Haemophilus influenzae is a small, Gram-negative bacterium recognized as a significant cause of various clinical syndromes. This organism can cause serious, life-threatening infections in children and adults, including meningitis, epiglottitis, and pneumonia. Rapid and accurate identification of H. influenzae in a clinical laboratory setting is necessary for guiding timely and effective patient treatment. Biochemical testing provides a precise method for distinguishing this species from other similar bacteria.
Essential Growth Requirements (X and V Factors)
The Haemophilus genus is characterized by its fastidious nature, meaning the bacteria have complex nutritional requirements and will not grow on standard laboratory media. H. influenzae requires two distinct accessory growth factors to fuel its metabolic processes: X factor (Hemin) and V factor (Nicotinamide Adenine Dinucleotide, or NAD).
Hemin (X factor) is an iron-containing component needed to synthesize essential respiratory enzymes, such as cytochromes and peroxidase. NAD (V factor) functions as a coenzyme in oxidation-reduction reactions, acting as an electron carrier within the organism’s metabolic pathways. Since H. influenzae cannot synthesize these compounds independently, they must be supplied externally in the culture medium.
This unique growth requirement is demonstrated by the classic Satellite Phenomenon test. When plated on a standard blood agar medium, H. influenzae will not grow alone because the X factor is bound within red blood cells and the V factor is absent or quickly degraded. If another bacterium, such as Staphylococcus aureus, is streaked nearby, H. influenzae colonies will appear as tiny “satellites” clustered closely around the helper organism. The helper organism excretes the V factor and releases the X factor by lysing the surrounding red blood cells, making both necessary nutrients available.
Primary Differentiation Tests (Oxidase and Catalase)
Clinical labs use rapid enzymatic tests to quickly narrow down the identity of an unknown bacterial isolate before performing specific factor-requirement assays. These general tests assess the presence of common bacterial enzymes that help differentiate large groups of microorganisms. The Catalase test and the Oxidase test are the primary initial screening tools for H. influenzae identification.
The Catalase test checks for the catalase enzyme, which allows the bacterium to break down hydrogen peroxide into water and oxygen gas. This enzyme helps the organism survive in an oxygen-rich environment by neutralizing toxic byproducts. H. influenzae is positive for this reaction, indicated by the rapid formation of bubbles upon adding the reagent.
The Oxidase test detects cytochrome c oxidase, an enzyme complex that is part of the bacterial electron transport chain. A positive result indicates that the organism utilizes oxygen as a terminal electron acceptor. H. influenzae strains consistently yield a positive result, which is visualized by a color change on the test strip or reagent within seconds.
Testing for Heme Synthesis (The Porphyrin/ALA Test)
The Porphyrin test, also known as the ALA test, provides definitive species identification by testing the organism’s ability to synthesize its own heme. This test differentiates H. influenzae from closely related species like Haemophilus parainfluenzae. The procedure supplies the bacteria with delta-aminolevulinic acid (ALA), the first precursor in the heme biosynthetic pathway.
If a Haemophilus strain possesses the necessary enzymes, it converts the supplied ALA into porphyrins, which are intermediate products leading to heme. These porphyrins accumulate and are excreted by the bacteria, serving as a marker for the organism’s ability to produce its own X factor. A positive result is identified by a strong red-orange fluorescence when the culture is examined under long-wave ultraviolet light (366 nm).
The interpretation of the ALA test is based on the organism’s growth factor requirement. A positive fluorescent result indicates the bacteria can synthesize their own heme and do not require exogenous X factor (Hemin); this result rules out H. influenzae. Conversely, H. influenzae lacks the enzyme needed for this conversion and is unable to produce porphyrins, resulting in a negative test (no fluorescence). This negative result confirms the organism’s dependence on the X factor and supports the identification of H. influenzae.
Classification by Metabolism (Indole and Urease Biotyping)
Once an isolate is confirmed as H. influenzae, additional biochemical tests categorize the strain into a specific biotype. Biotyping is useful for epidemiological tracking and outbreak investigations, classifying strains into categories designated I through VIII based on metabolic activity.
The Indole test detects the enzyme tryptophanase, which breaks down the amino acid tryptophan. A positive result shows the production of indole, visualized by adding a specific reagent that produces a red color.
The Urease test determines if the organism produces the urease enzyme, which hydrolyzes urea into ammonia and carbon dioxide. The ammonia byproduct raises the pH of the test medium, causing a color change in the indicator to signal a positive result. Both the Indole and Urease tests yield variable results among H. influenzae strains, creating a unique biochemical fingerprint for the clinical laboratory record.