52 
MALARIA 
examining thick films because of the loss of 
erythrocyte outlines and a slight change in 
parasite contour and size. The advantages 
of the technique, however, fully recompense 
one for the time spent in becoming profi¬ 
cient. 
Enumeration of parasites. To obtain a 
definite idea of the degree of infection a 
quantitative thick smear may he made and 
examined by the following modification of 
Earle’s method (Earle and Perez 1932). 
Five cubic millimeters of blood are taken in 
a special pipette and spread evenly over an 
accurately measured 3 x 15 mm area on a 
clean slide. (This may be marked on the 
slide by ruling instruments having diamond 
points set at the exact distances.) When 
dry the smear is stained like any other thick 
film. Preliminary to enumeration the mi¬ 
croscope is calibrated as follows: A Howard 
disc with a ruled square is inserted in the 
ocular. One side of the large square is 
measured with a slide micrometer and the 
area of the field covered by the square com¬ 
puted in square millimeters. The number 
of fields to be counted in order to cover one 
square millimeter is ascertained by dividing 
one square millimeter by the area of one 
square microscopic field. For example, 
since 5 cubic millimeters of blood are de¬ 
posited on 45 square millimeters of space, 
5 -r 45 = 0.11, which is the portion of one 
cubic millimeter of blood spread over one 
square millimeter. Then also since 1^-0.11 
cubic millimeter = 9 (the number of square 
millimeters carrying a blood volume equal 
to one cubic millimeter), the number of 
parasites counted in the required number 
of microscopic fields to equal one square 
millimeter is multiplied by 9 to obtain the 
number of parasites per cubic millimeter. 
The requisite number of fields is selected 
from different parts of the smear to make 
the count as representative as possible. If 
counts run high, fewer fields are examined 
and an estimation made for the required 
number of fields. 
Sporogonous Cycle 
Experimental Infection 
In the experimental infection of mos¬ 
quitoes the maturity of the gametoevtes 
should be ascertained prior to the applica¬ 
tion of the mosquitoes to the patient, as¬ 
suming that satisfactory densities of these 
cells are present. While mosquitoes may be 
lightly infected when fed on vivax patients 
with submicroscopic densities of gameto- 
cytes, it is desirable to limit their applica¬ 
tion to patients who, in the case of vivax, 
have counts of not less than 5 males and 5 
females per cmm. In falciparum patients 
a much higher density of gametocytes is 
necessary, approximately 100 of each sex 
to secure reasonably adequate infections. 
An exflagellation smear should also be made 
at this time. For this purpose a rather 
thick “thin” smear is prepared, over which 
the breath is exhaled. Before desiccation 
begins this smear is placed in the saturated 
air of a Petri dish having moistened filter 
paper in the bottom, and allowed to stay 
for about 30 minutes at a cool temperature. 
At the end of this time the smear is re¬ 
moved, dried and stained as are ordinary 
smears. If exflagellating microgametocytes 
are found one can be sure of the maturity 
of the gametocytes present in the donor. 
By treating a preparation in this manner 
the same development can be initiated 
which normally takes place in the stomach 
of the mosquito. 
The mosquitoes were previously placed 
Plate II— Phases of Malarial Parasites in the Mosquito Host {Continued) 
7. P. vivax. Cross section of lobe of salivary gland of A. quadrimaculatus, showing sporozoites within 
secretory cells of gland. Hematoxylin stain. 
8. P. vivax. Individual sporozoites extruded from crushed gland. Wet fixation, Giemsa stain. 
9. P. malariae. A 12 day old cyst, 8.4 micra diameter. 
10. P. malariae. A 23 day old cyst. Beginning formation of sporoblasts. 
11. P. malariae. Sporozoites extruded from crushed gland. Wright stain. 
12. P. falciparum. Cyst showing early stage of sporoblasts. Nine days old. 
13. P. falciparum. A cyst 17 days old showing sporozoites budding from sporoblasts. 
14. P. falciparum. Group of sporozoites. Wet fixation, iron-hematoxylin stain. 
