60 
Transactions of the Royal Society of South Africa, 
amount of brilliant green in the presence of which the haemolytic effect is 
produced with the minimum of serum. 
Excess of the dye, therefore, may inhibit the action of the serum and 
necessitate larger amounts of serum being used to produce lysis. In fact, 
treatment of the serum ]:>y brilliant green before the addition of the 
corpuscles may abolish the haemolytic action altogether, as shown in Table V. 
In carrying out these experiments, it was found that smaller amounts of 
serum were capable of producing haemolysis if the corpuscles were treated 
with the brilliant green for about a quarter of an hour before the addition of 
serum. Thus a certain length of time was required for the absorption of the 
dye by the corpuscles and the consequent physical or chemical alterations 
which rendered them susceptible. If the suspension which had been treated 
with brilliant green was centrifugalised, it was found that the bulk of the 
added dye was present in the supernatant fluid, and that when this was 
pipetted off and the corpuscular sediment washed with salt solution several 
times (till the supernatant fluid showed no tinting), and then re-suspended, 
the cells were still susceptible to the action of serum, and smaller amounts 
of serum were required to produce lysis than in the case of the original 
suspension. A treated suspension prepared in this way showed practically 
no green tinting. This suspension was found to be quite stable, and did 
not undergo spontaneous lysis ; on the addition of serum, however, almost 
instantaneous lysis occurred. 
Table YI shows the results of an experiment in which corpuscles were 
treated in this way : 1 c.c. of a 0*] per cent, solution of brilliant green was 
added to 10 c.c. 5 percent, suspension of washed ox's corpuscles (i. e. 0*05 c.c. 
to 0*5 c.c. suspension). The suspension was allowed to stand at room 
temperature for a quarter of an hour, and was then centrifugalised ; the 
supernatant fluid was decanted and the sediment was washed several times 
with 0'85 per cent. NaCl till the washing fluid showed no tinting. The 
sediment after the last washing was suspended in 10 c.c. 0*85 per cent. NaCL, 
and showed practically no green coloration. The addition to 0-5 c.c. of this 
suspension of 0'025 c.c. of rabbit's serum produced immediate lysis. A 
parallel test was made with corpuscles to which brilliant green had been 
added (0*05 c.c. 0"1 per cent, solution to 0*5 c.c. suspension), as in the original 
experiments, and after the suspension had stood at room temperature for a 
quarter of an hour, varying amounts of serum were added to 0'5 c.c. of the 
suspension. Lysis only occurred in this case with larger amounts of serum. 
As a further proof of the inhibiting effect of excess of brilliant green on 
the haemolytic action of the serum, an experiment was carried out in which 
a fixed amount of heated rabbit's serum was mixed with varying quantities 
of brilliant green and then red blood-corpuscles treated with brilliant green 
were added. The result showed that serum in the presence of excess of 
brilliant green was incapable of exerting its haemolytic action (Table YII). 
