188 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 
is drawn well up into the pipette, the end is sealed in the flame of a 
Bunsen burner, and the charged pipette is placed in the incubator at 
37° C. This is the normal or control. 
A precisely similar preparation is made, using the serum of the 
patient in place of the pooled serum. 
Incubation is maintained for fifteen minutes. 
The ends of the pipettes are now broken ofi", and the contents of 
each pipette mixed as before. A large drop of each respective mixture 
is spread upon clean glass slides, using the same technique as that for 
preparing a blood smear, and air-dried. The preparations are stained 
with LoflBer's methylene blue, Wright's stain, or other stain suitable 
for the organism used. 
The number of bacteria in 50, 100, or 200 leukocytes is determined 
by direct count, and the average number of bacteria per leukocyte of 
the normal serum compared with the average number of bacteria per 
leukocyte in the pathological serum : 
Example. 
Bacteria in Bacteria per 
100 leukocytes. leukocyte. 
Staphylococcus suspension + pooled serum and leukocytes 750 7 . 5 
Staphylococcus suspension + patient's serum and leuko- 
cytes 250 2.5 
2.5 
Opsonic index, patient's serum = or 0.33 per cent. 
7.5 
Numerous observers have been unable to obtain uniform results 
with the technique of Wright for opsonic index determination, and 
this is not surprising when the many variable factors entering into 
the method are reviewed. Attempts have been made to eliminate 
or limit the variable factors: Simon proposed a dilution method in 
which the pooled and patient's serum are diluted 1 to 10, 1 to 100, 
etc., before incubation with the bacteria and leukocytes. That dilu- 
tion of serum at which phagocytosis practically ceases in the normal 
and the patient's serum respectively is taken as a basis for compari- 
son. Inasmuch as the opsonic index is rarely determined as a guide for 
treatment of bacterial disease with bacterial vaccines at the present 
time, however, a discussion of these modifications, which are too 
involved for practical use, is left for more pretentious volumes. 
The Nature of Opsonins.— There appears to be no doubt that the hypo- 
thetical substance or substances called opsonin by Wright exist in 
normal sera, and it is equally certain that they may be diminished 
during infection. Furthermore, opsonin may be increased either in 
amount or in potency by careful immunization. The relation of 
opsonins to other antibodies, normal or specific, is a subject of con- 
troversy at present. The researches of Neufeld and Rimpau,^ Hek- 
toen- and others indicate that the normal opsonins— those of normal 
> Deutsch. med. Wchnschr., 1904, 30, 1458. 
2 Jour. Infec. Dis., 1906, 3, 434; 1909, 6, 78; 1913, 12, 1. 
