• Although not a strict requirement, it is recommended that • mycology laboratories that culture for filamentous fungi and • manipulate those organisms be separate and isolated from • the main microbiology laboratory with negative air pressure
• moving into the room from the main laboratory Direct access• moving into the room from the main laboratory. Direct access • to a Class II biological safety cabinet (BSC) is critical for this • activity whether mycology work is conducted in a separateactivity whether mycology work is conducted in a separate • room or in an isolated section of the main laboratory. Most • mycology diagnostic work can be conducted in the biosafety
• level (BSL)‐2 laboratory.
7.1. Specimen Receiving and Log‐In/SetUp StationStation
• 7 1 1 Leaking containers7.1.1. Leaking containers• Guidelines for the general microbiology
laboratory apply also for the mycologylaboratory apply also for the mycology laboratory.
• No special recommendations are necessary.
7.1. Specimen Receiving and Log‐In/SetUp StationStation
• 7 1 2 Visible contamination on outside of7.1.2. Visible contamination on outside ofcontainer
• Guidelines for the general microbiology• Guidelines for the general microbiology laboratory apply also for the mycology
laboratorylaboratory. • No special recommendations are necessary.
7.1. Specimen Receiving and Log‐In/SetUp Station
• 7 1 3 Loose caps7.1.3. Loose caps• Guidelines for the general microbiology
laboratory apply also for the mycologylaboratory apply also for the mycology laboratory.
• No special recommendations are necessary.
7.2. Stains and Disposal7.2. Stains and Disposal
• 7 2 1 Gram stain7.2.1. Gram stain• The Gram stain is not the optimum stain for
fungus but iffungus, but if • used particularly for yeast, the same
id li h lguidelines that apply to • bacteriology/clinical microbiology are
followed for mycology.
7.2. Stains and Disposal7.2.2. Mycology stains
• •Calcofluor white is notconsidered a hazardoussubstance;
• no special safety measures are required. Use routinelaboratory procedures: i.e., after skin contact, washwith soap and water; after eye contact, rinse eye wellp ; y , ywith water; seekmedical attention if illness is reportedafter inhalation or swallowing.
• Follow accepted laboratory procedures applied toinfectious materials for handling and disposing of
slidesslides.
• Fungal cells stained with calcofluor white can remain gviable.
7.2. Stains and Disposal7.2.2. Mycology stainsy gy
If potassium hydroxide (KOH) is used with thecalcofluor white more stringent precautions mustcalcofluor white, more stringent precautions must
be taken.
• KOH 10%–15% solution is corrosive. Handle it with care.
• KOH may cause burns or irritation to skin, eyes, andrespiratory tract. Avoid eye/skin contact andinhalation or ingestion. Use gloves and eye protectionif there is a danger of splashing or aerosol formation.
l d h d• — KOH is not listed as hazardous waste.
7.2. Stains and Disposal7 2 2 Mycology stains7.2.2. Mycology stains
• Lactophenol cotton blue (lactophenol aniline blue or
• Poirrier’s blue) • lactophenol cotton blue is acidic; avoid contact with• — lactophenol cotton blue is acidic; avoid contact with
skin, eyes, and clothing.• — Rinse thoroughly with water if spilled.
• — For fungal slides stained with the fluid, follow accepted laboratory procedures for handling and
disposing of infectious materials.p g
• —Dispose as hazardous waste.
7.2. Stains and Disposal 7 2 2 Mycology stains7.2.2. Mycology stains
India inkIndia ink• —No special personal protection is required
under normal use conditions; however theunder normal use conditions; however, the yeast cells can remain viable.
• — India ink is not a regulated hazardous waste.
7.2. Stains and Disposal
7.2.2. Mycology stains
Acid‐fast stainAcid fast stain• Guidelines for the tuberculosis laboratoryapply also for the mycology laboratoryapply also for the mycology laboratory.
• No special recommendations are necessary.
7.2. Stains and Disposal 7 2 2 M l t i7.2.2. Mycology stains
Gomori methenamine silver stainGomori methenamine silver stain
di i i d• — Ingredients are toxic, corrosive, and harmful; avoid contact with skin and eyes.
• —Use with adequate ventilation; do not inhale.
• —Dispose of as a hazardous waste.
7.2. Stains and Disposal7 2 2 Mycology stains7.2.2. Mycology stains
• Giemsa stainGiemsa stain
f i h l Gi• — Because of its methanol content, Giemsastain is toxic by inhalation, absorption, ori i P i l d f lingestion. Protective gloves and safety gogglesare not required but are recommended.
7.3. Culture Reading at the Bench
A separate, closable room for mycology activities is recommended but not required.
•Conduct all culture manipulations in a BSC whether in a separate room or within awhether in a separate room or within a
designated space in the open microbiology laboratory.
• All mould colonies (filamentous, fuzzy, cottony) must be handled in a Class II BSCmust be handled in a Class II BSC.
• This applies as well to moulds growing on• This applies as well to moulds growing on bacteriology plates.
7.3. Culture Reading at the Bench (1,113)
Use shrink sealsor tape to seal petri dishes (especially if mould begins to grow) in order to prevent accidental opening and spread of hyphal
segments, conidia, or spores.
