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TRYPANOSOMATIDAE
See also Taylor and Baker (1978)
Medium M22 (L4NCS)
g per litre
(i) Basal solution: g per litre
Proteose peptOne (Oxoid L46) 15.0 NaCI 8.0
Liver digest (Oxoid L27) 2.5 KCI 0.2
" Yeast extract (Oxoid L20) 5.0 CaC12 0.2
NaC1 5.0 KH2PO4 0.3
This basal solution can be replaced by nutrient Glucose 2.
broth No. 2 (Oxoid CM67), 25 g per litre.
(ii) Foetal bovine serum - heat inactivated
(56°C, 30 min).
(iii)10% erythrocyte lysate - prepared by lysing
1 ml of packed erythrocytes from defibrinated
rabbit blood (or horse blood, Oxoid SR50) in
9 ml distilled water.
Complete medium:
Basal solution (i)
Serum (ii)
Lysate (iii)
20 parts by vol.
1 part by vol.
2 parts by vol.
Sterilize by filtration (0.22 micrometre pore size)
and store in a deep freeze.
Cultures in this medium should be incubated at
25-28°C if possible.
Cultures of trypanosomatids are usually despatched
in medium M22.
Medium M23 4N nutrient agar-blood medium
(i) Add 40 g blood-agar base No. 2 (Oxoid) to
1 litre of distilled water, mix and dissolve
by steaming or autoclaving. Dispense 5 ml
aliquots while molten into screw-capped glass
bottles (30 ml capacity) and autoclave if
necessary.
(ii) When cooled to about 45°C, add aseptically to
each bottle 20 drops (ca 1 ml) of fresh
rabbit blood and allow to set in a slant at .
the base of the bottle. Alternatively, add
5.6
1 ml of sterile defibrinated horse blood
(Oxoid SR50) previously inactivated at 56°C
for 30 min.
(iii)Add aseptically to each bottle'l ml of
sterile Hanks's solution, or the following
modification of Locke's solution:
.. -
Antibiotics (penicillin, 200 iu and streptomycin,
2 micrograms/m1) can be included if desired.
Cultures in this medium should be incubated at
25-28°C if possible.
Smaller aliquots (eg 2 ml) can be used in test tubes
with air-tight caps; the volumes of blood and saline
added should be reduced proportionately. Store the
medium at 4°C (not frozen).