hr, at 0 minus 24 hr or at 0 plus 24 hr relative to the 
start of the 96-hr cadmium-exposure period. Second 
“booster’’ injections of SRBC were given 7 days after 
each initial injection. Serum was drawn from each 
fish 6 or 7 days after the second SRBC injection. 
Blood was removed from the ventral aorta using a 
Pasteur pipette equipped with a small, rubber finger 
bulb after nicking the artery with a scalpel blade. 
Blood was allowed to clot at room temperature and 
the clots to retract at 4°C. After removing red cells 
from the serum by centrifugation, sera were stored at 
—25°C until assayed. 
Hemagglutination Assay 
The microtiter system of the Cook Engineering Co., 
Alexandria, Va., was used in making dilutions and 
setting up hemagglutination assays. Serial twofold 
dilutions of fish serum were made in PBS, pH 7.2, 
containing normal rabbit serum (NRS) at 1/100 con- 
centration (the rabbit serum was preabsorbed with 
SRBC). Washed SRBC were suspended to 1% concen- 
tration (v/v) in the PBS-NRS diluent and added to 
each serum dilution (0.025-ml serum plus 0.025-ml 
SRBC suspension). Controls included normal nonim- 
mune fish serum, known positive serum from im- 
munized fish and PBS alone. After 2-hr incubation at 
room temperature and overnight at 4°C, the titers of 
each serum were read by examining the degree of 
SRBC agglutination based on a 0 to 4+ rating scale. 
The last dilution causing a 2+ agglutination of SRBC 
was taken as the titer. A 2+ rather than a 1+ end- 
point was used because it gave more reproducible 
results. Heat inactivation was not done because it 
created a gel in the serum. Hemolysis of red cells was 
not a problem because it occurred only after 48-hr in- 
cubation—a time long after final readings had been 
made. 
Growth and Injection of Bacteria 
Cells of Bacillus sp (biochemical tests consistent 
with Bacillus cereus) were grown well into stationary 
phase culture (72 hr) in Trypticase soy broth. The 
medium in the culture vessel was constantly agitated 
by an air-driven magnetic stirrer while the vessel was 
held in a water bath at 37°C. After incubation the 
culture consisted of about 70% single cells and 30% 
cells attached in pairs (with less than 0.1% spores) by 
phase-contrast microscopy. Cells were diluted in 
physiological saline, containing 0.1% peptone, and 
counted by the pour plate method in Trypticase soy 
agar. The following day the bacteria were washed 2X 
at 3°C with 0.15 M PBS, pH 7.2, and resuspended to 
about 5 X 10° cells/0.1 ml in PBS based on the counts 
of the previous day. 
Fish which had been held for 24 or 48 hr in running 
seawater after termination of 96-hr exposure to 0 ppm 
or 12 ppm Cd’* were injected intracardially on a 
vol/wt basis with the bacterial suspension. For exam- 
ple, a 40-g fish received 0.1 ml, a 60-g fish received 
0.15 ml, etc. Actual numbers of viable bacteria in- 
jected per 40-g fish varied from 5 X 10° to 5 X 108 
because of some cell loss and death during washing in 
the PBS diluent. Actual numbers were determined by 
bacterial counts on the suspension after injecting all 
the fish on a particular day. 
Measurement of Bacterial Clearance 
Intracardially injected fish were placed in 4-gal 
polyethylene pails containing seawater at ambient 
temperature (23°C). After 30 or 90 min, bacterial 
counts were made of the blood, liver, and spleen using 
the following procedures: Blood was removed from the 
ventral aorta with a premarked, heparinized Natelson 
blood collecting pipette; blood was drawn to the mark 
(0.125 ml) using mouth suction on the end of a short 
rubber tube with mouthpiece (after first wetting the 
heparin with a small amount of blood). Blood was 
diluted immediately into a tube of physiological 
saline containing 0.1% peptone. In order to minimize 
the amount of standing blood (containing bacteria) in 
the organs, aspiration of blood was continued until 
the fish was bled dry. The liver and spleen were 
removed, weighed to three places, and each diluted in 
an aqueous solution of 0.5% peptone. In many in- 
stances, blood from the pericardial space spilled onto 
the liver during removal of that organ. When this 
happened, the liver was washed with sterile, distilled 
water and blotted on sterile paper toweling prior to 
weighing. Each liver and spleen was ground in a 
sterile, motor driven tissue grinder with teflon pestle 
and further diluted serially in 0.1% peptone-saline 
diluent. Plate counts of the blood, liver, and spleen 
were made in Trypticase soy agar within 10 min of 
organ removal. Values were recorded as percent of the 
initial bacterial dose present in each organ. 
Calculations for total bacteria in the blood stream 
were based on a blood volume of 3% of the fish body 
weight (this was approximated from values given by 
Thorson, 1961). 
RESULTS 
Antibody Response to SRBC Injections 
In order to examine the possibility that cadmium 
could affect protein formation or cell division in newly 
produced, immunocompetent cells, fish were given 
priming doses of SRBC followed by a second antigen 
dose 7 days later. Production of antibody was mea- 
sured by ability of fish serum to agglutinate washed 
SRBC. Table 1 compares the reciprocal hemagglutina- 
tion titers of cadmium-treated fish and fish receiving 
no cadmium during the 96-hr holding period. Al- 
though antigen was injected into fish on the day be- 
fore, the day of, or the day after the start of cadmium 
