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MYCOLOGY SPECIMEN COLLECTION & 	HANDLING
Specimen collection & transport Important considerations: Proper collection Rapid delivery to the laboratory Prompt and correct processing Inoculation into proper and appropriate medium Incubation at a suitable temperature
Specimen collection & transport Transport of Specimen: Antibiotics may be incorporated in body fluid specimens to prevent proliferation of bacteria: ,[object Object]
100,000 units of Streptomycin
0.2 mg of Chloramphenicol,[object Object]
Dermatological: 15 – 30 OC
Others:		     4 OC,[object Object]
Specimen collection & transport BLOOD and BONE MARROW    Transport medium: at 1:10 proportion TSB or TSA (biphasic agar or broth) BHI transport medium Thioglycollate broth Volume:	10 ml
CEREBROSPINAL: Transport immediately. Do NOT refrigerate.  For suspected Cryptococcus, Coccidioides infections, containers must be leak proof and lab manipulations should be done under a hood Specimen collection & transport
Specimen collection & transport DERMATOLOGICAL SPECIMENS SKIN LESIONS Sterilized area with 70% alcohol or sterile water Collect at the the active border
Specimen collection & transport NAILS Clean with 70% alcohol If: Dorsal plate: scrape the deeper portion Nail plate:	 	scrape beneath the nail plate Whole nail or clippings
Specimen collection & transport HAIR    Collect from: Areas of scaling Alopecia Hair that fluoresce under Wood’s lamp
Specimen collection & transport EXUDATES & PUS  Undrained or unruptured abscess Aspirate using sterile syringe, recap needle and transport to lab immediately Failed aspiration, do skin biopsy
Specimen collection & transport URINE First early morning Transport and perform test ASAP within 2 hours If not possible, refrigerate specimen.
Specimen collection & transport VAGINAL SECRETIONS ,[object Object]
Put in transport medium or primary isolation broth immediately (ex: TSB),[object Object]
MYCOLOGY METHODS OF IDENTIFICATION
A.  DIRECT FUNGAL MICROSCOPY Clinical significance: Provide an immediate presumptive diagnosis Aid in the selection of appropriate culture media Aid in decision of what’s  best inoculation technique to use It will provide evidence of infection despite negative culture
A.  DIRECT FUNGAL MICROSCOPY Macroscopic Examination (physical exam): Note for: caseous material Purulent exudate Necrotic material Granules Punch biopsies Layers of skin that are broken vertically (fissures) Obtain specimens for microscopy and culture fro
Preparation for Microscopic examination: Mince or grind hard specimens Centrifuge for 3-5 minutes fluid specimens Pulvorize nail clippings Volume for fluid specimens: 0.5 ml Assemble a wet chamber for incubation A.  DIRECT FUNGAL MICROSCOPY
REAGENTS used for DIRECT MICROSCOPIC STUDY KOH 10-20% Routinely used 10% = skin and soft tissues, body fluids 20% = nail and hard tissues Calcoflour white Green flourescense India ink “Dark field” microscopy for Cryptococcus neoformans A.  DIRECT FUNGAL MICROSCOPY
A.  DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Lactophenol Blue very popular for quick evaluation of fungal structures stains the chitin in cell walls of fungi blue Use for following up fungal culture growths Wright’s/Giemsa stain (Diff quick) For rapid staining of blood and bone marrow fungi (ex: Histoplasmacapsulatum) Modified Acid-Fast Stain  used to differentiate the acid-fast Nocardia from other aerobic Actinomyces Gram Stain  generally fungi are gram positive Actinomyces and Nocardia are gram variable
A.  DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Stains for tissue mycoses: Periodic Acid - Schiff Stain (PAS)  stains certain polysaccharide in the cell walls of fungi Fungi stain pink-red with blue nuclei.  GomoriMethenamine Silver Stain  silver nitrate outlines fungi in black due to the silver precipitating on the fungi cell wall. The internal parts of hyphae are deep rose to black, and the background is light green.  Gridley Stain  Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow.
