Mycology: Specimen Collection

• Mycology is a specialized branch of biology focused on fungi, including their taxonomy, environmental effects, and biochemical traits.
• The MYCV lab protocols include biosafety practices and methods for isolating and growing various organisms.

Specimen Collection

• Accurate fungal infection diagnoses rely heavily on the type of sample taken.
• Correct specimen collection and prompt delivery to the lab are vital for effective fungal isolation.
• Delays can compromise the specimen's integrity or cause organism death.
• Specimens may contain infectious agents alongside bacteria that can overgrow and hinder the isolation of pathogenic fungi.
• Antibiotics can be added to culture media to suppress bacterial growth, especially when testing for fungi.

Hair, Skin, and Nails

• Hair, skin, and nail specimens are commonly tested for dermatophytes, which colonize these areas.
• Skin and nail samples are collected by scraping with a scalpel, while hairs are plucked with forceps.
• Samples are placed in sterile containers to prevent bacterial contamination.
• Mycosel agar, containing chloramphenicol and cycloheximide, is suitable for dermatophyte cultivation.
• Dermatophyte cultures should be incubated for 21 days at 30°C before a negative result is declared.

Skin Scraping

• Select the appropriate site or lesion
• 70% alcohol disinfects the site.
• Scrape the lesion's edge with a scalpel.
• Examine scrapings microscopically with a glass slide mount

Hair/Scalp Collection

• Use sterile forceps to pluck hair strands.
• Place cut hairs in a petri dish or KOH mount.
• Perform a wet mount or KOH preparation for direct microscopy.
• A calcofluor stain mount may be needed
• Inoculate specimens on sabaroud dextrose agar with chloramphenicol and gentamicine, incubate for at least 4 weeks
• Swabs are placed or smeared on sterilized slides for gram staining
• Specimens for bacterial analysis are placed on glass slides with antibiotics
• Maintain bacteria cultures for 4 weeks or 21-28 days.

Hair Analysis

• Wood's light (>365 nm UVL) can highlight Microsporum audouinii fluorescence on the scalp

Skin Inoculation

• Aseptic techniques are essential.
• Directly inoculate fungal media like SDA or PDA.

Nail Scraping

• Clip the entire nail thickness; disinfect the area with 70% alcohol
• Deep scrapings are needed for KOH mount
• Use sterile scissors to cut nails to avoid contamination.

Vaginal Swabs

• Collect vaginal samples within 24 hours using suitable transport swabs
• Preservation or immediate testing is crucial because they can easily degrade
• Keep swabs in moist, sterile tubes to prevent drying.
• Maintain ambient moisture for wet mounts.
• Fungi need moisture, low light, temperature, and nutrients.
• Plate both selective and inhibitory agars
• Incubate cultures to screen for yeasts for 7 days at 30°C.
• Yeasts thrive in higher temperatures, while molds are less temperature-sensitive
• Some yeasts require higher temperatures, such as 45°C

Blood

• Blood cultures are accurate for disseminated fungal infections
• Use automated systems like BACTEC, BacT/ALERT, or VersaTREK.
• Inoculate concentrate from the isolator onto media to detect fungi within 4 days, but H. capsulatum may take 10-14 days.
• Incubate blood cultures at 30°C for 21 days (21-28 days).

Blood Isolators

• The lysis centrifugation system lyses leukocytes and erythrocytes, inactivates plasma components, and combats certain antibiotics
• Lysing cells releases microorganisms, which centrifugation concentrates
• This concentrate is inoculated on culture media.
• Use 10mL per tube and maintain cultures at 30°C for 3-4 weeks (21-28 days).
• Labs that frequently recover dimorphic fungi should use lysis centrifugation
• Optimal for isolating H. capsulatum and other filamentous fungi
• Lyses RBCs and WBCs, concentrating them before culturing.
• It is the most sensitive recovery method.
• Sediments are inoculated onto culture media.
• Blood culture bottles are not recommended and tubed blood is preferred

Tissue and Sterile Body Fluids

• Mince all tissues or place in a high-speed laboratory blender before culturing.
• Cytoplasmic contents are extracted by rapidly moving blades against the tissue in a broth
• After processing, spread at least 1 mL of specimen on suitable agar and incubate for 21 days at 30°C.

CSF

• Concentrate specimens by centrifugation
• Plate 1 mL of specimen onto the surface of the appropriate media

Procedures

• Use one drop of concentrate for India ink or latex agglutination for Cryptococcus.
• Inoculate the rest of the specimen onto media.
• For tissue cultures, aseptically tease apart specimens in a sterile petri dish
• Exercise caution when inoculating certain fungi, as not all specimens should be minced as zygomycetous fungi will not survive this process
• If zygomycosis is suspected, gently tease tissues apart and inoculate directly onto isolation media
• If unsure about a specimen, keep it in saline or BHI broth to examine after frozen sections
• Tease apart specimens with zygomycetous hyphae; otherwise, mince and plate

Zygomycetes

• Mucor and Rhizopus species are examples
• Instead of homogenizing, gently tease the tissue and inoculate it directly onto the isolation media or the fungi will not survive.

