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Laboratory Diagnosis of Diphtheria (Isolation of corynebacterium…
Laboratory Diagnosis of Diphtheria
Isolation of corynebacterium diphtheriae
Direct smear
Gram stain
:- C. diphtheriae appear as irregular stained club shaped gram +ve bacilli of 3-6 μm length 🈯️ or cuneiform appearance
Albert's Stain
:- more specific , Appear as green bacilli with blues black metachromatic granules
Culture media
Selective medium
Hoyle's potassium tellurite agar (PTA)
contain lysed horse blood agar and potassium tellurite (0.04%) as inhibiting agent
C. diphtheria reduce tellurite to metallic tellurium which get incorporated into colonies given them black colour; C. ulcers and C. pseudo tuberculosis can also grow on PTA producing black coloured colonies
Advantage
:- throat commensals are inhibited
Disadvantage
:- colonies appear only after 48 hours of incubation
Tinsdale medium
Modified PTA added with cysteine, black-coloured colonies are produced surrounded by brown halo due to break down of cysteine by cysteinase enzyme produced by organism
Enriched medium
Blood agar
Colonies are small circular, white and haemolytic
Loeffler's serum slope
Composed of nutrient broth with horse or sheep serum and glucose
colonies appear as small, circular, glistening and white with yellow ting in 6-8 hours
Advantage
:- (1) growth detected early 6-8 hour (2) best medium for metachromatic granule production
Disadvantage
:- if incubated beyond 6-8 hours, it supports growth of other throat commensals
Specimen
(1) throat swab containing fibrinous exudate (2) A portion pseudomembrane (3) nose or skin specimen
Biochemical identification
Hiss's serum sugar media
Diphtheria bacili ferment glucose and maltose( with all biotypes) and starch (by only graves biotype), with production of acid
As it is fastidious only enriched sugar media such as Hiss's serum sugar media can be used for testing sugar fermentation test
pyrazinamidase test
+ve for C. diphtheriae, C. ulcerance and C. pseudotuberculosis which produce pyrazinamidase enzyme that break down pyrazinamide
Urease test
C. diphtheria dose not hydrolysed urea it differs this from C. ulcers and C. pseudo tuberculosis which are urease +ve
Corynebacterium is catalase +ve but oxidase -ve and nonmotile
Diphtheria Toxin Demonstretion
In vivo Tests (animal inoculation)
Subcutaneous test
0.8 ml of culture broth of test stain is injected subcutaneously into two guinea pig one of which has been protected with 500 units of diphtheria antitoxin on previous day
if the strain is virulent the unprotected animal will die within 4 hours
Intracutaneous test
In Vitro tests
Elek's gel precipitation test
Other in vitro tests