Fastidious Species

Specialized media are essential for isolating and identifying fastidious bacterial species that require specific growth conditions or nutrients not found in routine culture media. These media are formulated to provide the necessary growth factors, inhibit competing organisms, and aid in the identification of these challenging bacteria

Specialized Media for Fastidious Bacteria: Meeting Unique Growth Requirements

What are Fastidious Bacteria?
- Fastidious bacteria are microorganisms with complex nutritional requirements that are difficult to grow in standard culture media
- These bacteria require specific growth factors, such as vitamins, amino acids, or other organic compounds, that are not typically found in routine media
Why are Specialized Media Important for Fastidious Bacteria?
- Optimal Growth: Specialized media provide the specific nutrients and growth factors that fastidious bacteria need to grow and reproduce
- Selective Isolation: Some specialized media contain selective agents that inhibit the growth of competing organisms, making it easier to isolate the target bacteria
- Accurate Identification: Specialized media can help to differentiate between different species of fastidious bacteria based on their growth characteristics
- Detection of Pathogens: Many fastidious bacteria are important human pathogens, and specialized media are essential for their detection and identification
General Strategies for Culturing Fastidious Bacteria
- Enrichment: Adding specific nutrients or growth factors to the media
- Selective Inhibition: Using selective agents to suppress the growth of competing organisms
- Atmospheric Control: Providing the appropriate atmospheric conditions (e.g., anaerobic, microaerophilic)
- Temperature Control: Maintaining the optimal temperature for growth
- Moisture Control: Providing adequate humidity to prevent desiccation

Specific Specialized Media and Their Applications

Chocolate Agar

Composition: Enriched with lysed red blood cells, which release intracellular nutrients such as hemin (Factor X) and nicotinamide adenine dinucleotide (NAD, Factor V)
Target Organisms: Haemophilus species, Neisseria species
Applications
- Isolation of Haemophilus influenzae from respiratory specimens
- Isolation of Neisseria gonorrhoeae and Neisseria meningitidis from genital and CSF specimens
Rationale
- Haemophilus species require hemin and NAD for growth, which are released from lysed red blood cells
- Chocolate agar provides a rich source of these growth factors
Key Considerations
- Chocolate agar should be incubated in a humidified atmosphere with 5-10% CO2
- Modified Thayer-Martin agar is a selective version of chocolate agar used for Neisseria species

Modified Thayer-Martin (MTM) Agar

Composition: Chocolate agar base with added antibiotics (vancomycin, colistin, nystatin, and trimethoprim) to inhibit the growth of Gram-positive bacteria, Gram-negative bacteria, and fungi
Target Organisms: Neisseria gonorrhoeae, Neisseria meningitidis
Applications
- Selective isolation of Neisseria gonorrhoeae from genital specimens
- Selective isolation of Neisseria meningitidis from respiratory specimens
Rationale
- The antibiotics in MTM agar inhibit the growth of competing organisms, allowing Neisseria species to grow selectively
Key Considerations
- MTM agar should be incubated in a humidified atmosphere with 5-10% CO2
- Proper storage and handling of MTM agar are essential to maintain the activity of the antibiotics

Loeffler’s Serum Slant

Composition: Enriched with coagulated serum (typically bovine or horse serum) and dextrose
Target Organisms: Corynebacterium diphtheriae
Applications
- Isolation and identification of Corynebacterium diphtheriae from throat swabs
Rationale
- Loeffler’s serum slant provides a rich source of nutrients for Corynebacterium diphtheriae
- The high protein content enhances the metachromatic staining of volutin granules, which are characteristic of C. diphtheriae
Key Considerations
- Loeffler’s serum slant should be incubated aerobically at 35-37°C
- Gram stain and specialized staining techniques (e.g., Albert’s stain) are used to confirm the identification of C. diphtheriae

Buffered Charcoal Yeast Extract (BCYE) Agar

Composition: Enriched with yeast extract, charcoal, L-cysteine, and iron salts, buffered to a pH of 6.9
Target Organisms: Legionella species
Applications
- Isolation of Legionella pneumophila from respiratory specimens and environmental samples
Rationale
- Legionella species require L-cysteine and iron salts for growth
- Charcoal helps to remove toxic byproducts and enhance growth
- The buffered pH provides an optimal environment for Legionella
Key Considerations
- BCYE agar should be incubated in a humidified atmosphere at 35-37°C
- Legionella colonies typically appear as gray-white or blue-green with a ground-glass appearance
- Confirmation of Legionella identification requires serological testing

