70
seen. Only 2-5 x 10 cells ivere isolated from Hypaque-Ficoll isolated
preparations of whole spleen cell suspensions.
Small aggregates of lymphoid cells were present in glandular tis
sues found in the orbital sinus and the area of the cloaca. However
further histological studies are necessary before these tissue can be
defined as lymphoid equivalents of the Harder's Gland or Bursa found
in birds. In vitro studies of these tissues were not possible due to
the very few cells isolated by Hypaque-Ficoll gradient centrifugation.
No gut associated lymphoid tissue or lymph nodes were found.
Separation Technique
Hypaque-Ficoll (p = 1.077) was used to isolate relatively pure
lymphocyte preparations from heparinized whole blood or organ cell sus
pensions. White cell differential counts of fractionated and unfrac
tionated blood are presented in Table 12 and illustrate the efficiency
of the technique for isolating lymphocytes. Hypaque-Ficoll isolates
routinely contained only about 5% granulocytic cells (predominately
basophilic staining cells by May-Grunwald-Giemsa stain), and about 5%
%
red blood cells. Approximately 2-3% of the granulocytes were phagocytic
(assayed by collodial carbon uptake). Examination of the cells recov
ered from the interface and within the Hypaque-Ficoll gradient showed
> 99% of the lymphocytes were present at the interface. A photomicro
graph of a representative isolate is presented in Figure 7.
Culture Conditions
Various sera were tested to determine a suitable supplement with
MEM for in vitro studies. Ten percent alligator, human, calf, fetal
calf, and rabbit sera or all combinations of equimixtures (5% per serum)