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1. Laboratory Diagnosis Specimen Throat swab containing fibrinous exudates, portion of pseudomembrane , nose or skin specimens Direct Smear Microscopy Gram stain Albert Stain Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

2. Laboratory Diagnosis...Culture Fastidious, aerobe and facultative anaerobe Blood agar - Small circular, white Loeffler’s serum slope - Nutrient broth with horse or sheep serum and glucose - Colonies in 6–8 hours - Best medium for metachromatic granules production - Disadvantage – If incubated beyond 6–8 hours  growth of throat commensals Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

3. Laboratory Diagnosis...Culture Selective Medium: Hoyle’s potassium tellurite agar (PTA) - L ysed horse blood agar & potassium tellurite as inhibiting agent - Reduces tellurite to metallic tellurium  black color Tinsdale medium - Modified PTA with cysteine. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

4. Laboratory Diagnosis Specimen Throat swab containing fibrinous exudates, portion of pseudomembrane , nose or skin specimens Direct Smear Microscopy Gram stain Albert Stain Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

5. Laboratory Diagnosis...Culture Fastidious, aerobe and facultative anaerobe Blood agar - Small circular, white Loeffler’s serum slope - Nutrient broth with horse or sheep serum and glucose - Colonies in 6–8 hours - Best medium for metachromatic granules production - Disadvantage – If incubated beyond 6–8 hours  growth of throat commensals Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

6. Laboratory Diagnosis...Culture Selective Medium: Hoyle’s potassium tellurite agar (PTA) - L ysed horse blood agar & potassium tellurite as inhibiting agent - Reduces tellurite to metallic tellurium  black color Tinsdale medium - Modified PTA with cysteine. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

7. Biochemical Identification Corynebacterium is catalase positive but oxidase negative ™ Hiss’s serum sugar media - Enriched sugar medium for testing sugar fermentation - Diphtheria bacilli ferment glucose and maltose (all biotypes) and starch (gravis biotype), with production of acid but no gas. ™ Urease test - C. diphtheriae Negative. C. ulcerans & C.pseudotuberculosis are urease positive

8. Toxin Demonstration As the pathogenesis is due to diphtheria toxin, mere isolation of bacilli does not complete the diagnosis. Toxin demonstration should be done following isolation In vivo Tests (animal inoculation) - Guinea pig - Subcutaneous inoculation : Leads to death of the test animal within four days - Intra-cutaneous inoculation : Leads to necrosis of the inoculated site of the test animal in 48–72 hours. - Advantage -no death of animals and many strains can be tested at a time on the same animal. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

9. Toxin Demonstration ...In Vivo Tests ™ Elek’s gel precipitation test: Immunodiffusion in gel Filter paper soaked in diphtheria antitoxin - 20% horse serum agar - test strain at right angle to the filter paper strip Toxigenic strain - Precipitation Relatedness between the strains „Detection of Tox gene or Toxin Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

10. Albert staining technique Albert staining technique aims at detecting the presence of metachromatic granulated bodies of  Corynebacterium diphtheriae .  Albert stain is made up of two staining solutions; Albert Solution 1  and  Albert Solution 2 Albert Solution 1: toluidine blue, malachite green, glacial acetic acid, and alcohol Albert solution 2: Iodine and Potassium iodide in water Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

11. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

12. The metachromatic granules stain bluish black while the rest of the microbial cell stains green Corynebacterium diphtheriae   cytoplasmic membrane contains volutin granules( metachromatic granules) The staining by Albert solutions, stains the granules making the appear as round-shaped blue-black dots at the bottom of L-shaped or V-shaped green Bacilli. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

13. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers

14. Essentials of Medical Microbiology by Apurba S Sastry © 2018, Jaypee Brothers Medical Publishers