Lactophenol Cotton Blue (LPCB) - Principle and Morphology

What is LPCB?

Composition and Principle

Safety Note

Methods of Preparation

1. Cellophane (adhesive) tape mount - A piece of clear tape is briefly pressed onto the colony surface midway between the center and outer margin, then placed sticky-side down onto a drop of LPCB on a glass slide. This preserves the spatial arrangement of conidial structures.
2. Teased preparation - Mycelium is teased apart with inoculating needles, placed in a drop of LPCB on a slide, covered, and examined. The established standard method.
3. Slide culture - A square of agar (Sabouraud, potato dextrose, or potato flake) is suspended on a glass rod in a humid Petri dish and overlaid with a coverslip. After a few days of incubation, the coverslip is removed and placed in LPCB to observe undisturbed conidial structures in their original spatial relationships - used for easily disrupted reproductive structures.

Morphological Structures Visible on LPCB

1. Hyphae

• Septate hyphae - hyphae divided by cross-walls (septa); seen in most pathogenic molds (Aspergillus, dermatophytes)
• Aseptate (pauciseptate) hyphae - broad, ribbon-like, mostly without septa; characteristic of the Mucorales (Rhizopus, Mucor)

2. Conidia (Asexual Spores)

• Epidermophyton floccosum: blunt-ended, club-shaped macroconidia in aggregates along hyphae; no microconidia present
• Trichophyton mentagrophytes: elongated, pencil-shaped macroconidia lined along hyphae, with small microconidia alongside
• Microsporum canis: large, spindle-shaped, thick-walled, rough-walled macroconidia (black arrow) with microconidia (red arrow) - see image below

3. Conidiophores and Specialized Structures

• Aspergillus spp. - conidiophore swells at the tip into a vesicle; phialides arise from the vesicle surface and bear chains of conidia parallel to the long axis; some species also have metulae (supporting sterile cells between vesicle and phialides)
• Rhizopus spp. (Mucorales) - sporangiophores support a round sporangium containing sporangiospores; rhizoids (root-like structures) arise from stolons near the base of the sporangiophores; columella is a dome-like extension of the sporangiophore tip inside the sporangium
• Phialides - flask-shaped conidiogenous cells that release conidia from a collarette at the tip (enteroblastic conidiogenesis); seen in Aspergillus and Phialophora
• Annellides - conidiogenous cells that leave successive rings (annellations) at the apex as each conidium is released; "rings on a tree" appearance

4. Yeast Structures

• Budding yeast cells - round to oval cells with single or multiple buds
• Pseudohyphae - chains of elongated budding yeasts with constrictions at junctions (Candida spp.)

Representative LPCB Photomicrographs

Summary Table: Key LPCB Morphology by Organism

Sources: Henry's Clinical Diagnosis and Management by Laboratory Methods; Tietz Textbook of Laboratory Medicine, 7th Edition; Medical Microbiology 9e (Murray)

Dermatophytes - Culture Methods

Overview

Step 1 - Specimen Collection

Hair (Tinea capitis / Tinea barbae)

• Clean the area of alopecia with alcohol (does not affect fungal viability)
• Pluck (do not cut) infected hairs from the root - cut hairs are rarely infected and often bacterial-contaminated
• Collect black dots (hair stubs in endothrix infection) or scalp scale from the area of alopecia
• For Microsporum infections, a Wood's lamp (365 nm) can be used first - fluorescing hairs (yellow-green pteridine fluorescence) are preferentially selected for culture
• Use a toothbrush, scalpel, gauze square, or cotton-tipped applicator

Skin (Tinea corporis, cruris, pedis, manuum)

• Clean the advancing edge of annular lesions with alcohol
• Scrape the active advancing margin (not the center - the center is "healed" and devoid of viable fungi)
• For intertriginous mycoses: collect from the dry scaling edge - avoid moist macerated central material (contaminated and devoid of viable fungi)
• For vesicular lesions: collect the vesicle roof - vesicular fluid and vesicle base are usually devoid of fungi

Nails (Onychomycosis)

• Distal subungual type: trim back the nail to the leading edge of infection (discard the distal portion) - then collect keratinaceous debris from beneath the remaining trimmed nail plate edge (the actual fungal reservoir)
• Proximal subungual type: collect from the active edge closest to the distal end
• White superficial type: simply scrape the surface area of involvement
• Note: Simple nail clippings and whole-removed nail plates are less ideal

Step 2 - Culture Media

Primary Isolation Media

DTM - How the Color Change Works

• Dermatophyte proteolytic activity raises the pH to 8 or above → medium turns RED
• Saprophyte growth produces acidic byproducts → medium turns yellow
• Amber color with no change = most saprophytes / no dermatophyte
• Important caveat: DTM may alter colony morphology, so direct identification on DTM may not be reliable. Subculture to SDA is recommended for definitive species identification. - Fitzpatrick's Dermatology

Note on cycloheximide: Some clinically relevant non-dermatophyte nail pathogens (e.g., Scopulariopsis brevicaulis, Aspergillus spp., Fusarium spp.) do not grow on cycloheximide-containing media and may be missed if only selective media are used, especially for nail specimens. - Dermatology 5e (Bolognia)

Secondary/Identification Media (for subculture)

Step 3 - Incubation Conditions

• Temperature: 20°C to 25°C (room temperature)
• Duration: Incubate for at least 4 weeks before reporting no growth
• Growth rates vary by species:
  • Epidermophyton floccosum: 5-7 days
  • Most Trichophyton spp.: 1-2 weeks
  • Trichophyton verrucosum: up to 4 weeks (slowest grower)

