1. Lab. No. 3

3. Gram’s +ve Bacilli Spore forming Non spore forming AerobicAnaerobic Bacillus Clostridium Corynebacterium

4. Most Important Species: Most Important Species:  C. diphtheriae causes diphtheria.

5. Identification of Corynebacteria (I) Morphology (microscopical examination):  Gram +ve, nonspore forming nonmotile bacilli  club-shaped (Coryne= club) arranged at acute angles or parallel to each other → a Chinese letters appearance  Beaded (metachromatic granules)

6. (II) Cultural characteristics: Growth occurs best on media containing blood or serum. Growth occurs best on media containing blood or serum. 1. Loeffler’s serum agar:  Used to enhance the characteristic microscopical appearance of Corynebacteria. 2. Mcloed’s blood agar (blood tellurite medium):  It is a selective medium (K tellurite) for isolation of diphtheria bacilli from the pharynx (The best isolation medium).

7. (III) Biochemical Reactions: 1- Catalase test:  All corynebacterium species are catalase +ve.

8. 2- Carbohydrate Fermentation Test: Principle: EEEEach species of corynebacteria has its specific carbohydrate fermentation pattaern.  C C C C.diphtheriae can be differentiated from other corynebacterium species by fermentation of glucose and maltose but not sucrose. With production of acid without gas. Procedure: 1. Inoculate three tubes of sugar medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism GlucoseMaltose Sucrose 2. Incubate the tubes at 35 o C for 24 hrs.

9. Results: C. xerosis C. diphtheriae GlucoseMaltose Sucrose GlucoseMaltose Sucrose Sugar fermentation can be indicated by change of color of the medium from red to yellow. +ve - ve +ve+ve

10. (IV) Toxigenicity testing of C.diphtheriae strains: Elek’s Toxigenicity Test: Elek’s Toxigenicity Test: Principle: It is an Ag-Ab reaction, in which, toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin. Procedure: 1. Place a strip of filter paper saturated with diphtheria antitoxin on a serum agar plate. 3. Incubate the plate at 35 o C for 24 hrs. 2. Streak the test organism across the plate at right angle to the filter paper.

11. Results: Positive test: formation of four radiating lines resulting from the precipitation reaction between exotoxin and diphtheria antitoxin.