Haemophilus

1. Haemophilus - Microscopic appearance - Cultural characteristics - Identification of Haemophilus species - Serological methods for detection of H.influenzae

3. H. influenzaeis a small non-motile Gram negative coccobacillusor short rod

4. coccobacillusor short rod appearance of H. influenzaeby Electron Microscope

5. - Media used to grow H. influenzaemust contain haemin or other iron-containing porphyrin and nicotinamide adenine dinucleotide (NAD) or its phosphate (NADP). • The porphyrin requirement is referred to as growth • factor X and the NAD or NADP requirement as • growth factor V.

6. - Factor X is used by H. influenzaeto produce essential respiratory enzymes such as cytochromes, catalase, and peroxidase. - Factor V is used as an electron carrier in the organism’s oxidation-reduction system..

7. in a moist carbon dioxideatmosphere, capsulated H. influenzaestrains producemucoidcolonies, 1.5 mm or more in diameter. Cultures have a distinctive smell.

8. H. influenzaegrows well on chocolate agarbecause it contains factors X and V. Heating blood agar to 75 ºC inactivates serum NADase and releases extra factor V from the red cells.

9. Addition of bacitracin (300 mg/litre) provides a selective medium to recover H. influenzaefromsputum. This is NOT needed when culturing c.s.f?!?!?

10. H. influenzaeproduces very small colonies on horse or rabbit blood agar (colonies may appear beta-haemolytic). There is usually no growth on sheep blood agar. If, however, S. aureuswhich produces factor V inexcess of its own needs, is cultured on a blood agar plate with H. influenzae, the factor V and the haemin releasedby staphylococcal haemolysins help the growth of H. influenzae.

11. This ‘help’ given by S. aureus, forms thebasis of the • satellitismtest which is a simple way of • recognizing H. influenzae. • - S. pneumoniaealso produces factor V and causes • H. influenzae to show satellitism

12. How to perform satellitism test ?? 1- Mix a loopful of suspect Haemophilusgrowth in about 2 ml of sterile physiological saline or sterile peptone water. Make sure none of the chocolate agar medium is transferred. 2- Using a sterile swab, inoculate the organism suspension on a plate of nutrient agar, and a plate of blood agar.

13. 3- Streak a pure culture of S. aureusacross each of the inoculated plates. 4- Incubate both plates in a carbon dioxide enriched atmosphere at 35–37 ºC overnight. 5- The following morning examine the cultures for growth and satellitecolonies.

14. H. Influenzae shows growthon the blood agar plate but not on the nutrient agar plate, and the colonies near the column of S. aureus growth are larger than those furthest from it.

15. Identification of H. influenzaeusing X, V, XV discs 1- Make a saline suspension (approx. 0.5 McFarland turbidity) of the test organism from a primary culture. Using a swab, inoculate the suspension on a plate of nutrient agar.

16. 2- Place the factor tablets or discs 10–20 mm in from the side of the plate, positioning each disc as follows: Factor X. . . . . . . . . . . . . . . at ‘12 o’clock’ Factor V. . . . . . . . . . . . . . . at ‘4 o’clock’ Factor XV . . . . . . . . . . . . . at ‘8 o’clock’

19. 3- Set up a control plate using a known H. influenzae strain. 4- Incubate the plates overnight in a moist carbon dioxideatmosphere at 35–37 ºC.

21. H. influenzae: Growth around factor XV and slight growth between X and V ?? H. parainfluenzae: Growth around factor XV and factor V

22. Serology - H. influenzaeorganisms are divided into six serogroups, a–f. • The majority of strains that cause meningitis • belong to invasive serogroupb. Very occasionally • meningitis is caused by groups a, e, and f. - Most of the strains that cause chronic bronchial disease are non-capsulated.

23. Slide coagglutination reagents are commercially available for the rapid immunological detection of specific polysaccharide H. influenzae bantigen inc.s.f. rapid Easy to perform specific sensitive

24. Brucellae - Microscopic appearance - Cultural characteristics - Identification of Brucella species - Serological methods for detection of Brucella antibodies.

26. Brucella Brucellae are coccobacillior short rods

27. Brucellae are small Gram negative non-capsulated coccobacilli or short rods

31. - Tryptonesoya (tryptic soy) diphasicmedium (Castaneda) is recommended for the isolation ofBrucellaspecies.

32. Several commercially produced blood culture systems are also suitable and some provide rapid isolation.

34. Brucellaeare aerobic with B. abortusrequiring a • carbon dioxide enrichedatmosphere. • Cultures should be kept for 4 weeks with sub- • culturing every few days. When subcultured on solid • agar, colonies usually appear 2–3 days after incubation.

35. - A variety of colonial forms are produced by Brucellastrains including smooth, mucoid, and rough colonies. They may be colourless or greywhite.

36. B.abortus produce smooth grey-white colonies on Blood agar

37. B.melitensis produce smooth grey-white colonies on Blood agar

38. Urease Test The test organism is cultured in a medium which contains urea and the indicator phenol red. When the strain is urease producing, the enzyme will break down the urea (by hydrolysis) to give ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline as shown by a change in colour of the indicator to pink-red.

39. Agglutination test for diagnosis of Brucellosis