50 
MALARIA 
press). It is believed that stains based on set of brom-thymol blue color standards are 
this second study will soon be on the market used for testing. 
(formula “M” below) : Staining. For the staining of thin films 
Formula “A” 
Formula “ M ’ ’ 
Nocht 
formula 
(cc) 
Pure 
dye 
weight 
Total 
dye 
weight' 
1: 1000 
solution 
mg 
mg 
Azure B (80% est.) 
2.0 
200 
250 
Azure B eosinate 
250 mg 
Azure A 90% 
0.5 
50 
55 
Azure A ‘ ‘ 
50 “ 
Methylene blue 87% 
2.7 
270 
310 
Meth. blue ‘ ‘ 
200 “ 
Eosin T 93% 
5.0 
500 
537 
Methylene blue chlo¬ 
ride (87%) 
100 “ 
Glycerine 
50 
CC 
Glycerine 
50 cc 
Methyl alcohol 
50 
cc 
Methyl alcohol 
50 cc 
Total 
100 ec stain 
Total 100 cc stain 
For thin films Wright’s and Leishman’s 
stains made from standard formulae will 
also give satisfactory results. 
Buffer solutions. To obtain a pH of from 
7.0 to 7.2 in the water used for the dilution 
of the stock stain, as well as for washing 
the stained slides, buffer solutions should 
be added to the distilled water. Disodium 
phosphate and either sodium or potassium 
acid phosphate in M/15 solutions are used 
and are prepared by dissolving the salts as 
follows: Na 2 HP0 4 (anhydrous), M/15 = 9.5 
grams per liter; NaH 2 P0 4 • H 2 0, M/15 = 9.2 
grams per liter; and KH 2 P0 4 , M/15 = 9.07 
grams per liter. The stock solutions are 
kept in separate glass-stoppered pyrex bot¬ 
tles from which are removed the following 
quantities to make the indicated amount of 
the buffered water: 
M 
M 
pH — NaJEPO* 
— NaH/PO., • H 2 0 
Distilled 
or 
M 
15 EH * P °‘ 
H 2 0 
7.0 61.1 cc 
38.9 cc 
900 ee 
7.2 72.0 cc 
28.0 cc 
900 cc 
The pH of the buffered water is then 
tested and adjustments made if necessary. 
Brom-thymol blue indicator solution and a 
with Wright’s or Leishman’s stains, which 
are alcoholic solutions, the red cells are 
fixed by placing the undiluted stain directly 
on the blood films for a given period of 
time. Then the stain is diluted with the 
amount of buffered water found to give best 
staining results, and the slide washed by 
flushing away the stain with additional 
water. The amounts and time for each step 
must be ascertained for different lots of dye. 
To use Giemsa stain on thin films, the blood 
smears must be previously fixed with ace¬ 
tone-free, absolute methyl alcohol, then 
stained in the manner described below for 
thick films, except that they are washed by 
dipping quickly in clear, buffered distilled 
water. 
Thick films should never be fixed by chem¬ 
ical or other means, since lysis of the eryth¬ 
rocytes is necessary to make such smears 
transparent. The portion of the slides con¬ 
taining the smears is vertically immersed, 
in a solution of one part stock Giemsa stain 
solution diluted with 50 parts of neutral 
distilled water for 45 minutes. Then they 
are removed and washed for a sufficient 
time to clear the background and differ¬ 
entiate the parasite colors. This varies with 
the age and thickness of smears—from sev¬ 
eral dips in neutral distilled water to a five 
minutes ’ immersion. Older and thicker 
