Charts C-33

2. The medium should be dispensed in tubes and coagulated-sterilized in the autoclave as follows:
a. When the suspension is uniform, dispense in tubes.
b. Arrange the tubes in a slant position not more than four deep with several layers of newspaper
or paper towels below and above the tubes to prevent rapid coagulation.
c. Tightly close the autoclave, turn on the steam, and allow pressure to remain at 10 psi for
20 minutes.
d. During this time, allow no air or steam to escape.
e. Adjust the steam-inlet valve and open the air-escape valve such as to maintain a pressure of
10 psi. Abrupt changes in pressure may cause the medium to bubble.
f. Close the outlet valve when all air has been replaced by steam and allow the pressure to reach
15 psi and hold there for 15 minutes.
g. Allow the autoclave to cool slowly. When properly prepared, the slants are smooth and grayish
white. Before inoculation, the slants should be incubated for 24 hours at 35°C as a sterility check.
III. Procedure
1. When C. diphtheriae is suspected, inoculate the Loeffler’s medium as soon as possible after collection
of the specimen.
2. Examine the slants for growth after 8–24 hours of incubation.
3. Prepare smears and stain with methylene blue.
IV. Results
A. Interpretation
See Chart 14-1 for interpretation of these stained smears.
V. Comment
Because Loeffler’s medium is difficult to prepare, purchase of commercially prepared medium in sealed
tubes is recommended.
VI. Bibliography
BBL. Manual of Products and Laboratory Procedures. 5th Ed. Cockeysville, MD: BBL, Division of Becton,
Dickinson, and Co, 1973:118–119.
Buck T. A modified Loeffler’s medium for cultivating Corynebacterium diphtheriae. J Lab Clin Med
1949;34:582–583.

14-3 Tinsdale’s Agar (as Modified by Moore and Parsons)

I. Principle
Tinsdale’s medium supports the growth of all species of Corynebacterium while inhibiting the growth of
normal inhabitants of the upper respiratory tract. Moore and Parsons modified the original Tinsdale’s
formula, simplifying the composition, but retaining the specificity for the recovery of C. diphtheriae and
C. ulcerans.
The potassium tellurite is deposited within the colonies of Corynebacterium, turning them black. Tin-
sdale’s medium is cystine-sodium thiosulfate tellurite, which is specifically helpful in the identification of
C. diphtheriae, colonies of which are surrounded by a brown halo (Color Plate 14-3G).
II. Media and Reagents
A. Formula (Modified Tinsdale’s Base)
Thiotone peptone 20 g
Sodium chloride 5g
Agar 14 g
l-Cystine 0.24 g
Sodium thiosulfate 0.24 g
Distilled water 1L
B. Preparation
1. Suspend 39 g of powder in 1 L of distilled water and heat with agitation. Boil for 1 minute. Auto-
clave for 15 minutes at 121°C.
2. Cool the modified base to 50°C and add:
Sterile bovine serum 100 mL
Tellurite solution, 1% 30 mL
3. Pour 15–20 mL of this medium into petri dishes and allow to harden.
4. The Tinsdale’s agar base medium without serum and tellurite is stable indefinitely if stored in
closed screw-capped tubes or bottles. However, the medium is ordinarily stable for only 2 or 3 days
when stored in the refrigerator following the addition of serum and tellurite.

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