Stains for tissue mycoses … Fluorescent Antibody Stain  simple, sensitive, and extremely specific method of detecting fungi in tissues or fluids. Applications for many different fungal organisms.  Mayer Mucicarmine Stain  will stain capsules of Cryptococcus neoformansdeep rose. Papanicolaou Stain  good for initial differentiation of dimorphic fungi Works well on sputum smears also A.  DIRECT FUNGAL MICROSCOPY
KOH Wet Mounts Principle: KOH softens most tissues, dissolves fat droplets, bleaches many pigments and dissolves the “cement” that holds keratinized cells together; glycerine clears tissue debris, thus making it easier to demonstrate presence of fungal elements.  Reagents: 10 – 20 % KOH:	 KOH pellets 		10 – 20 grams Glycerine (optional)	10 ml Distilled water		90 ml
KOH Wet Mounts Procedure: Place a small amount of specimen on a clean glass slide place 1-2 drops of KOH on the specimen and overlay a cover slip Allow the preparation to stand for 10-30 minutes in a wet chamber.  You can gently heat preparation to hasten the action of KOH Do not over heat for it may crystallize the KOH Examine preparation under low then high magnification. Take note for the presence of fungal elements (hyphae and/or spores)
INDIA INK PREPARATION aka:	Nigrosin stain Principle: Specimen placed in a drop of India ink becomes darkly colored because of the carbon particle in the ink.  Hyaline structures such as capsules and cell walls will be highlighted against a dark background of inked colored specimen creating an illusion of darkfield microscopy. Reagent:	1:1 dilution of the ink
India Ink Preparation Procedure: Place a drop of the specimen (body fluid or from culture) on a clean glass Put a drop of India Ink, mix and overlay a cover slip Examine under low power and high power with a bright field microscope Result: India ink creates a dark background against which hyaline fungal cell wall and capsules can se seen Limitation:	wbc may be confused as fungi
Lactophenol Cotton Blue  Principle: The morphology of fungal elements are preserved and stained better. Reagents: Lactic acid & Phenol Kills the organism	 Glycerin Prevents easy dehydration Cotton blue Dye or stain
DIAGNOSIS OF MYCOSES by UNSTAINED & STAINED MICROSCOPY
A. SKIN or DERMATOMYCOSIS KOH for superficial involvement, look for: Spaghetti & meat balls (lung aspirate) Malasseziafurfur Pseudohyphae and yeasts (vaginal secretions) Candida species
A. SKIN or DERMATOMYCOSIS KOH & LPCB for superficial involvement, look for: Hyaline septatehyphae 	ex: Dermatophytes Dematiaceousseptatehyphae 	ex: Tineanigra Alternaria
A. SKIN or DERMATOMYCOSIS H & E stain for Oral Candidiasis on Skin biopsy of tongue, look for: Pseudohyphae yeasts
B. DRAINING SINUS for        MYCETOMAS & ACTINOMYCOSIS KOH, look for Various colored granules Actinomycosis/ Nocardiosis GMS stain, look for  Various granules Mycetoma
C. EYE SCRAPINGS & ASPIRATE 	for KERATOMYCOSIS KOH & LPCB, look for Septate hyaline hyphae Aspergillus species Fusarium species Coenocytic hyaline hyphae Mucor species Pseudohyphae and yeasts Candida species
D. NASOPHARYGNEAL ASPIRATES f	for RHINOSPORIDIOSIS KOH, look for Large sporangium with spores (lacrimal gland aspirate) Rhinosporidium species
E. HAIR for DERMATOMYCOSES 	& ALOPECIA KOH, look for Endothrix spores/hyphae Trichophyton Ectothrix spores/hyphae Trichophytonmentsgrophytes
E. HAIR for PIEDRA KOH, look for: Hard, brown, compact nodules (Black piedra) Piedraiahortae Soft, off-white, concretions/nodules 	(White piedra)	 Trichosporonbeigeli
F. NAILS for ONYCHOMYCOSIS KOH & LPCB, look for Septate, hyaline hyphae Dermatophytes Epidermophyton Trichophyton Microsporon Pseudohyphae and yeast cells Candida species
G. SYSTEMIC MYCOSESSpecimens: blood, CSF, sputum, other body fluids KOH & Mucicarmine stain systemic involvement, look for: Pseudohyphae and yeast cells (CSF) Candida species Broad based buds 	(brain and CSF) Blastomyces species
SYSTEMIC MYCOSES GMS for systemic involvement, look for: Spherules/sporangia (CSF) Coccidioidesimmitis PAS stain for systemic involvement, look for: Dematiaceousseptatehyphae (brain tissue)
SYSTEMIC MYCOSES H & E stain for systemic involvement, look for: Endospores (CSF brain tissue) Coccidioides species Fission/sclerotic bodies Chromomyces species