Step 4 - Colony Identification

Macroscopic Features Examined

• Surface texture: powdery, granular, velvety, cottony, waxy, heaped
• Pigmentation (surface and reverse)
• Growth rate and colony size
• Radial grooves, folds, topography

Species-Specific Colony Features (Key Examples)

Step 5 - Microscopic Identification (LPCB)

Conidia - Genus-Level Key

Step 6 - Supplementary Biochemical Tests

Wood's Lamp as Pre-Culture Screening (Hair)

• Wavelength: 365 nm
• Ectothrix Microsporum species (M. canis, M. audouinii): yellow-green fluorescence (pteridines) → fluorescing hairs selected for culture
• T. tonsurans (most common tinea capitis in the US): does NOT fluoresce → limits usefulness of Wood's lamp alone
• Fluorescing hairs should always be sent for culture, not relied upon solely for diagnosis

Important Caveat: Non-Dermatophyte Molds in Nails

Sources: Fitzpatrick's Dermatology (Vol 1-2); Medical Microbiology 9e (Murray); Pfenninger & Fowler's Procedures for Primary Care; Tietz Textbook of Laboratory Medicine 7e; Dermatology 5e (Bolognia); Henry's Clinical Diagnosis and Management by Laboratory Methods

Dermatophyte Test Medium (DTM) - Criteria and Interpretation

What is DTM?

Composition and Rationale of Each Component

Principle - How the Color Change Works

Dermatophytes (Positive Result - RED)

1. Dermatophytes preferentially metabolize proteins first (proteolytic activity)
2. Protein catabolism releases alkaline metabolites (ammonia/nitrogen-containing byproducts)
3. pH rises from 5.6 to ≥8 (phenol red turns red at ≥8.2)
4. Medium turns red simultaneously with or before colony growth
5. When proteins are exhausted, dermatophytes shift to carbohydrate metabolism → acidic byproducts → medium may gradually revert to yellow if read late

Saprophytes/Contaminants (Negative - AMBER/YELLOW stays)

1. Most saprophytes preferentially ferment carbohydrates first
2. Carbohydrate fermentation → acidic byproducts → medium stays yellow or turns more yellow/amber
3. As a contaminant uses up carbohydrates, it may eventually shift to protein metabolism → late red color change (beyond day 10-14) - this is the basis of false positives

DTM Interpretation Criteria

POSITIVE (Dermatophyte present)

The simultaneous or early color change with white aerial hyphae = strong presumptive positive for dermatophyte. - [Hardy Diagnostics DTM package insert]

NEGATIVE (No dermatophyte)

FALSE POSITIVE (Red color change but NOT a dermatophyte)

Dermatophytes = white/buff aerial hyphae + early red color change (within 10-14 days, simultaneous with growth)

Saprophytes = dark/colored colonies (green, black, brown) + late red color change (after day 14 if at all)

Yeasts = white creamy bacterial-looking colonies ± possible red change

Step-by-Step Interpretation Protocol

Day 1-14: Check daily

Is there COLONY GROWTH?
        │
        ├─ NO → Continue incubating up to 14-21 days
        │
        └─ YES
              │
              ├─ Is the medium turning RED?
              │       │
              │       ├─ YES (simultaneously or before colony growth)
              │       │       │
              │       │       ├─ Are colonies WHITE/BUFF aerial hyphae?
              │       │       │       └─ YES → PRESUMPTIVE POSITIVE for dermatophyte
              │       │       │                (confirm with LPCB microscopy)
              │       │       │
              │       │       └─ Are colonies dark/greenish/black?
              │       │               └─ YES → FALSE POSITIVE (saprophyte contaminant)
              │       │
              │       └─ NO red change (amber/yellow)
              │               └─ Likely NOT a dermatophyte
              │
              └─ Is color change appearing AFTER day 14?
                      └─ Likely FALSE POSITIVE regardless of colony color

Important Limitations of DTM

1. Presumptive only - DTM cannot give definitive species identification. Colony morphology is often altered in DTM, making species-level ID unreliable. Always subculture to SDA or PDA for speciation.
2. Cycloheximide-sensitive pathogens missed - Clinically significant non-dermatophyte nail pathogens (Scopulariopsis brevicaulis, Aspergillus spp., Fusarium spp., Candida tropicalis, Trichosporon spp., Scedosporium spp.) are inhibited by cycloheximide and will not grow on DTM. For nail specimens especially, always culture on non-selective SDA in parallel.
3. Reading window is strict - Must read within 14 days; beyond this, false positives increase significantly as even saprophytes eventually oxidize proteins.
4. Loose cap required - The cap must be kept loosely applied during incubation to ensure proper gas exchange and optimal dermatophyte recovery. A tight cap can inhibit growth.
5. Confirm positives microscopically - All color-change positives must be confirmed by LPCB microscopic examination of the colony (tape mount or teased preparation) to identify macroconidia/microconidia morphology and confirm genus/species.

Summary Table: DTM Color Change Interpretation at a Glance

Sources: Fitzpatrick's Dermatology (Vol 1-2); Tietz Textbook of Laboratory Medicine 7e; Hardy Diagnostics DTM package insert; Pfenninger & Fowler's Procedures for Primary Care; OSU CVM Veterinary Clinical and Professional Skills Center; microbeonline.com DTM review