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  Section: Microbiology Methods » Diagnostic Microbiology In Action
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Diagnostic Microbiology In Action
  Microbiology of the Respiratory Tract
      Isolation and Identification of Staphylococci
      Staphylococci in the Normal Flora
    Streptococci, Pneumococci, and Enterococci
      Isolation and Identification of Streptococci
      The CAMP Test for Group B Streptococci
      Identification of Pneumococci
      Identification of Enterococci
      Streptococci in the Normal Flora
    Haemophilus, Corynebacteri and Bordetella
    Clinical Specimens from the Respiratory Tract
      Laboratory Diagnosis of a Sore Throat
      Laboratory Diagnosis of Bacterial Pneumonia
      Antimicrobial Susceptibility Test of an Isolate from a Clinical Specimen

The genus Haemophilus contains a number of species of fastidious, gram-negative bacilli. Most of these are found as normal flora of the upper respiratory tract. Haemophilus species can cause infections in a variety of sites in the upper respiratory tract and elsewhere in the body. Laboratory diagnosis is made by identifying these organisms in clinical specimens appropriately representing the area of infection (throat swab, sinus drainage, sputum, conjunctival swab, spinal fluid, blood, or other). A direct smear of the specimen may be useful, particularly for spinal fluid or an exudate from the eye, in providing rapid, presumptive information. (Smears of material from the upper respiratory tract, with its mixed flora, may have little value unless the organisms are present in large numbers.) Latex antibody tests can also be performed directly with certain patient body fluids to detect Haemophilus antigen. Until an effective vaccine came into widespread use in the early 1990s, most serious Haemophilus disease was caused by H. influenzae serogroup b (H. influenzae strains are divided into serogroups a–f on the basis of their antigenic polysaccharide capsule). This organism is seldom isolated in the clinical laboratory today, but other Haemophilus species and H. influenzae serogroups other than serogroup b are occasionally encountered.

The fastidious Haemophilus organisms require specially enriched culture media and microaerophilic incubation conditions. “Chocolate” agar is commonly used for primary isolation of Haemophilus from clinical specimens. This medium contains hemoglobin derived from bovine red blood cells as well as other enrichment growth factors. Because the hemoglobin is dark brown, the agar in the plate has the appearance of chocolate.

Two special growth factors, called X and V, are required by some Haemophilus species. Some require one but not the other. The X factor is hemin, a heat-stable derivative of hemoglobin (supplied in chocolate agar). The V factor is a heat-labile coenzyme (nicotinamide adenine dinucleotide, or NAD), essential in the metabolism of some species that lack it. Yeast extracts contain V factor and are one of the most convenient supplements of chocolate agar or other media used for Haemophilus. Organisms other than yeasts elaborate V factor. Staphylococci, for example, when growing on an agar plate secrete NAD into the surrounding medium. Haemophilus species that need V factor may grow in the zone immediately around the staphylococci but not elsewhere on the plate. This growth of the dependent organism is described as “satellitism” (see colorplate 34). X and V factors can also be incorporated directly into agar media that do not contain these factors, or alternatively, they can be impregnated in filter-paper disks that are pressed on the surface of X and V factor–deficient media. In the latter case, the growth factors diffuse into the agar in a manner similar to diffusion from disks impregnated with antimicrobial agents (see Experiment 15.1).

Purpose To identify Haemophilus species in culture
Materials Sheep blood agar plate
Chocolate agar plate
Nutrient agar plate
X and V disks
Haemophilus ID Quad Plate
Chocolate agar plate cultures of Haemophilus influenzae and Haemophilus parainfluenzae
Demonstration blood agar and nutrient agar plates showing satellitism

  1. Describe the microscopic morphology of the two Haemophilus species you Gram stained, indicating any distinctions you observed between them.
  2. Complete the following chart, describing the Gram-stain appearance of the two Haemophilus species and indicating any morphological distinctions you observed between them. Describe the colonial morphology of each Haemophilus species.

  3. Diagram the appearance of the growth of each Haemophilus species on the nutrient agar plate with X and V disks and interpret.

  4. Diagram the appearance of the growth of each Haemophilus species on the Quad Plate and interpret (see colorplate 35).


  5. Diagram the appearance of the demonstration plates and interpret.


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