MYCOLOGY

• Culture Media:

  • Any material containing essential nutrients to support the growth of fungi
  • Should have a slightly acidic pH 5.6 to neutral pH
  • 2 cultures must be incubated in different temperatures:
  • 22°C (Room Temperature) to observe molds
  • 37°C inside incubator to observe dimorphism/yeast
• Inhibitory Agents (Antibiotics):

  • Cycloheximide: inhibits fungal contaminants
  • Chloramphenicol: inhibits both gram positive and gram negative organisms, especially if the sample comes from an unsterile area
  • Gentamycin: prevents gram negative bacteria
  • Streptomycin: prevents gram-positive bacteria
• Growth Rate:

  • Rapid Growers (1-3 days): Dermatophytes
  • Intermediate/Moderate Growers (5-9 days): Opportunistic Fungi
  • Slow Growers (2 weeks - 8 months): Systemic & Subcutaneous Fungi
• Safety:

  • Use biosafety cabinet type II
  • Wear PPE because fungi have spores that are carried out through the air (airborne)
  • Do not use an ordinary mask, use n95/respiratory mask
• Tube Media vs Plated Media:

  • Tube Media:
  • Handling spores
  • Advantages: prevent spore release into the environment, prevent dehydration, easily handled and stored
  • Plated Media:
  • Examining colonial appearance, texture, and topography
  • Advantage: easily examine (larger surface area for observation)
• Culture Media Used for Fungi:

  • Sabouraud Dextrose Agar (SDA): used for pathogenic (dimorphic) and saprophytic fungi, contains dextrose, casein, and animal tissues in the form of peptones, pH 5.6 (acidic)
  • Emmon’s Modification Media: used for pathogenic and non-pathogenic fungi, contains 24 grams of dextrose, pH 6.9 (neutral)
  • Brain Heart Infusion (BHI): used for systemic mycoses, can be cultured in BHI
• Culture Media:

  • Any material containing essential nutrients to support the growth of fungi
  • Should have a slightly acidic pH 5.6 to neutral pH
  • 2 cultures must be incubated in different temperatures:
  • 22°C (Room Temperature) to observe molds
  • 37°C inside an incubator to observe dimorphism/yeast
• Inhibitory Agents (Antibiotics):

  • Cycloheximide: Inhibits fungal contaminants
  • Chloramphenicol: Inhibits both gram positive and gram negative organisms, especially if the sample comes from an unsterile area
  • Gentamycin: Prevents gram negative bacteria
  • Streptomycin: Prevents gram-positive bacteria
• Growth Rate:

  • Rapid Growers (1-3 days): Dermatophytes
  • Intermediate/Moderate Growers (5-9 days): Opportunistic Fungi
  • Slow Growers (2 weeks - 8 months): Systemic & Subcutaneous Fungi
• Safety:

  • Use biosafety cabinet type II
  • Wear PPE because fungi have spores that are airborne
  • Do not use an ordinary mask, use n95/respiratory mask
• Tube Media vs Plated Media:

  • Tube Media:
  • Handling spores
  • Advantages: Prevent spore release into the environment, prevent dehydration, easily handled and stored
  • Plated Media:
  • Examining colonial appearance, texture, and topography
  • Advantage: Easily examine (larger surface area for observation)
• Culture Media Used for Fungi:

  • Sabouraud Dextrose Agar (SDA): Used for pathogenic (dimorphic) and saprophytic fungi, contains dextrose, casein, and animal tissues in the form of peptones, pH 5.6 (acidic)
  • Emmon’s Modification Media: Used for pathogenic and non-pathogenic fungi, contains 24 grams of dextrose, pH 6.9 (neutral)
  • Brain Heart Infusion (BHI): Used for systemic mycoses, can be cultured in BHI
• Sabouraud Dextrose Agar (SDA):

  • Contains dextrose since yeast & molds love sugar
  • Peptones provide amino acids and nitrogenous compounds essential for fungal growth
  • Inhibitory Substances Added: Cycloheximide, Chloramphenicol, Chloramphenicol & Gentamicin, Chloramphenicol & Tetracycline
• Emmon’s Modification Media:

  • Same as SDA but does not contain Casein
• Brain Heart Infusion (BHI):

