Demonstration of yeasts and filamentous fungi
Diagnosis Presumptive Definitive
Laboratory Diagnosis Mycological Diagnosis Histopathology Serology Laboratory Animal
Laboratory Diagnosis a. Specimen collection b. Direct microscopy c. Culture d. Identification
a. Specimen collection 1.In superficial mycoses the scales or infected hairs may be stored into small sterile glass Petri dishes. 2.Infected nail or skin scrapings are taken from the edges with a blunt scalpel. 3.Specimens from the mucous membranes or from orifices should be collected with dry swabs or preferably swabs soaked in Sabouraud’s broth. http://bluesplayer.co.uk/youtube_cartoons/video/4J1T2mQMxXM/Mycology-Specimen-Collection-Part-1.html
4. For the diagnosis of bronchopulmonary infection morning sputum should be collected in a sterile container. 5. For systemic mycosis, pus swab from an ulcer or aspiration from unruptured abscess, or biopsy during surgical operation are collected by strict aseptic technique. 6. For urinary tract infection, mid-stream urine samples are collected into a wide mouth sterile container.
7. For cerebrospinal infections, a lumbar puncture should be performed to collect CSF into sterile test tubes. 8. For Pleural and Peritoneal Effusions, a sample is collected by needle aspiration into sterile container.
Culture The agar medium most commonly used is: Sabouraud’s Dextrose Agar (SDA) which has the following composition: Dextrose 20 g Peptone 10 g Agar 20 g H2O 1 L Autoclave at 121 C for 10 minutes (pH 5.4)
2. SDA is the most suitable medium because fungal growth is favoured by a high sugar concentration and is relatively tolerant to acidity (pH 5.4) 3. The agar is prepared as slopes in test tubes stoppered with cotton-wool as most of the fungi are aerobic 4. If the specimens are contaminated e.g. sputum, incorporate antibiotics such as chloramphenicol (0.5 g/l) in the media used for isolation 5.Cycloheximide (Actidione*) can also be incorporated for the isolation of dermatophytes in order to get rid of saprophytic fungi
6. Petri-dishes should not be used for isolation of pathogenic fungi; the possibility of having a confusing contaminant is increased. Also the use of petri-dishes is hazardous if Dimorphic fungi are present 7. The pathological material is spread upon the surface of agar slopes. The fragments of skin, hair or nail are planted with a firm straight pointed wire. Incubate slopes at temperature of 25°C/ 37°C
Recto/verso
Recto/verso
LPCB Lactophenol cotton blue Lactic acid – clears debris, kills fungi Phenol – kills fungi, aids penetration of stain Glycerol – fixative (semi-permanent) Cotton blue – dye imparts color
Staining techniques Gram stain LPCB stain Calcofluor white Giemsa stain Gomori methanamine silver nitrate stain Mayer’s Mucicarmine stain Nigrosin stain India Ink KOH Mount + LPCB Periodic Acid Schiff stain
India Ink prep of CSF
Penicillium marneffei
Penicillium marneffei
Black Piedra
White Piedra
Paracoccidioidomycosis brasiliensis Budding yeast 29
Daisies
S. schenckii budding yeast and asteroid body in tissue (PAS stain)
Trichophyton mentagrophytes http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Trichophyton/mentagrophytes.html
Trichophyton rubrum http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Trichophyton/rubrum.htm
Trichophyton violaceum http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Trichophyton/violaceum.html
Trichophyton verrucosum
Epidermophyton floccosum http://www.doctorfungus.org/thefungi/epidermophyton.htm http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Epidermophyton/
Microsporum canis Teleomorph: Arthroderma otae http://www.doctorfungus.org/thefungi/microsporum_canis.htm http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Microsporum/Microsporum_canis.html
Microsporum gypseum http://www.doctorfungus.org/thefungi/microsporum_gypseum.htm http://www.mycology.adelaide.edu.au/Fungal_Descriptions/Dermatophytes/Microsporum/Microsporum_gypseum.htm
END of ‘My’cology Innings