Skirrow’s Agar

Composition: Blood agar base with added antibiotics (vancomycin, polymyxin B, and trimethoprim) to inhibit the growth of competing organisms
Target Organisms: Campylobacter jejuni, Campylobacter coli
Applications
- Selective isolation of Campylobacter species from stool samples
Rationale
- The antibiotics in Skirrow’s agar inhibit the growth of most bacteria, allowing Campylobacter species to grow selectively
Key Considerations
- Skirrow’s agar should be incubated in a microaerophilic atmosphere (5% O2, 10% CO2, 85% N2) at 42°C
- Campylobacter colonies typically appear as gray-white or pinkish and may be moist or mucoid

Cefsulodin-Irgasan-Novobiocin (CIN) Agar

Composition: Selective and differential medium containing cefsulodin, irgasan, and novobiocin to inhibit the growth of competing organisms, as well as mannitol and neutral red to differentiate mannitol-fermenting bacteria
Target Organisms: Yersinia enterocolitica
Applications
- Selective isolation of Yersinia enterocolitica from stool samples
Rationale
- The antibiotics in CIN agar inhibit the growth of most bacteria, allowing Yersinia enterocolitica to grow selectively
- Yersinia enterocolitica ferments mannitol, producing colonies with a pink or red center (“bull’s-eye” appearance)
Key Considerations
- CIN agar should be incubated at 25-30°C for optimal recovery of Yersinia enterocolitica
- Confirmation of Yersinia enterocolitica identification requires biochemical testing

Bordet-Gengou (BG) Agar

Composition: Enriched with potato infusion and glycerol, and supplemented with 15-20% defibrinated sheep blood
Target Organisms: Bordetella pertussis, Bordetella parapertussis
Applications
- Isolation of Bordetella pertussis and Bordetella parapertussis from nasopharyngeal swabs
Rationale
- BG agar provides a rich source of nutrients for Bordetella species
- The potato infusion and glycerol enhance growth, while the blood provides additional growth factors
Key Considerations
- BG agar should be incubated in a humidified atmosphere at 35-37°C
- Bordetella colonies typically appear as small, glistening, and gray-white
- Confirmation of Bordetella identification requires serological testing or PCR

Lowenstein-Jensen (LJ) Medium

Composition: Enriched with egg, glycerol, and mineral salts, and contains malachite green to inhibit the growth of most bacteria
Target Organisms: Mycobacterium tuberculosis, other Mycobacterium species
Applications
- Isolation of Mycobacterium tuberculosis from respiratory specimens
- Isolation of other Mycobacterium species from clinical specimens
Rationale
- LJ medium provides a rich source of nutrients for Mycobacterium species
- Malachite green inhibits the growth of most other bacteria, allowing Mycobacterium to grow selectively
Key Considerations
- LJ medium should be incubated at 35-37°C in a humidified atmosphere with 5-10% CO2
- Cultures should be incubated for up to 8 weeks to allow for slow-growing Mycobacterium species to grow
- Confirmation of Mycobacterium identification requires biochemical testing, molecular methods, or lipid analysis

Key Considerations and Best Practices

Specimen Collection: Collect specimens properly to minimize contamination and ensure adequate sample volume
Transport: Transport specimens promptly to the laboratory in appropriate transport media
Media Selection: Choose the appropriate specialized media based on the suspected pathogens and the specimen type
Incubation Conditions: Follow the recommended incubation conditions for each medium, including temperature, atmosphere, and duration
Quality Control: Perform quality control testing on all media to ensure proper performance
Identification: Use appropriate identification methods to confirm the identity of isolated bacteria
Documentation: Document all media selection, incubation conditions, and identification results

Key Terms

Fastidious Bacteria: Microorganisms with complex nutritional requirements
Specialized Media: Culture media formulated to support the growth of specific microorganisms
Enrichment: Adding specific nutrients or growth factors to the media
Selective Inhibition: Using selective agents to suppress the growth of competing organisms
Atmospheric Control: Providing the appropriate atmospheric conditions (e.g., anaerobic, microaerophilic)
Growth Factors: Organic compounds that are required by some microorganisms for growth
Selective Agents: Substances that inhibit the growth of certain microorganisms
Differential Agents: Substances that allow different types of microorganisms to be distinguished
Quality Control: A set of procedures designed to ensure the accuracy and reliability of laboratory test results
Standard Operating Procedure (SOP): A detailed written instruction to achieve uniformity of the performance of a specific function

Selection

Mycobacteria & Fungi