Haemophilus Microbiology Notes

The Genus Haemophilus

  • Overview of the Genus Haemophilus

    • Composed of Gram-negative bacilli.

    • Characteristics:

    • Facultative anaerobes.

    • Non-motile.

    • Non-endospore forming.

    • Oxidase positive, indicating the presence of cytochrome C oxidase in the electron transport chain (ETC).

Classification and Importance of Haemophilus

  • Family: Pasteurellaceae.

  • Species Classification:

    • Includes fastidious species, requiring enriched media for growth.

  • Normal Microbiota:

    • Haemophilus species are commonly found in the normal microbiota of the upper respiratory tract.

  • Clinical Significance:

    • Can lead to infections in the upper respiratory tract, lower respiratory tract, or meninges.

    • Clinical specimens may include:

    • Throat swab

    • Sinus drainage

    • Conjunctival swab

    • Sputum

    • Cerebrospinal fluid (CSF)

    • Blood

    • Direct smear of specimens can provide quick presumptive information (e.g., in meningitis or eye exudate).

Haemophilus influenzae

  • Historical Context:

    • Until the early 1990s, H. influenzae serogroup b (Hib) was a significant cause of meningitis in young children.

  • Capsule Structure:

    • H. influenzae strains are categorized into serogroups a-f based on their antigenic polysaccharide capsule.

  • Preventative Measures:

    • Conjugate vaccines are effective in preventing Hib disease in children.

  • Non-Encapsulated Strains:

    • Known as non-typable strains (NTHi); cause infections like eye infections, otitis media, and sinusitis in children.

Culture Media and Incubation Conditions

  • Media Requirements:

    • Fastidious species of Haemophilus require enriched media for primary isolation.

    • Commonly used media include:

    • Chocolate agar (contains hemoglobin and yeast extract providing growth factors).

    • Haemophilus ID Quad Plates.

      • Uses disks with X (hemin) and V (NAD) factors.

  • Chocolate Agar:

    • A complex medium essential for Haemophilus growth.

    • Contains:

    • Hemoglobin from bovine red blood cells.

    • Yeast extract for V factor support.

Testing Growth Factor Requirements

  • Quad Plate Usage:

    • Crucial for determining requirements for X and V factors.

    • Design:

    • Two quadrants represent only X or V factors.

    • One quadrant includes both X and V factors.

    • Fourth quadrant includes blood (5% blood agar).

  • Growth Interpretation for H. influenzae:

    • Grows in the quadrant containing both X and V factors, indicating dependency on both.

Satellitism Phenomenon

  • Description:

    • Observed colonies of H. influenzae grow around Staphylococcus streak on Blood Agar Plates (BAP).

    • Mechanism:

    • The medium provides the X factor, while Staphylococcus releases the V factor into the surrounding medium.

Laboratory Exercises Overview

  • Objectives:

    • Each student performs a Gram stain.

    • Review of the Gram stain method through a video tutorial (link provided).

  • Observation After Staining:

    • Students will describe their observations post-staining (specific observations are left to student input).

Oxidase Test

  • Purpose:

    • Used to detect cytochrome c oxidase within the electron transport chain.

  • Method:

    • Place filter paper on a glass slide.

    • Add a drop of TMPD (tetramethyl-p-phenylenediamine dihydrochloride).

    • Smear bacterial cells onto the drop with a sterile wooden toothpick.

    • Observe color change indicating a positive test.

Streaking Techniques for H. influenzae

  • Materials Required:

    • Chocolate agar plates.

    • Bunsen burner, wire inoculating loop, candle jar or CO2 gas pak system, standard incubator.

  • Process:

    • Perform streaking techniques to isolate H. influenzae colonies.

    • Increase CO2 levels for optimal growth.

  • Expected Results After Incubation (35-37°C for 48 hours):

    • Expect translucent, gray, moist colonies with a musty odor.

Streaking H. influenzae on TSA plates

  • Initial Steps:

    • Use TSA plus X (hemin) and V (NAD) disks for isolation.

    • Divide the bottom of TSA plates into three sections marked for X, V, and XV factors.

  • Plating and Incubation Protocol:

    • Streak with a sterile inoculating loop and use sterile tweezers to place disks.

    • Incubate plates at 35-37°C for 48 hours with increased CO2.

Analysis of Simulated Clinical Specimens

  • Throat Swab Scenario:

    • Exercise involving BAP and throat swab methodology to isolate Streptococcus pyogenes causing strep throat.

    • Incubation guidelines: Invert BAP plates for 24-48 hours at 35-37°C.

  • Expected Outcomes Post-Incubation:

    • Hemolytic colonies exhibit clear halos on BAP.

    • Perform Gram stain reaction, morphology, catalase activity, bacitracin susceptibility (CAMP test), and serological testing to identify group beta-hemolytic Streptococcus.

  • Sputum Sample Scenario:

    • Similar analysis for identifying Staphylococcus pneumoniae and Staphylococcus aureus.

    • Interpret MSA and BAP results post-incubation (24 hours at 35-37°C).

Summary of Expected Results for Specimens

  • Throat Swab (Streptococcus pyogenes):

    • Hemolysis type, Gram stain reaction, morphology assessed for identification.

  • Sputum Sample:

    • Mannitol fermentation on MSA, hemolytic pattern, Gram stain reaction, catalase, coagulase activity, and positive serological tests for surface proteins assessed for identification.