18
exposure of the blood to a high temperature for a longer time than is absolutely essential to
ensure mixing.
(b) In this laboratory sodium citrate was not added to the medium. The addition of
citrate was found to interfere with the growth of the parasite and had an inhibitive effect in
the proportion in which it was used.
A small quantity of sodium citrate in solution has, however, to be used in drawing the
blood, to prevent it from clotting.
(2) The presence in the culture tubes of an adequate quantity of condensation fluid, is
an essential factor in the successful cultivation of these flagellates. It was often found
necessary to ensure a sufficient quantity of this fluid, by having recourse to the simple means
of decanting out the contents of two or more tubes into one, with strict precautions against
contamination.
(3) The growth of these parasites takes place only when a constant temperature of
22°C. is successfully maintained, even a slight rise above this point affecting the growth
adversely. Difficulties were experienced in maintaining a constant temperature with the
Hearson's cool incubator that was used here. The temperature was fluctuating as a result of
(a) the water used in the feeding tanks becoming warm and passing in that condition
through the ice jacket and (b) saw dust getting in and blocking the pores of the ice tank.
These two difficulties were overcome (1) by storing water for the feeding tanks for about 1 2
hours previously in the cool room of the Refrigerator and (2) by providing a cylindrical funnel
with wire gauze screen to the ice tank of the cold incubator.
(4) The next point relates to the technique of cultivation. Peripheral blood contains
the parasite in such small numbers that in attempting to grow them on artificial media with a
small amount of condensation fluid, great care has to be taken in inoculating just enough
blood into the tubes which would on the one hand contain at least one parasite and on the
the other would not unduly dilute the condensation fluid. From some experiments conducted
with suitable controls, it would appear that the centrifugalising of about a c.c. of blood
would help to get over this difficulty, inasmuch as the deposit can be relied upon to ensure
a satisfactory culture and avoid errors on the side of too much or too little of blood.
(5) Once the parasite is obtained in culture on N.N.N. medium, it is comparatively
easy to carry it on through several generations ; and during these transfers slight variations
in the technique, composition of the media and temperature do not seem to exert any
deleterious effect on its vitality. They do not however grow at all on any other media.
(6) As the N.N.N. culture tubes inoculated with the patients' blood have to be
incubated for a fairly long time, and subjected to periodical examinations it becomes
necessary to guard against their contamination with spores and other organisms and against
evaporation of the condensation fluid so essential for their growth.
This, in practice, is best achieved by soaking the cotton wool plugs in hot melted paraffin
before plugging the tubes and by providing rubber caps on top of the cotton wool plugs.
Judging from the results of the examination of this limited number of cases, although
it might be hazardous to definitely contradict the findings of other workers on peripheral
blood cultivations, it can be stated that this method cannot replace the liver and spleen
puncture methods which are of more certain diagnostic value.
If, however, the factors governing the egress of the parasites into the peripheral blood
at stated times or after certain provocative treatment, are definitely established, then the
peripheral blood cultivations will mark a definite step in the direction of advance in our
knowledge of Kala Azar.