58 
MALARIA 
to 5 minutes in each. It is then cleared in 
carbol-xylol and mounted in balsam under¬ 
neath a cover slip. 
Formulae for the stains mentioned are as 
follows: 
Mayer’s Acid Haemalum 
Dissolve 1 gram of haematin in 50 ee of 90 per 
cent alcohol and warm. Add this solution to a 
solution of 50 grams of aluminum potassium sul¬ 
phate in 1000 ce of distilled water dissolved by 
heating. Mix warm, cool, and filter. Add 2 cc of 
glacial acetic acid to 100 cc of haemalum solution. 
Bonin’s Fixative 
Picric acid, saturated aqueous solution . 30 parts 
Formalin (40 per cent) . 10 parts 
Acetic acid . 2 parts 
This should be prepared daily. 
Salivary glands. The cover slip is gently 
raised from the crushed gland and inverted. 
The material on both the slide and the cover 
slip is allowed to dry, is then fixed by flood¬ 
ing with absolute methyl alcohol, and is 
stained with Giemsa stain exactly as a blood 
smear. Forceps for holding the cover slips 
are very convenient in staining. The sur¬ 
face of both the slide and cover slip is ex¬ 
amined with a low power lens and the one 
to which the glands have adhered is saved. 
If on the cover slip, the slip is attached, 
gland side upward, to a slide by a drop of 
balsam (Boyd 1932). 
Examination of Stained Preparations 
A well-stained stomach preparation shows 
the cysts slightly darker than the stomach 
tissue, with the pigment showing as in a 
fresh preparation. Large or mature cysts 
are readily recognized by their internal con¬ 
tent of sporoblasts or sporozoites. Small 
cysts are identifiable by their content of 
pigment, the recognition of which is indis¬ 
pensable for diagnosis of the body as a cyst. 
The only criterion for the specific identifica¬ 
tion of the cysts is by the characteristics of 
the pigment, which is retained from the 
macrogametocytes from which it developed. 
The characteristics of the pigment in the 
different species are shown in the accom¬ 
panying tabulation (Table I). Pigment is 
rarely observed in the large cysts. It is not 
certain whether its disappearance is appar¬ 
ent or real, i.e., whether it is masked by the 
increased internal structure of the cyst or 
has been metabolized. The stained sporo¬ 
zoites exhibit the characteristic colors of a 
malarial parasite. The reddish chromatin 
is usually a central oval mass or may appear 
as three or four small, round granules. The 
cytoplasm has the familiar blue of a para¬ 
site. The sporozoites of P. vivax and P. falci¬ 
parum are morphologically indistinguish¬ 
able, but one could probably differentiate 
the sporozoites of P. malariae, as these are 
definitely larger and coarser, with splotchy 
chromatin. Sporozoites from different mos¬ 
quitoes vary greatly in their size, although 
those derived from any particular insect 
show relatively slight difference (Boyd 
1935a). 
TABLE I 
Differential Diagnosis in Sporogonous Cycle 
Produce 
sporozoites at 
20? C in 
P. vivax 
P. malariae 
P. falciparum 
Cysts 
16-17 days 
30-35 days 
22-23 days 
Pigment in small 
cysts 
Fusiform crystals 
frequently in a 
chain 
Rounded, angular 
masses, golden 
brown 
Rectangular blocks 
dense black 
Sporozoites 
Size 
Variable, probably 
dependent on de¬ 
gree of infection 
in mosquito 
Definitely largest 
and coarsest 
Variable, probably 
dependent on de¬ 
gree of infection 
in mosquito 
Romanowsky 
staining 
Indistinguishable 
from 
P. falciparum 
Chromatin displays 
the diffuse char¬ 
acter typical of 
P. malariae 
Indistinguishable 
from P. vivax 
