Práctica de Micología
Laboratory Techniques for Mycology
Overview of Materials and Methods
- The materials used in mycology laboratories are similar to those in bacteriology, with a wider inoculation needle called the mycological spatula. Culturing fungi requires selective media such as Sabouraud agar and differential media like DTM for distinguishing between different fungi.
Types of Fungi
- Fungi can be categorized into two types based on their microscopic appearance: yeasts and filamentous fungi. Yeasts produce opaque, creamy colonies while filamentous fungi exhibit rough, cottony, or powdery growths.
Cultivation Techniques
- For suspected yeast species, inoculation is performed by exhaustion similar to bacteria. A pure culture of fungal mycelium is obtained by inoculating three equidistant points on a plate; if identical growth appears at all points, it indicates a pure culture. Samples from skin lesions or nails should be directly seeded onto TM medium.
Incubation Conditions
- Incubation temperatures vary based on sample origin; surface samples require lower temperatures (22-25°C). Identification of yeasts follows similar procedures as bacteria but includes specific tests like auto-gram and cimo-gram for physiological characteristics.
Microscopic Observation Techniques
- Morphological characteristics are crucial for identifying filamentous fungi both macroscopically (coloration of the mycelium) and microscopically (cell shape and size). Two main techniques for microscopic observation include adhesive tape technique and micro-cultivation technique using blue methylene stain. Necessary sterile materials include slides, cover slips, scissors, adhesive tape, tweezers, etc.
Adhesive Tape Technique Steps
- To perform the adhesive tape technique:
- Place a drop of blue methylene stain on a slide.
- Cut a piece of adhesive tape (~1 cm²) and press it onto the fungal growth.
- Transfer the tape to the stained slide and cover with another slip before observing under an optical microscope at various magnifications up to 40x.
Micro-Cultivation Technique Steps
- In micro-cultivation:
- Use sterile materials including glass petri dishes.
- Place affected tissue on an agar square in the center of a petri dish.
- Inoculate around this square with fungal samples using the mycological spatula.
- After incubation at appropriate temperatures (25–28°C), extract adhered material for microscopic examination with blue methylene stain at magnifications up to 40x.
Identification of Dermatophytes