Laboratory diagnosis of Haemophilus influenzae

a. General considerations

  • Most Haemophilus species are normal inhabitants of upper respiratory tract of humans and other animals.
  • The species of Haemophilus that most frequently cause human infections are H. influenzae (respiratory and invasive infections), H. aegyptius (acute conjunctivitis) and H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. aprophilus, H. paraphrophilus and H. segnis (abscesses and infective endocarditis).

Find more information about Infectious disease associated with Haemophilus spp and specimen for culture

  • These are pleomorphic Gram negative coccobacilli.
  • The laboratory diagnosis of H. influenzae is based on growth and colony morphology in Chocolate Agar, and cell morphology on Gram staining.
  • These are confirmed by the haemophilic character of the genus that reflects a requirement for either or both of the two factors called X and V. The X factor comprises haemin or other iron containing porphyrins and V factor comprises nicotinamide adenine dinucleotide (NAD).

b.   Culture and identification for Haemophilus spp

Direct Examination

  • Perform Gram stain
  • Gram staining shows: Gram negative pleomorphic thin rods or coccobacilli.


  • Inoculate samples onto chocolate agar media (Think why not  Blood Agar or any other media)
  • Incubate at 37°C in aerobic atmosphere containing 5-10% CO2 for 24-48 hours.
Flow chart Haemophilus influenzae identification
Flow chart Haemophilus influenzae identification

Colony morphology on Chocolate Agar

  • large flat, colourless to gray or opaque colonies.
  • Colonies are 0.5 – 1mm circular, low convex, smooth, pale grey and transparent.
  • With a characteristic “mouse nest” odour. No haemolysis or discolouration is seen.
  • Encapsulated strains appear more mucoid (watery) and non capsulated strains appear as compact grayish colonies.

 Note:    Growth is enhanced on chocolate agar and  satellitism  around Staphylococcus aureus is seen on blood agar.

Biochemical reactions for differentiation.

            Confirmatory tests for X and V factor requirements.

X and V Factor: Growth is seen around XV Factor only.
X and V Factor: Growth is seen around XV Factor only.
  • Inoculate a single suspected colony from chocolate agar onto Mueller Hinton agar plates.
  • Place commercially available X, V, and XV factor discs/strips on streaked plates.
  • Incubate plates at 37°C in 5-10% CO2 atmosphere for 18-24 hours.
  • Observe growth around the discs and H. influenzae will only grow around the combined XV disc.

Note that if only X and V factor discs (without XV) are applied, place them at least 2 cm apart and H. influenzae will grow between the two discs.

Serological identification (serotyping) of Haemophilus influenzae

Slide agglutination test.

  • Agglutinating antisera for serotypes “a” to “f” are available commercially. Such sera contain antibodies directed towards somatic antigens present in patient’s sera which result in agglutination.
  • Apply one drop of normal saline on a slide and make a homogenous suspension with a single suspected colony of H. influenzae.
  • Add one drop of specific antiserum and mix thoroughly.
Anti-microbial Sensitivity Testing of H. influenzae in Chocolate Agar
Anti-microbial sensitivity testing of H. influenzae in Chocolate Agar


  • Observe for agglutination (visible clumping) within 1 minute.


  • A visible clumping within 1 minute is indicative of a positive reaction.

 Antimicrobial susceptibility testing.

  • Perform antimicrobial susceptibility test against a selected group of antimicrobials by disk-diffusion method
  • Follow the standard techniques as provided in the manual.

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