SYSTEMIC MYCOSES 	Wright’s/Giemsa stain (Diff quick) & LPCB for systemic involvement, look for: Small, intracellular budding yeast (CSF) Histoplasma species Small, intracellular yeast dividing by fission (CSF) Penicillin species
SYSTEMIC MYCOSES Mucicarmine stain & India Ink for systemic involvement, look for: Encapsulate yeast (CSF) Cryptococcus neoformans
SYSTEMIC MYCOSES LPCB & Fluorescent Antibody stain for systemic involvement, look for Large yeast with multiple buds called “mariner’s wheel” Paracoccidioidesbraziliensis
SYSTEMIC MYCOSES Calcoflour mounts for systemic mycoses , look for (flourescence) Pseudohyphae and yeasts (blood) Candida species Septate, hyaline at right degrees angle (bronchial lavage) Aspergillus species

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2 specimen collection

  • 2. Specimen collection & transport Important considerations: Proper collection Rapid delivery to the laboratory Prompt and correct processing Inoculation into proper and appropriate medium Incubation at a suitable temperature
  • 3.
  • 4. 100,000 units of Streptomycin
  • 5.
  • 7.
  • 8. Specimen collection & transport BLOOD and BONE MARROW Transport medium: at 1:10 proportion TSB or TSA (biphasic agar or broth) BHI transport medium Thioglycollate broth Volume: 10 ml
  • 9. CEREBROSPINAL: Transport immediately. Do NOT refrigerate. For suspected Cryptococcus, Coccidioides infections, containers must be leak proof and lab manipulations should be done under a hood Specimen collection & transport
  • 10. Specimen collection & transport DERMATOLOGICAL SPECIMENS SKIN LESIONS Sterilized area with 70% alcohol or sterile water Collect at the the active border
  • 11. Specimen collection & transport NAILS Clean with 70% alcohol If: Dorsal plate: scrape the deeper portion Nail plate: scrape beneath the nail plate Whole nail or clippings
  • 12. Specimen collection & transport HAIR Collect from: Areas of scaling Alopecia Hair that fluoresce under Wood’s lamp
  • 13. Specimen collection & transport EXUDATES & PUS Undrained or unruptured abscess Aspirate using sterile syringe, recap needle and transport to lab immediately Failed aspiration, do skin biopsy
  • 14. Specimen collection & transport URINE First early morning Transport and perform test ASAP within 2 hours If not possible, refrigerate specimen.
  • 15.
  • 16.
  • 17. MYCOLOGY METHODS OF IDENTIFICATION
  • 18. A. DIRECT FUNGAL MICROSCOPY Clinical significance: Provide an immediate presumptive diagnosis Aid in the selection of appropriate culture media Aid in decision of what’s best inoculation technique to use It will provide evidence of infection despite negative culture
  • 19. A. DIRECT FUNGAL MICROSCOPY Macroscopic Examination (physical exam): Note for: caseous material Purulent exudate Necrotic material Granules Punch biopsies Layers of skin that are broken vertically (fissures) Obtain specimens for microscopy and culture fro
  • 20. Preparation for Microscopic examination: Mince or grind hard specimens Centrifuge for 3-5 minutes fluid specimens Pulvorize nail clippings Volume for fluid specimens: 0.5 ml Assemble a wet chamber for incubation A. DIRECT FUNGAL MICROSCOPY
  • 21. REAGENTS used for DIRECT MICROSCOPIC STUDY KOH 10-20% Routinely used 10% = skin and soft tissues, body fluids 20% = nail and hard tissues Calcoflour white Green flourescense India ink “Dark field” microscopy for Cryptococcus neoformans A. DIRECT FUNGAL MICROSCOPY
  • 22. A. DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Lactophenol Blue very popular for quick evaluation of fungal structures stains the chitin in cell walls of fungi blue Use for following up fungal culture growths Wright’s/Giemsa stain (Diff quick) For rapid staining of blood and bone marrow fungi (ex: Histoplasmacapsulatum) Modified Acid-Fast Stain used to differentiate the acid-fast Nocardia from other aerobic Actinomyces Gram Stain generally fungi are gram positive Actinomyces and Nocardia are gram variable
  • 23. A. DIRECT FUNGAL MICROSCOPY STAINS for MICROSCOPIC STUDIES: Stains for tissue mycoses: Periodic Acid - Schiff Stain (PAS) stains certain polysaccharide in the cell walls of fungi Fungi stain pink-red with blue nuclei. GomoriMethenamine Silver Stain silver nitrate outlines fungi in black due to the silver precipitating on the fungi cell wall. The internal parts of hyphae are deep rose to black, and the background is light green. Gridley Stain Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow.
