Culture and Identification of Fastidious Bacteria

Neisseriae AND Haemophilus Species

Neisseriae & GONORRHEAL DISEASES

Diseases: Gonorrhea.

Etiologic Agents: Neisseria gonorrhoeae

Source: Human genital tract by sexual contact or by birth.

Pathogenesis: Infections of mucous membranes lined by nonsquamous epithelium. Local epithelial cell destruction with PMNs.

Laboratory Diagnosis: Isolation of etiologic agent. Gram stain. Antigen detection by enzyme immunoassay. Several manufactures have identification kits that give rapid results like Dupont's "Gonocheck II"

NEISSERIAL DISEASES (other than gonorrhea)

Diseases: Megingitis (epidemic CNS fever), acute otitis media, maxillary sinusitis, bronchopulmonary infections

Etiologic Agents: Neisseria meningitidis, lactamica & sicca; Moraxella catarrhalis

Source: Commensal bacterial flora of the oropharynx, human respiratory aerosol transmission, acquired by inhalation.

Pathogenesis: Neisseria meningitidis - Central nervous system responses reflect acute inflammatory changes. Septic manifestations include disseminated intravasuclar coagulation and damage leading to shock.

Laboratory Diagnosis: M. catarrhalis infections - Gram-stained smear and culture; Neisseria spp. - Gram stain and culture from blood, CSF, or transtracheal aspirates isolation.

Adapted from Laboratory Diagnosis of Infectious Diseases, ed. A. Balows

Representative of the genus Neisseria are Gram-negative cocci which are oxidase positive and catalase positive.

Materials:

Neisseria lactamica culture BAP and Chocolate agar plates

Oxidase & Catalase reagents Nasal culture (sterile swab needed)

I. Morphological studies:

STAINING CHARACTERISTICS:

 1. Prepare a Gram stain of these organisms.
2. Study the morphological and staining characteristics.

CULTURE:

1. Streak the nasal swab for the isolation of Neisseria spp. from the normal flora on each of BAP and Chocolate agar plates.

2. Incubate in CO2 incubator at 37oC for 24 hours.

3. Study and record the colony characteristics.

II. Identification:

A. Oxidase Test


This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram-negative bacteria. It is a hallmark test for the Neiserria. It is also used to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other Enterobacteriaciae.

MATERIALS:
  • Bacterial colonies from nasal or oropharyngeal swab culture growing on Chocolate Agar Plates (CA)
  • Oxidase reagents
  • Sterile toothpicks
  • Neisseria culture and an unknown culture
PROCEDURES:

  1. Pick a colony for testing using a sterile wooden toothpick. Transfer the colony to the surface of one of the four grid areas on the Oxidase Test slide.
  2. The test slide surface is impregnated with the reagent tetramethyl-p-phenylenediamine dihydrochloride. This reagent causes a dark purple color to appear in the presence of cytochrome oxidase.
  3. Observe the color change. The reaction color will change from pink to maroon to dark purple.
  4. Read the test results within 10 seconds. Some organisms may show slight positive reactions after this period and such results are NOT considered definitive.

B. Catalase test

1. Transfer a large isolated colony from the culture dish to a clean

microscope slide.

2. Add two drops of 3% hydrogen peroxide reagent to the colony.

3. Observe for immediate bubbling (gas liberation) and record the result.

Identification of Neisseria spp.using Gonochek II

The Gonochek II ID Test utilizes three chromogenic substrates contained in a single tube to detect preformed enzymes associated with three pathogenic Neisseria species. The active chemical ingredients used in the tube and the enzyme reactions detected are:

1). 5-bromo-4-chloro-indoyl-ß-D-galactopyranoside (50 ug).
Hydrolysis of the ß-D-galactoside bond by ß-galactosidase yields a blue color.

2). Gamma-glutamyl-para-nitroanilide (50 ug).
Hydrolysis of this substrate by gamma-glutamyl aminopeptidase releases yellow p-nitroaniline.

3). ß-naphthyl amino acid derivative (50 ug).
Hydrolysis of this substrate by hydroxypyroly aminopeptidase releases colorless free ß-naphthylamine derivative. Complexing of the ß-naphthylamine derivative with a diazo dye coupler (O- amino-toluene diazonium salt) contained in the red stopper of the Gonochek II tube produces a pink or red color.

PROCEDURE:

1. Only oxidase-positive isolates should be used.

2. Remove the paired stoppers from the tube.

3. Dispense four drops of PBS into the Gonocheck II tube.

4. Harvest 5-10 medium to large size colonies from a single plate and emulsify well into the Gonochek II tube. Recap tube with paired stoppers.