Respiratory Tract

• Respiratory specimens: sputum, induced sputum, bronchial washings, bronchoalveolar lavage, and tracheal aspirations—are common for fungal cultures
• Use antibiotics or inhibitors in plating media for proper isolation and to avoid contamination
• The respiratory tract is a common sample source along with blood because some fungi, like tuberculosis, share similar characteristics
• In x-rays, there can be opacities similar to TB, and fungal infection is suspected due to TB-like symptoms
• Aspergillus is similar with characteristics of TB
• Antibacterial antibiotics are used in plating media to prevent contamination.
• Cycloheximide is used to prevent the rapid growth of molds
• Use 0.5 mL of specimen to inoculate each medium
• Thick viscous samples can be concentrated using N-acetyl-L-cysteine, to digest viscous fluids and act as an antimicrobial agent

Oropharyngeal

• Viscous samples are softened with N-acetyl-L-cysteine
• The agent acts as as mucolytic and antimicrobial.
• Swabs are used to streak or inoculate after processing.
• These sample types are collected by rolling sterile swabs and scraping suspected Candida areas with a sterile tongue depressor
• Send specimens to the lab as soon as possible
• Delays longer than two hours at room temperture can impede growth; store at 4 degrees Celsius if necessary
• Collect samples in the morning for overnight fungal growth in the lungs
• Patients should not eat before collection
• Bronchial washings and induced sputum should be performed in the morning

Sputum Contamination

• Samples are not accepted after 24 hours because of bacterial and fungal overgrowth
• Bronchial washing and expectorated sputa are often contaminated with normal throat flora, necessitating antibiotics/inhibitors

Sputum, Washings, and Aspirations

• Sputum Washings and Aspirations which are not too viscous are plated with a sterile pipette otherwise are streaked/inoculated onto media

Urine Processing

• Process urine for fungal culture quickly, avoid 24-hour collections
• Concentrate and centrifuge to isolate colonies using a wire loop
• Use antibacterial agents because urine is often contaminated with gram-negative bacteria
• First morning urine specimens are preferred because they are more concentrated
• Samples should be stored at 4°C if delays are expected.
• Centrifuge urine for 10–15 minutes at 2000 rpm, decant the supernatant, and collect the sediment.

Specimen Direct Examination

• Specimens containing keratin are tested with KOH or calcofluor white in a wet mount.
• Hyphal elements are observed for clarity, hyaline or dematiaceous nature, and septation patterns.

Cultures

• Incubate specimens at room temperature (22-25°C) or higher (30°C) for suspected dimorphic fungi
• Macroscopic characteristics indicate if it is yeast or mold
• Germ tube media and biochemical tests identify yeast-like colonies on special/selective media
• Cornmeal agar identifies blastoconidia and chlamydospores, while other media like birdseed and caffeic acid help determine carbon source use

Microscopic ID

• Color, texture, pigment and growth rate
• Lactophenol cotton glue is used to prep the slide
• Size
• Shape
• Arrangement

Microscopic Observations

• Conidia presence dictates slide culturing
• Color, sepate or sparse areas, mycelium nature

Staining

• Path is magenta on pink or green element

The germ tube will either not grow

• Growth above 42 is candida albicans
• Below 42 C is dubliensis

Microscopic Examinations

• To examine karatinized

Procedures

• Mix drops of solutions on a clean slide
• Then plate the specimen and cover with a slip
• Heat gently and microscopically examine for bright or florescent elements
• A combination of M27-A3 and M38-A2 documents are used for antifungal testing

M27A3/M38A2

• Reference to all microbiology related tests
• Tests the susceptibility to yeast
• Uses broth to dilute

RPM1640

• Helps grow nutrients and buffer is used
• Base is used to dilute for 2 glucose procedures

YMB

• Provides sustainable source for Carbohydrate Assimilation
• Incubated for 48-72 hours
• Growth = assimilation
• Glucose all for cell growth

Bird Seed Agars

• Differential medium created for preliminary ID
• Guizotia provides enrichment for biphenyl and enzyme
• Colonies darken to rich brown from enzyme accumulation
• Colors change based on phenol
• Other colonies remain colors
• Selective agar prevents spread

Chromogenic Agar

• Accurate id in clinical labs
• Contains chloramphenicol to prevent bacteria
• Selects to dextrose
• Creates yellow and blue green colors from alban colonies
• incubate for 48

Hair Perforation test

• Add hair stands to extraction
• Observe at a weekly interval
• Used if unsure if similar to mentagroytes

Fungal Culturing in Tubes

• Media reduces spoilage

SDA

• Limits bacterial growth
• Prevent bacterial growth

Fungal growth

• Room temperature prevents Nascordia, Spp from growing

Fungal morphology

• Can grow but some may be limited
• Tecture, cotonny or wooly
• Fungal can be colorless ###Vegetative mycelium, provides nutrients
• Aerial spores can show case studies