PROGRESS IN PROTOZOOLOGY

2.32 I. CUNNINGHAM
growth of the organisms comparable to that obtained in more complex
media containing serum.
The complexity of the blood-enriched and semi-defined media had
prevented studies on the nutritional requirements of the in vitro grown
trypanosomes. This was overcome by the development of the defined
medium HX-25 of Cross and Manning (1973) originally designed
for the cultivation of procyclic forms of T. brucei. When T. cruzi was
cultivated at 28°C in HX-25, the population increased about 10-fold
in 10 days, but inocula somewhat larger than those used for T. brucei
were necessary for good growth. Preliminary data on amino acid utilization
indicated threonine uptake and glycine excretion (Cross et al.
1975). Medium HX-25 was also successful in the hands of An d e r s o n and
Krassner (1975) who were able to adapt T. cruzi orginally grown in
a beef heart infusion (BHI) to HX-25 in cultures incubated at 25-26°C.
The omission from medium HX-25 of HEPES; KH2P04; NaHC03; Coenzyme
Q6 and Ql0 and linoleic acid, and the addition of beta-glycerophosphate
Na salt; Na stearate; KC1 and Na2P04 • 12HaO was reported by
Azevedo and Roitman (1977) to improve the cultures which
attained yields up to 1.6 X 10 7 organisms/ml incubated at 28°C. The defined
medium of Avila et al. (1979) contained bovine liver catalase,
horseradish peroxidase, lactoperoxidase and bovine haemoglobin maintained
continuous propagation of cultures grown at 27°C. About 95°/o of
the populations consisted of epimastigotes.
The forms which occur in the mammalian host can be grown in
association with various cell cultures derived from mammals or man.
Of all the cell culture systems tested, human heart cells were considered
to be best at supporting good growth of intracellular amastigotes
(Brener 1973).
Using two liquid media (F-29 and F-32) Pan (1971) developed
a system of cultivation of axenic T. cruzi amastigote forms in the absence
of host cells. Medium F-29 consisted of TC 199, trypticase, haemin
and foetal bovine serum. Medium F-32 was F-29 enriched with chicken
plasma. The inocula for serial cultivation in both media contained over
99% epimastigote forms. The proportion of the structural types remained
the same during the first 10 passages at 29°C. However, at second
passages in medium F-29 at 35.5°C, most of the organisms became amastigotes,
and after several more subcultures, they comprised 92% of the
population. During the original passage in medium F-32 at 35.5°C over
90% of the parasites were trypomastigotes with a terminal kinetoplast.
After further passages, and regardless of incubation temperatures
(29.5-35.5°C), over 94% became amastigotes. In both F-29 and F-32
media a seven-fold increase of amastigotes could be achieved after
4 days at 35.5°C.
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