186 ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
OPSONINS. TROPINS. BACTERIAL VACCINES. 
A most important contribution to the literature of immunity is 
tlie work of Denys and his associates, ^ who showed that the sera of 
rabbits immunized to Streptococcus pyogenes possessed two prop- 
erties not exhibited by the serum of a normal animal, namely, the 
property of restricting the development of the organism, and the 
property of stimulating phagocytosis. Their ver>' comprehensive 
studies demonstrated that the leukocytes of normal animals, suspended 
in the serum of immunized animals, phagocytized streptococci ener- 
getically, but the leukocytes of immunized animals suspended in 
normal serum failed to exliibit specific phagocytic activity. Their 
conclusion was that the immunity of rabbits to the streptococcus 
resides in the serum. These observations not only added materially 
to the restricted field in which they were cast— they brought sharply 
into focus the interrelation of the humoral and cellular aspects of 
immunity. 
Wright and Douglas,^ using a modification of the technique of Leish- 
man,^ were able to study phagocytosis in litro; by an ingenious series 
of experiments they showed that normal serum contains substances 
—opsonins— which prepare bacteria for phagocytosis, as described 
in a preceding section (Cellular Immunity). The technique of meas- 
uring the potency of opsonins in the sera of normal and infected 
individuals, as practised by Wright and his associates, consisted 
essentially in mixing intimately equal volumes of bacterial emulsion, 
serum and leukocytes; after incubation at body temperature the 
mixture was spread evenly upon microscopic slides, stained and 
examined with the microscope. The average number of bacteria per 
polymorphonuclear leukocyte was determined by direct count. A 
comparison, under parallel conditions, of the phagocytic activity 
of leukocytes for a specific organism in the serum of a normal indi- 
vidual and that of an individual infected with the specific organism, 
according to the technique outlined below, was called by Wright the 
opsonic index. 
Procedure. — 1. Leukocyte Suspension.— About 0.5 cc. of blood, 
drawn from the lobe of the ear or the tip of the finger, is collected 
in a centrifuge tube containing 10 cc. of sterile physiological salt 
solution in which has been dissolved 1 per cent of sodium citrate; 
this mixture is centrifuged at moderate speed until a sharp separation 
of blood cells and clear supernatant fluid is obtained. The super- 
natant fluid is carefully poured off and the top layer of blood cells, 
which contains practically all the leukocytes, is removed to a fresh 
centrifuge tube containing 10 cc. of physiological salt solution. 
1 Denys and Le Clef: La Cellule, 1895, 11, 175; Bull de I'Acad. roy. de Belgique, 1895; 
Denys and Marchand: Ibid., 1896. Van de Velde: Ann. Inst. Pasteur, 1896, 10, 580. 
Denys: Centralbl. f. Bakteriol., 1898, 24, 685. 
- See Constable (Studies in Immunization, 1909) for bibliography. 
3 British Med. Jour., 1902, i, 73. 
