OSPE DIPHTHERIA

Question 1

Specimen collection

Answer 1

1. Throat swab (one or two) containing fibrinous exudates
2. A portion of pseudomembrane
3. Nose or skin specimens (if infected)

Question 2

Direct Smear Microscopy

Answer 2

gram stain:
* Club- shaped
* Gram +ve- purple
* Arranged in V- or L-
shaped

Albert’s stain:
green bacilli with bluish black metachromatic granules at the poles

Question 3

Culture characteristics-

Answer 3

• C.diphtheriae-fastidious,aerobeorfacultative
• enriched medium
  1. 5% sheep Blood agar
  2. Loeffler’s serum slope
  3. Chocolate agar
  4. Selective medium- Potassium tellurite agar

incubatedat37°Caerobically

Question 4

Blood Agar

Answer 4

Colonies are small circular, white and sometimes hemolytic (mitis biotype)

Question 5

Loeffler’s serum slope

Answer 5

Colonies appear as small, circular, glistening, and white with a yellow tinge in 6–8 hours

Advantages:
(1) Growth can be detected as early as 6–8 hours.
(2) Best medium for metachromatic granules production

Question 6

Potassium tellurite agar(PTA)

Answer 6

black colonies of C. diphtheriae
Advantages: Commensals are inhibited

Question 7

Biochemical tests

Answer 7

-Sugar fermentation test using Hiss’s serum sugar media (+ve)

• Methyl red +ve
• Pyrazinamidase test (-ve)
  Human pathogenic C. diphtheriae, C. ulcerans and C. pseudotuberculosis, DO NOT produce pyrazinamidase while other corynebacteria members produce
• Indole, Citrate, Urease test, VP (-ve)

Question 8

Automated identification systems

Answer 8

MALDI-TOF
VITEK

Question 9

Toxin Demonstration (In Vitro Test)-to test for toxigenic strain

Answer 9

Elek’s gel precipitation test:
-immunodiffusion in gel
1. Cross-over with each other—indicates unrelated strain
2. Spur formation—indicates partially related strain
3. Fused with each other—indicates identical strain

Question 10

Other Toxin Demonstration (In Vitro Test)

Answer 10

• Detection of Tox gene by PCR
• Detection of diphtheria toxin by ELISA or immunochromatographic test
  (ICT)
• Cytotoxicity produced on cell lines.