1. Aerobic Non-Spore Forming Gram-Positive Bacilli
Corynebacterium

2. Gram Positive Bacilli Gram positive rods Spore forming
Non spore forming
Anaerobic
Aerobic
Clostridium spp
Bacillus spp
Corynebacterium

3. Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Facultative anaerobic
Breakdown glucose by oxidative and fermentative i.e. O+/F+
C. diphtheriae is fastidious while diphtheriods are non-fastidious
Catalase positive
Oxidase negative

4. Species of Corynebacteria
It caused diphtheria
C. diphtheriae is the only pathogenic members of this genus
Normal flora of RT, urethra, vagina, Skin

5. Diphtheria Cased by C. diphtheriae
Mycocarditis, neuroitis, palate perforation
Acute, Toxin mediated
Recovery or complication & death (if more toxin absorbed)
Childhood disease affect upper respiratory tract
Diphtheria
Respiratory obstruction due to extensive membrane formation
Transmitted by droplet infection
2-6 days I.P.
Sore throat, Pharyngitis
2-3 days, Bluish white adherent pseudo membrane

6. Specific treatment must be never delayed for laboratory results
To confirm the clinical manifestation

7. Symptomatic patient
Asymptomatic

8. Laboratory diagnosis of case
Specimen: A throat swap
Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37C for 24.
On Loeffler's serum medium:
Corynebacteria grow much more readily than other respiratory pathogens
Used to enhance the characteristic microscopical appearance of corynebacteria
The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges
Loefflers serum

9. Cultural characteristics
On blood tellurite agar (Mcloed’s blood agar)
It is selective medium for isolation of C. diphtheriae (Potassium tellurite)
3 biotypes of C. diphtheriae are characterized on BTA
i.e. Gravis, mitis and intermedius biotypes
The most severe disease is associated with the gravis biotype
Colony of gravis biotype is large, non-hemolytic & grey.
Colonies of mitis biotype are small, hemolytic and black
Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

10. Morphology Stain Gram +ve, nonspore forming nonmotile bacilli
Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance)
Beaded (metachromatic granules)
Stain
Gram stain:
C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped
Polychrome methylene blue stain:
C. diphteriae appears beaded due to the presence of intercellular “Metachromatic or volutin" granules
By stain, the granules appear red while the rest of organism appears blue.

11. Gram stain of C. diphtheriae
C. diphtheriae on BTA

12. Biochemical Reaction
All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

13. 2- Carbohydrate Fermentation Test:
Principle:
Each species of corynebacteria has its specific carbohydrate fermentation pattern
C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only
Procedure:
Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism
Glucose
Maltose
Sucrose
Incubate the tubes at 37oC for 24 hrs.

14. Results: C. diphtheriae C. xerosis
Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH
Glucose
Maltose
Sucrose
Glucose
Maltose
Sucrose
+ve
+ve
+ve
+ve
+ve
- ve
C. diphtheriae
C. xerosis

15. Swap from throat & nose
Inoculation on Loeffler’s
Or BTA for 24 h/37 C
Diphtheria like M.O.

16. One is used as Test The second is used as Control If both GP live
Two guinea pigs are used
One is used as Test
The second is used as Control
Injected with diphtheria antitoxin
Both test and control injected with isolated MO
If both GP live
If control live & test die
Diphthrioids 0r non-toxigenic
C. diphtheriae
i.e. non pathogenic
C. diphtheriae
i.e. produce exotoxin

17. In Vitro: Elek’s Test Principle: It is toxin/antitoxin reaction
Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin
Procedure:
A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar
The organism is streaked at right angels to the filter paper
Incubate the plate at 37C for 24 hrs

18. Filter paper saturated with diphtheria antitoxin
Resuls:
After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism
Lines of precipitations
Inoculated M.O.
Positive Elek’s Test