had been removed. Complete lysis on each occasion was again given by 15 per
cent of the serum so no loss of complement was demonstrable. The serum was
not tinged with hæmoglobin as a result of the intravenous injection. No anaphy-
lactic symptoms were observed after the second intravenous inoculation.
Rabbi t 101 r ecei ved 1 cc washed goat M RBCs subcut aneousl y dai l y f or 6 days.
Forty-five days later 6 . 5 per cent of its serum in a total of 2 cc fluid lysed o .o5 cc goat
M RBCs completely. It then received 6 cc washed goat M RBCs intravenously.
Blood drawn 20 and 40 minutes after the injection contained hæmoglobin, clotted
very slowly and very little serum separated from the clot. While 6 . 5 per cent. gave
complete lysis of goat M RBCs before the injection, 50 per cent. or 7 times the
quantity gave no lysis either 20 or 40 minutes after.
This rabbit contained probably about 130 cc blood or, say, 65 cc serum. If
0 . 13 cc before injection lysed 0.05 RBCs, 15 . 6 cc would be required to lyse the 6 cc
injected, leaving about 50 cc unaffected. Theoretically, the lytic power would have
been reduced by less than by the injection, and, instead of lysis of . 0 .05 RBCs
being accomplished by 0.I 3 cc, 0. I 7 cc would have been needed. Actually, however,
1 .0 cc or nearly six times that quantity gave no appreciable lysis. The injection,
therefore, put out of action far more lysin than was really needed for the complete
solution of all the RBCs it contained.
This intra vitam experiment has its counterpart in vitro in the experiment de-
tailed on p. 16 in which. 1 cc immune serum dissolved o . 6 c c R B C s i n 1 7 m i n u t e s ,
but, when the RBCs were added in successive lots of 0 . 2 cc each, lysis was not
complete in 3 hours.
The retardation of the clotting of the rabbit's blood may have been due to the
partial removal of some essential element such as calcium from the plasma by the
lytic reaction, or to leucopenia.
Chart No. 10 shows the lysin content of the rabbit's serum during the period
of its immunisation, and Chart No. 12 that of sheep R.
Sheep R received daily for 6 days 7 cc washed goat M RBCs subcutaneously.
Forty-five days later its serum gave partial lysis with goat M RBCs (1.95 cc serum
and o .o5 cc RBCs). It then received 38 cc washed goat M RBCs intravenously,.
and blood was drawn 10, 40 and 9.0 minutes later. As in the case of the rabbit
there was haemoglobinaemia, clotting was very slow and very little serum separated.
There was probably not enough. blood in the sheep to lyse completely all the
goat RBCs injected, judging by the amount of lysis given by the sample taken
immediately before inoculation, though, had the injection been given 8 days after
the last subcutaneous inoculation, there would have been ample lysin present to
have dealt with the whole inoculum. The lytic power of sheep R's serum deterio-
rated for some unknown reason very rapidly after reaching its maximum on the 8th
day, and on the 45th day was less than 1/10 its previous maximum power.
The intravenous injection rapidly removed all the available lysin from sheep.
R serum, so that 10 minutes afterwards no lysis of goat M RBCs could be accom-
plished by it, even with. the aid of added complement. The sheep passed hæmo-
globin in its urine 12 hours after the injection, but grazed and showed no sign of
discomfort 4 hours later. During the night, however, it died. The autopsy showed
the spleen to be soft, but not enlarged ; the kidneys hyperæmic; the lungs œdematous
and the pleurae full of blood-stained fluid, which had no lytic action on goat M RBCs
even with. the aid of added complement. Differential counts were made of the
leucocytes in the blood of the sheep before, 10, 40 and 90 minutes after the intra-
venous injection. The results are shown in Chart No. 11. The marked fall in the
percentage of polymorphonuclears immediately following the injection and the
succeeding rise have a remarkable resemblance to the counts described by
Andrewes in his Croonian Lectures (Lancet, July 1910) after the intravenous
injections of bacterial suspensions into previously immunised animals. The total
number of leucocytes was not estimated, so it is not known whether actual leuco-
penia co-existed.