How should a biopsy specimen with suspected fungal infection be planted for culture, including media and incubation conditions?

Fungal Culture Protocol for Biopsy Specimen with Septate Hyphae

The biopsy specimen showing septate hyphae with acute-angle branching should be cultured on Sabouraud dextrose agar with antibiotics (gentamicin and chloramphenicol) at both 30°C and 37°C for at least 72 hours, with extended incubation up to 3 weeks if needed.

Primary Media Selection

• Sabouraud dextrose agar (SDA) with antibiotics is the gold standard for primary isolation 1
  • Add gentamicin and chloramphenicol to inhibit bacterial growth
  • Use high-quality commercial formulations to ensure consistent results 2
  • Brain Heart Infusion (BHI) agar should also be used as it can help recover some isolates that may not grow well on SDA 1

Specimen Processing

1. Direct inoculation of the biopsy material onto culture media is essential for maximum yield 1
2. Process specimen rapidly to prevent overgrowth by bacteria and other contaminants 1
3. Divide specimen to allow for:
   • Histopathologic examination with GMS (Gomori methenamine silver) and PAS (periodic acid-Schiff) stains
   • Direct microscopic examination with fluorescent dyes (Calcofluor White or Blancophor)
   • Culture on multiple media 1

Incubation Conditions

• Temperature: Incubate at both 30°C and 37°C simultaneously 1
• Duration: Minimum 72 hours, but maintain cultures for up to 3 weeks 1
• Humidity: Maintain adequate humidity to prevent drying of media
• Examination frequency: Check cultures daily for the first week, then every 2-3 days

Special Considerations

• Morphologic identification is critical - the acute-angle branching septate hyphae described are characteristic of Aspergillus species 1
• Extended incubation may be necessary as some filamentous fungi grow slowly, particularly if the patient has received antifungal therapy 1
• If growth is poor or atypical, consider:
  • Using specialized media like potato dextrose agar (PDA) for better sporulation 1
  • Water agar supplemented with sterile distilled water and 0.2% yeast extract to induce sporulation of difficult species 3

Identification Methods

1. Macroscopic examination: Colony morphology, color, texture, and growth rate
2. Microscopic examination: Prepare slide cultures or tape preparations to observe characteristic conidiophores and spore arrangements 1, 4
3. Species identification: For isolates with atypical growth or concerns for resistance, molecular methods should be employed 1

Quality Control

• Include positive control strains to verify media performance
• Use the highest quality commercial SDA formulations, as significant differences exist between manufacturers 2
• Document all growth characteristics systematically

Common Pitfalls to Avoid

1. Inadequate specimen quantity - ensure sufficient material is collected for both histopathology and culture 1
2. Delayed processing - transport specimens to the laboratory immediately 1
3. Premature termination of incubation - some molds require extended incubation periods 1
4. Failure to use antibiotics in media - bacterial overgrowth can mask fungal growth 1
5. Using anesthetics with preservatives during biopsy collection, which can inhibit fungal growth 5

The morphological description of septate hyphae with acute-angle (dichotomous) branching strongly suggests Aspergillus infection, which is consistent with the clinical presentation of a neutropenic patient with fever and pulmonary infiltrates 1. Proper culture techniques are essential for species identification and potential antifungal susceptibility testing, which will guide optimal therapy for this life-threatening infection.