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Haemophilus. - Microscopic appearance. - Cultural characteristics. - Identification of Haemophilus species. - Serological methods for detection of H.influenzae. H. influenzae is a small non-motile Gram negative coccobacillus or short rod.

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- Microscopic appearance

- Cultural characteristics

- Identification of Haemophilus species

- Serological methods for detection of H.influenzae


- 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.

- Factor X is used by H. influenzaeto produce essential

respiratory enzymes such as cytochromes, catalase, and


- Factor V is used as an electron carrier in the

organism’s oxidation-reduction system..


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


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.


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


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.


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

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.


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.


H. Influenzae

shows growthon the blood agar plate but not on

the nutrient agar


and the colonies

near the column

of S. aureus

growth are larger

than those furthest

from it.


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.


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’


3- Set up a control plate using a known H. influenzae


4- Incubate the plates overnight in a moist carbon

dioxideatmosphere at 35–37 ºC.


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

H. parainfluenzae: Growth around factor XV and factor V



- 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.


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


Easy to perform





- Microscopic appearance

- Cultural characteristics

- Identification of Brucella species

- Serological methods for detection of Brucella




Brucellae are coccobacillior short rods


- Tryptonesoya (tryptic soy) diphasicmedium

(Castaneda) is recommended for the isolation



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


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.

- A variety of colonial forms are produced by

Brucellastrains including smooth, mucoid, and

rough colonies. They may be colourless or



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.