effect of infection of the filarial parasite brugia malayi - Pondicherry ...

CHAPTER 4.2
CHANGES IN THE ACTMTY OF MEMBRANE BOUND ENZYMES AND
HAEMATOLOGICAL PARAMETERS IN MASTOMYS NATALENSIS INFECTED
4.2.1 MATERIALS AND METHODS
WITH BRUGlA MALAY/
M. natalensis of the same age group (6-8 weeks) and weight (35-40 grn) were
divided into two groups, one served as the control while the other was infected with B.
rnalayi infective larvae as described earlier. At 0, 30, 60, 90 and 120 days of post-
inoculation, brain homogenate of the animals were prepared as described earlier and used for
the determination of the activity of different enzymes. Blood samples were collected at 0,30,
60, 90 & 120 days post infection and used for haematological and biochemical
investigations.
The erythrocyte membrane was isolated according to the procedure of Dodge et al.
(1%3). Blood plasma was removed and the remaining packed cells were washed three times
with isotonic phosphate buffer, pH 7.4. The washed RBC suspension was haemolysed with
hypotonic buffer (20 milliosmolar, pH 7.2) in the ratio of buffer: cells of 14:l. The ghost
cells were sedimented in a high speed refi-igerated centrifuge at 20,Wg for 40 minutes. The
supernatant was decanted carefully and the ghost button was resuspended by swirling.
Sufficient buffer of the same strength was added to reconstitute the original volume. The
mtio of cells to washing solution was approximately 1:3 by volume. The ghosts were washed
three times subsequent to haemolysis. The supernatant after the last wash was either pale
pink or colourless. The pellet of erythrocyte membrane was suspended in 10 ml of 0.32 M
sucrose solution and hornogenised. Aliquots of this homogenate were used for enzyme
assays and estimation of protein.