5. Incubate in 37oC incubator for 30 minutes.

6. If the Gonochek II is blue or yellow do not continue. If there is no color change, remove the red stopper and then the translucent stopper. Discard the translucent stopper and recap tube with the red stopper. Invert the tube to allow the liquid in the tube to come into contact with the reagent on the red stopper. Return the tube to an upright position. The color of the liquid will turn either pink/red or remain unchanged.

RESULTS:

Gonochek II Species

Pink/red after inverting tube= N. gonorrhea

Yellow= N. meningitidis

Blue= N. lactamica

No color change= B. catarrhalis

Haemophilus influenza DISEASES

Diseases: Pneumonia, meningitis, epiglottitis, septic arthritis, cellulitis, otitis media, conjunctivitis, neonatal infection, and other clinical syndromes

Etiologic Agents: Haemophilus influenza (both serotypable and nonserotypable).

Source: Human respiratory and genital tract by direct contact or inhalation.

Pathogenesis: Infection by serotypable H. influenza results in a primary bacteremia followed by acute pyogenic infection. Infection by nonserotypable H. influenza results in isolated mucosal infection with an occasional secondary bacteremia.

Laboratory Diagnosis: Laboratory cultures of organisms from blood, CSF, joint fluid, and biopsy material.

INFECTIONS BY Haemophilus SPECIES
OTHER THAN H. influenza

Diseases: Conjunctivitis, genital ulcer, endocarditis, brain abscess, pneumonia, and other syndromes.

Etiologic Agents: H. parainfluenza and other Haemophilus species.

Source: Human respiratory and genital tract by direct contact or inhalation.

Pathogenesis: Acute pyogenic infections

Laboratory Diagnosis: Isolation of the organism from blood, sputum, tissue or mucosal swab.

Adapted from Laboratory Diagnosis of Infectious Diseases, ed. A. Balows

The bacterial species in this group can cause serious respiratory infections. Haemophilus also has species commonly found in the normal flora.

The genus Haemophilus contains a number of species of fastidious, Gram-negative coccobacilli. Gram-stained preparations of infected clinical materials show pleomorphic threadlike filaments, commonly admixed with the coccobacillary forms. These fastidious organisms require specially enriched culture media such as chocolate agar and microaerophilic conditions of incubation. Culture on both BAP and chocolate agar (heated blood agar), with growth only on the chocolate agar is characteristic of the Haemophilus group organisms. The use of blood agar also allows differentiation of H. haemolyticus from H. influenzae based on hemolysis produced by the former. Some species require Factor X, a heat-stable derivative of hemoglobin; others require NAD, also known as Factor V. Factor V is heat-labile. It can be derived from extracts of yeasts and is produced by certain bacteria such as Staphylococcus aureus. H. influenza requires both X and V factors. These culture characteristics will be reinforced during the satellitism demonstration (see next page).

 

CULTURE OF Haemophilus SPECIES

MATERIALS REQUIRED:

Haemophilus influenzae 2 BAP plates

Haemophilus parainfluenzae 2 Chocolate agar plates

Streak blood agar and chocolate agar plates with each species. Incubate within a candle jar at 37oC overnight or longer. Note differences in the two media’s ability to support growth, and in colony morphology.

 

 

Staphylococcus Streak Technique (Satellitism)

Material required:
Haemophilus influenzae 1 BAP
Haemophilus parainfluenzae
Staphylococcus aureus

PROCEDURE:

1. H. influenzae should be heavily streaked on a BAP.

2. Using an inoculating wire, a single narrow streak of a hemolytic Staphylococcus known to produce NAD (factor V) should be made through the area where the H. influenza had been inoculated.

3. Incubate plates in a candle jar at 37oC for 24 hours. Observe the results and record below:

V and X Factor Production: Strain Identification

Material required: (each group of four students)

2 NA plates Haemophilus influenzae

X, V & XV discs Haemophilus parainfluenzae

PROCEDURE:

1. Each of the Haemophilus strains should be inoculated heavily for confluent growth on the NA plates.

2. Divide each streaked plate in thirds (draw sections on the plate bottom with a marker). Place an X disc at the center of one of the thirds, a V disc at the center of a second section, and an XV disc at the center of the third section (place discs on the agar surface).

3. Repeat the procedure for the second strain. Both plates should be incubated in a candle jar at 35oC for 24 hours (this produces a microaerophilic condition).

4. A characteristic growth pattern should be the result of this exercise. Record your observations.

 
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Authored by Mary T. Johnson, Ph.D. Last modified November 27, 2007