in vitro culture and isoenzyme analysis of giardia lamblia

negative. Metronidazole, 20~g/ml (Flagyl; Rhone-Poulenc, Montreal) was
administered orally with a blunted feeding needle for three consecutive days to all
the mice. The animals were then mated.
4.2.2.2 Determination of a Reliable Infective Dose of Human Giardia Cysts in
C57BU6 Suckling mice.
Pelleted cysts (prepared as previously described in 2.2.3.3) were diluted to contain
different concentrations varying from 100, 1000 to 10 000 cysts/m I in 0,1 ml
distilled water. Three different litters of C57BLl6 mice (each consisting of 5, 6 and
9 three-day old neonatal mice born of mothers who were pre-treated with
Metronidazole) were intragastrically inoculated with the doses of cysts as follows:
Five neonatal mice inoculated with 1 OD-cysts/ ml; nine neonatal mice inoculated
with 1000 cysts/ml; and a dose of 10000 cysts/m I injected onto the stomachs of
six suckling mice. The mice were sacrificed ten days post inoculation (pi). The
entire small intestines were finely chopped with a fine pair of scissors in 1 ml of
cold TYI-S-33 in Petri dishes. A 0,01 ml aliquot was transferred to a glass slide
and a 20 x 40mm coverslip was applied. The area of the slide covered by the
coverslip was scanned for the presence of trophozoites by light microscopy. The
rest of the chopped intestines were transferred to an 8ml Kimax glass tube
containing 7ml TYI-S-33 medium and maintained as described in 4.2.3 below.
4.2.2.3 Determination of Peak Trophozoite Growth.
The optimum period (days after inoculation) during which maximum intestinal
trophozoite numbers are obtained had to be determined before the in vivo
experiments were initiated.
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