1. Nora Al-Kubaisi nalkubaisi@ksu.edu.sa

2. C LASSIFICATION OF G RAM -P OSITIVE Gram’s +ve bacilli Aerobic Non spore forming Corynebacterium Listeria Spore forming Bacillus Anaerobic Spore forming Clostridium

3. A EROBIC S PORE F ORMING B ACILLUS SPP. Bacillus Pathogenic B. anthracisB. cereus Non- pathogenic (Anthracoids) B. subtilis

4. B. cereus B. subtilisB. anthracis Bacillus

5. G ENERAL C HARACTERS OF B ACILLUS SPP. Very large Gram positive bacilli Arranged in long chains Motile except B. anthracis Spore forming (outside the host) Capsulated (inside the host) Facultative anaerobic Catalase positive It is found in soil habitats

6. C HARACTERS OF B ACILLUS SPP. Gram StainPositive Catalse Test Positive Microscopically appearance long chains Motility Motile except B. anthracis Outside the host Spore forming Inside the host Capsulated Habitat Soil

8. Anthrax; is an acute infectious disease in man & animal caused by : the spore-forming B. anthracis. Anthrax is zoonotic disease. Anthrax is occupational disease. Direct person-to-person spread of anthrax is extremely unlikely to occur.

9. Cutanoues Anthrax (Malignant Pustule). Pneumonic Anthrax (Woolsorters disease). Intestinal Anthrax.

11. B. cereus is a normal inhabitant of soil. Also isolated from food such as grains and spices. B. cereus causes Two Types of food poisoning.

12. E METIC FORM OR SHORT INCUBATION : It is caused by heat stable enterotoxin. N ausea, vomiting and abdominal cramps. Incubation period of 1-6 hrs. It ressembles S. aureus food poisoning.

13. D IARRHEAL FORM OR LONG INCUBATION : It is caused by heat labile enterotoxin Abdominal cramps and diarrhea Incubation period of 8-16 hrs It resembles food poisoning caused by; Cl. perfringens

14. EmeticDiarrheal incubation short incubation 1-6 hr. long incubation 8-16 hr. Caused by; heat stable enterotoxin heat labile enterotoxin symptoms 1.Nausea, 2. vomiting 3.abdominal cramps 1.Abdominal cramps 2.diarrhea resembles S. aureus Cl. perfringens B. cereus causes Two Types of food poisoning.

15. Similar point Normal inhabitant of soil No hemolysis -hemolysis Non-Motile Motile

16. I DENTIFICATION OF B ACILLUS S PP. Specimen –Pastular exudates in malignant pustule. –Sputum in pneumonic anthrax –Stool in intestinal anthrax (also in food poisoning by B. cereus ). Stool specimen is emulsified and heated to 80 C to kill non spore forming microorganism

17. I DENTIFICATION OF B ACILLUS S PP. Morphology –Macroscopical (Cultural characteristics). –Microscopical (Gram Stain, Spore Stain).

18. I DENTIFICATION OF B ACILLUS S PP. Cultural Characteristics; On ordinary medium. Grow aerobically at 37C with characteristic mucoid or smooth colonies, which indicates the pathogensity of organism (presence of capsule).

19. CulturalCharacteristics; Rough colonies are relatively a virulent. Stab culture on gelatin medium results in inverted fire tree appearance.

20. G ROWTH ON B LOOD A GAR Bacillus species grow well on blood agar showing a double zone of hemolysis. B. anthracis, which grows well on blood agar without any hemolytic effect.

21. C ULTURAL C HARACTERISTICS Nutrient Agar Blood Agar B. cereus B. anthracis

22. I DENTIFICATION OF B ACILLUS S PP. Morphology Microscopical Stain Gram Stain Gram positive bacilli Found in chains

23. Spore Stain Procedure; 1. Make a heat fixed smear of Bacillus. 2. Place the slide on the slide rack. 3. Cover the smear with malachite green stain. 4. Apply heat for 3-5 min without boiling and drying of the slide.

24. 5.Wash the slide gently in running water. 6.Counterstain with safranin for one minute. 7.Gently rinse with water. 8.Gently blot the slide dry, no rubbing, and let it air dry and examine with oil immersion optics. 9.Observe red vegetative cells and sporangia, and green endospores and free spores.

25. I DENTIFICATION OF B ACILLUS S PP. Spore Stain Bacillus spores are oval & central. By spore staining technique; (Malachite green & safranin), the spore appears green while the vegetative cells appear red.

27. B IOCHEMICAL T ESTS : 1- C ATALASE T EST All Bacillus species are catalase positive ( Remember staphylococci are catalase positive).

34. P RACTICAL W ORK Gram Stain Spore Stain Catalase Test Starch hydrolysis

35. Corynebacterium spp Gram positive bacilli. characteristic morphology (club shaped and beaded). Non motile. Non spore forming. Non capsulated. Facultative anaerobic. C. diphtheriae is fastidious while diphtheriods are non-fastidious. Catalase positive. Oxidase negative.

36. S PECIES OF C ORYNE BACTERIA Corynebacterium Pathogenic C. diphtheriae Commensal "Diphtheriods" C. hofmannii, C. xerosis, C. acne

38. 2.To confirm the clinical manifestation 1.Specific treatment must be never delayed for laboratory results Diagnosis of diphtheria 1.Clinical Diagnosis2.Laboratory Diagnosis

39. Laboratory diagnosis of case;

40. Corynebacteria grow much more readily than other respiratory pathogens. Used to : Enhance the characteristic microscopical appearance of corynebacteria

41. The colonies of C. diphtheriae are ; 1. Small 2. granular 3. grey 4. smooth 5. creamy with irregular edges.

42. C ULTURAL CHARACTERISTICS On blood tellurite agar ( Potassium tellurite) : It is selective medium for isolation of C. diphtheriae.

43. M ORPHOLOGY Gram stain: Gram +ve. Spore forming : Non. Motility : Non motile bacilli.

44. M ORPHOLOGY Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance). Beaded (metachromatic granules).

45. – P OLYCHROME 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.

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

47. B IOCHEMICAL R EACTION All Corynebacterium species are catalase positive. ( Also, Staphylococcus and Bacillus species are catalase positive)

49. Diagnosis of Carrier I- Isolation of organism Swap from throat & nose Inoculation on Loeffler’s Or BTA for 24 h/37 C Diphtheria like M.O. II- Detection of exotoxin Test for toxigenicity

51. I N V ITRO : E LEK ’ S T EST Principle: It is toxin/antitoxin reaction Toxin production by C.diphtheriae can be demonstrated by a precipitation between; exotoxin and diphtheria antitoxin

52. P ROCEDURE 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.

53. R ESULTS : 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