IV. INVESTIGATION OF C . DIPHTHERIAE
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XIII. SPECIAL TECHNIQUES AND MEDIA USED I N T H E
BACTERIOLOGICAL DIAGNOSIS OF C. DIPHTHERIAE
A. Staining of metachromatic granules
1. Preparation of alkaline Methylene blue solution (Loejler's formula) for
del Vecchio staining or for use as such
(a) the basic solution of Methylene blue is a 10% alcoholic solution kept
for 7 days in the incubator.
(b) The solution used for staining is a 10% aqueous solution prepared
from (a). T o this second solution O. Olyo potassium hydroxide is added.
After incubation for 1 week, the solution is filtered and is ready for use.
2. Staining for metachromatic granules
(a) Simple staining. The smears, dried and heat-fixed, are stained for 10
min with the alkaline Methylene blue solution and washed with tap water.
Microscopic appearance : metachromatic granules, red ; the bacillary
body, blue.
(b) Double staining by del Vecchio's method. Smears are dried and heatfixed and then stained as follows:
Loeffler's alkaline Methylene blue, 10 min
Wash with tap water
Lugol solution, 10 min
Wash with tap water
Microscopic appearance : metachromatic granules, brown ; the bacillary
body, yellowish brown.
B. Media
1. ECST enrichment j u i d medium (egg-cystine-serum-tellurite) (Calalb et al.,
1961)
(a) Formula
Normal bovine serum
150 ml
Nutrient broth with 0.2% glucose
50 ml
Potassium tellurite 2% distilled water 20 ml
L-Cystine solution 1%
2-2 ml
Egg yolks
2 each
(b) Preparation. Wash fresh hens eggs with soap and water and leave them
for ij h in 70" alcohol. Place each egg in a small sterile container. With
sterile scissors and forceps cut out the upper end of the egg shell and