  • 24. Stains for tissue mycoses … Fluorescent Antibody Stain simple, sensitive, and extremely specific method of detecting fungi in tissues or fluids. Applications for many different fungal organisms. Mayer Mucicarmine Stain will stain capsules of Cryptococcus neoformansdeep rose. Papanicolaou Stain good for initial differentiation of dimorphic fungi Works well on sputum smears also A. DIRECT FUNGAL MICROSCOPY
  • 25. KOH Wet Mounts Principle: KOH softens most tissues, dissolves fat droplets, bleaches many pigments and dissolves the “cement” that holds keratinized cells together; glycerine clears tissue debris, thus making it easier to demonstrate presence of fungal elements. Reagents: 10 – 20 % KOH: KOH pellets 10 – 20 grams Glycerine (optional) 10 ml Distilled water 90 ml
  • 26. KOH Wet Mounts Procedure: Place a small amount of specimen on a clean glass slide place 1-2 drops of KOH on the specimen and overlay a cover slip Allow the preparation to stand for 10-30 minutes in a wet chamber. You can gently heat preparation to hasten the action of KOH Do not over heat for it may crystallize the KOH Examine preparation under low then high magnification. Take note for the presence of fungal elements (hyphae and/or spores)
  • 27. INDIA INK PREPARATION aka: Nigrosin stain Principle: Specimen placed in a drop of India ink becomes darkly colored because of the carbon particle in the ink. Hyaline structures such as capsules and cell walls will be highlighted against a dark background of inked colored specimen creating an illusion of darkfield microscopy. Reagent: 1:1 dilution of the ink
  • 28. India Ink Preparation Procedure: Place a drop of the specimen (body fluid or from culture) on a clean glass Put a drop of India Ink, mix and overlay a cover slip Examine under low power and high power with a bright field microscope Result: India ink creates a dark background against which hyaline fungal cell wall and capsules can se seen Limitation: wbc may be confused as fungi
  • 29. Lactophenol Cotton Blue Principle: The morphology of fungal elements are preserved and stained better. Reagents: Lactic acid & Phenol Kills the organism Glycerin Prevents easy dehydration Cotton blue Dye or stain
  • 30. DIAGNOSIS OF MYCOSES by UNSTAINED & STAINED MICROSCOPY
  • 31. A. SKIN or DERMATOMYCOSIS KOH for superficial involvement, look for: Spaghetti & meat balls (lung aspirate) Malasseziafurfur Pseudohyphae and yeasts (vaginal secretions) Candida species
  • 32. A. SKIN or DERMATOMYCOSIS KOH & LPCB for superficial involvement, look for: Hyaline septatehyphae ex: Dermatophytes Dematiaceousseptatehyphae ex: Tineanigra Alternaria
  • 33. A. SKIN or DERMATOMYCOSIS H & E stain for Oral Candidiasis on Skin biopsy of tongue, look for: Pseudohyphae yeasts
  • 34. B. DRAINING SINUS for MYCETOMAS & ACTINOMYCOSIS KOH, look for Various colored granules Actinomycosis/ Nocardiosis GMS stain, look for Various granules Mycetoma
  • 35. C. EYE SCRAPINGS & ASPIRATE for KERATOMYCOSIS KOH & LPCB, look for Septate hyaline hyphae Aspergillus species Fusarium species Coenocytic hyaline hyphae Mucor species Pseudohyphae and yeasts Candida species
  • 36. D. NASOPHARYGNEAL ASPIRATES f for RHINOSPORIDIOSIS KOH, look for Large sporangium with spores (lacrimal gland aspirate) Rhinosporidium species
  • 37. E. HAIR for DERMATOMYCOSES & ALOPECIA KOH, look for Endothrix spores/hyphae Trichophyton Ectothrix spores/hyphae Trichophytonmentsgrophytes