  • Biphasic Culture Bottles for recovery of fungi in blood and bone marrow
  • Brain-Heart Infusion Agar (BHI) used for bacteria, yeast, saprobic & pathogenic fungi
• Dermatophyte Test Medium (DTM):

  • Used to recover dermatophytes
  • Sample is from cutaneous sample (hair strands, nails, skin scrapings)
  • Principle: Dermatophyte releases alkaline metabolites causing pH increase turning culture medium from yellow to pink
• Mycosel/Mycobiotic Agar:

  • Used for isolation of pathogenic fungi (dermatophytes) from samples with large amounts of normal flora
  • Inhibitory Agents: Cycloheximide & Chloramphenicol
• Littman Oxgall Agar:

  • For isolation of dermatophytes and Aspergillus flavus
  • Aspergillus flavus is the most pathogenic species of Aspergillus due to the presence of aflatoxin
• Lima Bean Agar:

  • For recovery of dermatophytes
  • Growth of colony starts from the center
• Phyton Yeast Extract Agar (PYEA):

  • For recovery of dermatophytes, specifically Trichophyton verrucosum
  • Inhibitory Agents: Chloramphenicol & Streptomycin
• Yeast Extract Phosphate Agar:

  • For recovery of systemic fungi such as Histoplasma capsulatum and Blastomyces dermatitidis from contaminated specimens
• Inhibitory Mold Agar (IMA):

  • Recovery of dimorphic pathogenic fungi except dermatophytes
  • Inhibitory Agents: Chloramphenicol & Gentamycin
• Birdseed (Niger Seed) Agar:

  • Demonstration of melanin
  • For Cryptococcus neoformans
  • Differentiating C. neoformans and C. albicans
• Nitrate Reduction Medium:

  • For C. neoformans
  • Nitrate reduces to nitrite
• Christensen Urea Agar/Urease Test:

  • Used to differentiate Trichophyton mentagrophytes from Trichophyton rubrum
  • Trichophyton mentagrophytes: Positive to Urease Test (purple)
  • Trichophyton rubrum: Negative to Urease Test
• Cornmeal Agar with Tween 80 (Chlamydospore Agar):

  • Stimulates the production of chlamydospore
  • Used for isolation of Candida albicans
• Other Culture Media Used for Differentiation of Candida spp:

  • Inoculation technique for CHROM Agar is 4 quadrant streaking pattern
  • Candida BCG Agar, BIGGY Agar, Pagano Levin Agar, CHROM Agar/Chromogenic Agar
• Trichophyton Agar (1-7):

  • Used to differentiate pre-identified Trichophyton spp
  • Different preparations with varying vitamin amino acids
• Rice Medium:

  • For isolation of Microsporum audouinii and Microsporum canis
  • M. audouinii: Brown discoloration
  • M. canis (+): Woolly texture
• Potato Dextrose Agar (PDA):

  • Used to differentiate T. mentagrophytes and T. rubrum
  • Alternative to Sabouraud Dextrose Agar
• Bromocresol Purple (BCP):

  • Isolation of dermatophytes
  • (+): Blue to purple (presence of dermatophytes)
• Cottonseed Agar/KT Medium/Kelly Agar:

  • Isolation of Blastomyces dermatitidis (systemic & dimorphic)
  • Yeast Form Conversion to Mold Form
• Czapek’s Agar:

  • Isolation of Aspergillus spp. and Penicillium
  • Utilization of Sodium nitrate as a carbon source
• Carbohydrate Yeast Assimilation Media:

  • Ability to utilize carbohydrates as a source of carbon
  • Growth: Purple to yellow, pH Indicator: Bromocresol Purple
• Carbohydrate Fermentation Broth:

  • Yeast and yeast-like fungi utilize carbohydrates by acid and gas production
  • Incubated under anaerobic conditions
• Acetate Ascospore Agar:

  • Isolation of yeast Saccharomyces cerevisiae
• Nutritionally Poor Media:

  • Recovering fungi from nonclinical samples
  • Includes Dilute Hay Infusion Agar, Soil Extract Agar, 2% Water Agar
• Laboratory Methods:

  • Wet Mount Preparation
  • Scotch Tape Preparation
  • Tease Mount Preparation
  • Slide Culture/Microculture Method
  • Hanging Drop Culture