequently
gather in clumps clothed in. carbon, preventing
thereby a clean collection.
No sterilized earth was tried to moderate the
faeces.
(ii) Importance of the petridish cover
The importance of the use of the petridish
cover cannot be overstressed whether the culture
be incubated at any definite temperature or set
on the laboratory bench.
Migration of infective larvae is possible only in
the presence of moisture on the wall of the cul-
ture dish. On a cold, humid morning, in the
absence of a petridish cover over the culture, the
dew falling may provide the requisite moisture to
facilitate migration of the infective larvae over
the wall of the culture dish; but there is the danger
of the culture being actually flooded and so prevent
migration. Similarly the use of the cover is a
necessity to maintain the correct humidity in
hotter days; otherwise the fall and rise of tem-
perature during the night and day, together with,
probably, the warmth and/or physical changes
produced in the culture itself, will affect adversely
the optimum conditions.
The author's experience of this culture method
is limited to observations made in Madras, on
bovine faecal cultures, raised on the laboratory