  • 38. E. HAIR for PIEDRA KOH, look for: Hard, brown, compact nodules (Black piedra) Piedraiahortae Soft, off-white, concretions/nodules (White piedra) Trichosporonbeigeli
  • 39. F. NAILS for ONYCHOMYCOSIS KOH & LPCB, look for Septate, hyaline hyphae Dermatophytes Epidermophyton Trichophyton Microsporon Pseudohyphae and yeast cells Candida species
  • 40. G. SYSTEMIC MYCOSESSpecimens: blood, CSF, sputum, other body fluids KOH & Mucicarmine stain systemic involvement, look for: Pseudohyphae and yeast cells (CSF) Candida species Broad based buds (brain and CSF) Blastomyces species
  • 41. SYSTEMIC MYCOSES GMS for systemic involvement, look for: Spherules/sporangia (CSF) Coccidioidesimmitis PAS stain for systemic involvement, look for: Dematiaceousseptatehyphae (brain tissue)
  • 42. SYSTEMIC MYCOSES H & E stain for systemic involvement, look for: Endospores (CSF brain tissue) Coccidioides species Fission/sclerotic bodies Chromomyces species
  • 43. SYSTEMIC MYCOSES Wright’s/Giemsa stain (Diff quick) & LPCB for systemic involvement, look for: Small, intracellular budding yeast (CSF) Histoplasma species Small, intracellular yeast dividing by fission (CSF) Penicillin species
  • 44. SYSTEMIC MYCOSES Mucicarmine stain & India Ink for systemic involvement, look for: Encapsulate yeast (CSF) Cryptococcus neoformans
  • 45. SYSTEMIC MYCOSES LPCB & Fluorescent Antibody stain for systemic involvement, look for Large yeast with multiple buds called “mariner’s wheel” Paracoccidioidesbraziliensis
  • 46. SYSTEMIC MYCOSES Calcoflour mounts for systemic mycoses , look for (flourescence) Pseudohyphae and yeasts (blood) Candida species Septate, hyaline at right degrees angle (bronchial lavage) Aspergillus species
  • 47. Interpretation of Direct Microscopic Findings (Summary)

Editor's Notes

  1. Specimen depends on clinical presentations and the organ system affected. Adequate amount & Sample from area most likely affectedAsepsis: to prevent contamination and for laboratory safety Work in a biological safety cabinet to prevent the dissemination of the highly mobile conidia & spores. Greatest hazard to lab personnel comes from the handing of dimorphic pathogens – C. immitis & H. capsulatumSwabs are NOT encouraged : Decreases viability and High risk for contaminationViability: few hours to 16 daysTemperature: Most MOLDS grow best at: 25 – 30 OC Most YEAST grow best at 35 – 37 OC
  2. Rinse mouth prior to collection
  3. Aseptic (alcohol-I2-alcohol prep); Container: yellow-top vacutainer
  4. bottle number 3 specimen; Do NOT refrigerate because the specimen is a good transport medium by itself
  5. Collect at the the active border (peripheral, erythematous, growing margin of the lesion)Scrape lesion using glass slide or scalpel blade; Place scrapings between two glass slides or in an envelope
  6. Dorsal plate: scrape surface & discard, scrape the deeper portionPut specimen in an envelope
  7. No cleaning of scalp neededEpilate 10 hairs including hair follicle; Put sample between 2 glass slides or envelope
  8. If not possible, refrigerate specimen to inhibit bacterial proliferation
  9. Hair, nails & skin scrapping: Clean and dry container Biopsy materials: Placed in sterile saline or on a transport medium ; Place in between sterile gauze wet with sterile NSS (to prevent dehydration) and place inside a screw-capped container Viability: 8 hours at ref temp