1. Demonstration of yeasts and filamentous fungi

2. Diagnosis
Presumptive
Definitive

3. Laboratory Diagnosis Mycological Diagnosis Histopathology Serology
Laboratory Animal

4. Laboratory Diagnosis a. Specimen collection b. Direct microscopy
c. Culture
d. Identification

5. 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.

6. 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. 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.

8. 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)

9. 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

10. 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

13. Recto/verso

14. Recto/verso

16. 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

18. 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

24. India Ink prep of CSF

25. Penicillium marneffei

26. Penicillium marneffei

27. Black Piedra

28. White Piedra

29. Paracoccidioidomycosis brasiliensis
Budding yeast 29

33. Daisies

34. S. schenckii budding yeast and asteroid body in tissue (PAS stain)

36. Trichophyton mentagrophytes

37. Trichophyton rubrum

38. Trichophyton violaceum

39. Trichophyton verrucosum

40. Epidermophyton floccosum

41. Microsporum canis Teleomorph: Arthroderma otae

42. Microsporum gypseum

43. END of ‘My’cology Innings