Never use plates if Coccidioides immitis is suspected or if a filamentous culture is to be mailed or otherwise transported to another
laboratory Use slants in screw‐cap tubeslaboratory. Use slants in screw cap tubes.
• —Observe all plates and slants for growth before opening.In general cultures growing yeast‐like colonies can be read on theIn general, cultures growing yeast‐like colonies can be read on the
open bench in a BSL‐2 laboratory; but if the isolate is suspected of being Cryptococcus neoformans(moist, mucoid colonies) or any
dimorphic fungus, move it to a Class IIA2 BSC.
Never sniff a fungal culture to determine whether it has an odor. Do not open plates containing moulds on the open bench, even if it is a
b t i l k t tibacteriology work station.
7.4. Personal Precautions
• 7.4.1. Biosafety cabinet
The Class IIA2 BSC is recommended forsome mycology work; i.e., all moulds y gy ; ,
(fuzzy, wooly, cottony, powdery, or velvety) must be handled in the BSC, never on the ,
open bench.
For mycology, follow the same BSCguidelines that apply to bacteriologyguidelines that apply to bacteriology.
7.4. Personal Precautions
• 7.4.2. Personal protective equipment
• Guidelines for the general microbiologylaboratory apply also for the mycology
laboratory.
• • Wear gloves and remove watches andbracelets when manipulating a mould culture
having the possibility of being a dermatophytehaving the possibility of being a dermatophyte. When the task is completed, wash hands and
wrists well.sts e
7.4. Personal Precautions7.4. Personal Precautions
• 7.4.3. Disinfection7.4.3. Disinfection• Recommendations for the general
microbiology laboratorymicrobiology laboratory • are sufficient for use in the mycology
laboratory; these includelaboratory; these include • guidelines for disinfection of countertops and
items such asitems such as • telephones, computers, equipment, and
hands‐free telephones.hands free telephones.
7.4. Personal Precautions
• 7.4.4. Decontamination and disposal of laboratory Waste
Follow the same guidelinesthat apply toclinical microbiologyclinical microbiology.
If an autoclave is unavailable and medicalIf an autoclave is unavailable and medicalwaste is handled off‐site, open plates and tubes containing Coccidioides spp. and
l l i h i 1 10 bl hcompletely immerse them in a 1:10 bleach solution overnight before disposing.
7.5. Clean versus Dirty Areas of the Laboratory
• Guidelines for the general microbiologyGuidelines for the general microbiology laboratory apply also for the mycology
laboratorylaboratory.
7.6. Select Agents and Pathogenic Moulds
Handle all mould‐likecolonies in a BSCHandle all mould likecolonies in a BSC.
Make a wet preparation of all cultured moulds before setting up a slideculture in order to detect structures that may indicate the possibility ofculture in order to detect structures that may indicate the possibility ofthe isolate being a highly pathogenic systemic fungus.
• Refrain from setting up slide cultures of isolates that on wet preparation are suggestive of H. capsulatum, B. dermatitidis, C. immitis,
C. posadasii, P. brasiliensis, P. marneffei, or • Cladophialophora bantiana.
•• Make every attempt to identify them by well‐prepared wet preps and
DNA probes if available.
7.6. Select Agents and Pathogenic Moulds
• If a laboratory‐isolated organism is identified as H. capsulatum, B. dermatitidis, Coccidioidesspp., or
Paracoccidioides brasiliensis, BSL‐3 practices and facilities , pare recommended for handling mould‐form cultures and environmental samples likely to contain infectious conidia.
Coccidioides spp. are the only fungi currently classified as aselect agent requiring registration with CDC/U.S.
To decontaminate Coccidioides isolates whenTo decontaminate Coccidioides isolates whenautoclaving is unavailable, soak the plates and tubes,overnight in a fresh 1:10 bleach solution that completelyimmerses the opened tubes and plates prior to disposalimmerses the opened tubes and plates prior to disposal.
7.7. Blood Culture Bench
d l f h l b l l b l l f hGuidelines for the general microbiology laboratory apply also for the mycology laboratory.
Plates growing mould‐like colonies must be examined in a BSC, not on the open benchnot on the open bench.
If a small‐celled yeast (~3µm) is detected, consider the possibility y ( µ ) , p yof H. capsulatum, and handle under BSL‐2 conditions in a Class 2 BSC.
7.8. Instrumentation
• Instruments used for mycology studies areInstruments used for mycology studies are most commonly those for continuously monitored blood culture and for yeastmonitored blood culture and for yeast
identification.
• Follow the same guidelines that apply to bac‐i l / li i l i bi lteriology/clinical microbiology.
7.9. Rapid Testing (Kits)
Apply the clinical microbiology guidelines for monomorphic yeasts in mycology.
If the isolate is a mold, it must be handled in the BSC.
7.10. Molecular Testing
• Follow the clinical microbiology safety guidelines for mycology with the additionalguidelines for mycology with the additional advisory that mold isolates must be handled in a BSC during extraction of nucleic acidsin a BSC during extraction of nucleic acids.