25
250. One attempt to reduce the high counts of control unstimulated
cultures obtained with some of the supplement pools was to dialyze
the plasma pools against 0.15MNaCl. The data obtained with four
bream plasma pools which elicited high background levels prior to
dialysis are presented in Table 2. In three of the four pools tested
in this experiment the control CPM dropped significantly (p < 0.05)
in the cultures incubated at 22C and thus resulted in increased stimu
lation indices with LPS. With three of four pools used with cells
maintained at 32C the background remained unchanged. In the other
case the background dropped as a result of dialysis and hence the
stimulation index obtained using PHA increased. In conclusion it can
be stated that dialysis of bream plasma did not significantly decrease
the responses in any cultures and in fact in some cases enhanced the
response. Thereafter all bream plasma were dialyzed before use as
culture medium supplements for mitogenic assays.
Dialysis of certain heterologous supplements that elicited high
control CPM was also tried. Dialyzed bass plasma was supportive as a
supplement in mitogenic assays in the sense that significant stimulation
indicies were obtained. However, these indices were never > 10 and
therefore bass plasma was not used routinely. Dialysis of human, calf,
fetal calf, and alligator sera did not improve the situation with re
spect to high levels of background counts.
To summarize the culture conditions discussed thus far, RPMI 1640
supplemented with 7% dialyzed bream plasma was found to be supportive
for iii vitro mitogenic stimulation.
During the course of several experiments involving different fish
it was observed that there were differences both in the types of Hypaque-