1. Practical medical mycology
بسم الله الرحمن الرحيم
Practical medical mycology
Lab 5 Direct examination of the specimen and other techniques ( part 2 )

2. Dimorphic fungal infection
Histopasmosis
Blastomycosis
Coccidiodomucosis
Paracoccidiodomycosis
"Pseudohyphae" are distinguished from true hyphae by their method of growth, relative frailty and lack of cytoplasmic connection between the cells.

3. A. skin or dermatomycosis
KOH for superficial involvement, look for:
1. Spaghetti & meat balls (lung aspirate)
Malassezia furfur
2. Pseudohyphae and yeasts (vaginal secretions)
Candida species.
3. Hyaline septate hyphae ex: Dermatophytes
4. Dematiaceous septate hyphae ex: Tinea nigra.
"Pseudohyphae" are distinguished from true hyphae by their method of growth, relative frailty and lack of cytoplasmic connection between the cells.

4. H & E stain ) Hematoxylin and eosin stain( for Oral Candidiasis on Skin biopsy of tongue, look for:
1. Pseudohyphae
2. yeasts

5. G. Systemic mycoses specimens: blood, CSF, sputum, other body fluids
KOH & Mucicarmine stain systemic involvement, look for:
1. Pseudohyphae and yeast cells (CSF)
Candida species
2. Broad based buds (brain and CSF)
Blastomyces species

6. B. Draining sinus for mycetomas & actinomycosis
KOH, look for:
1. Various colored granules Actinomycosis/Nocardiosis
GMS Silver stain, look for:
1. Various granules.

7. C. Eye scrapings & aspirate for keratomycosis
KOH & LPCB, look for:
1. Septate hyaline hyphae
Aspergillus species
Fusarium species
2. Coenocytic hyaline hyphae
Mucor species
3. Pseudohyphae and yeasts
Candida species

8. D. Nasopharyngeal aspirates for rhinosporidiosis
KOH, look for
1. Large sporangium with spores (lacrimal gland aspirate)
Rhinosporidium species

9. E. Hair for dermatomycoses & alopecia
KOH, look for
1. Endothrix spores/hyphae
Trichophyton
2. Ectothrix spores/hyphae

10. E. Hair for piedra
KOH, look for:
1. Hard, brown, compact nodules (Black piedra)
Piedraia hortae
2. Soft, off-white, concretions/nodules (White piedra)
Trichosporon beigeli

11. F. Nails for onychomycosis
KOH & LPCB, look for
1. Septate, hyaline hyphae
Dermatophytes )Epidermophyton, Trichophyton, Microsporom.
2. Pseudohyphae and yeast cells
Candida species

12. GMS for systemic involvement, look for:
1.Spherules/sporangia (CSF)
Coccidioides immitis
PAS stain for systemic involvement, look for:
1. Dematiaceous septate hyphae (brain tissue)

13. Wright’s/Giemsa stain (Diff quick) & LPCB for systemic involvement, look for:
1. Small, intracellular budding yeast (CSF)
Histoplasma species.
2. Small, intracellular mold dividing by fission (CSF)
Penicillin species.

14. India Ink for systemic involvement, look for:
1. Encapsulate yeast (CSF)
Cryptococcus neoformans

15. LPCB & Fluorescent Antibody stain for systemic involvement, look for
1. Large yeast with multiple buds called “mariner’s wheel”
Paracoccidioides braziliensis

16. Identification of Fungal Isolates
•Fungal Isolates can be identified by one of the following methods:
(1)Tease Mount Procedure (Lactophenol cotton blue).
(2) Scotch Tape Lactophenol Mount
(3) Slide culture technique

17. Tease mount
 For microscopic examination, fragment of fungal culture is teased out using two teasing needles and placed on a glass slide in drop of LCB stain
To study the morphology of hyphae, spores and other structures, tease mounts are prepared in lactophenol cotton blue (LCB) which contains lactic acid, phenol, glycerol and cotton blue
Microscopic characteristics that should be observed are the following:
Sepatate versus sparsely septate hyphae
Spores or conidia

18. Preparation of Lactophenol cotton blue (LPCB) slide mounts
Place a drop of 70%alcohol on a clean microscope slide.
Material from cultures of filamentous fungi should be removed using a stiff inoculating wire.
Remove a small amount of the culture. For fungal cultures, Immerse the fungal material in the drop of 70%alcohol
Before the alcohol dries out add one or at most two drops of the stain lactophenol cotton blue to the slide
Cover by cover slip& examine under the microscope
Make the initial examination using a low power objective lens. The thinner parts of the preparation, generally around the edges of the mounted material, will yield the best images.
Switch to a higher power 40X objective for more detailed examination of spores and other structures.

19.  Lactophenol Cotton blue mount of the isolated pathogen, showing yeast cells and septate hyphae

20. Aspergillus
Penicillium

23. 2-Scotch Tape Lactophenol Mount
•The Scotch tape mount is an easy and fast procedure that is used for the identification of filamentous fungi since most structures will be intact for observation thank to the gummed side of the tape.
• As with the lactophenol mount, the organism will be immersed in the solution, rendering the organism safe for handling outside of the biological safety hood.

24. •Limitations include:
- the tape will dissolve eventually so that it is not to be used for permanent mounts.
- The procedure can only be performed on molds growing from plates.

25. Procedure
(1)Cut a strip of Scotch transparent tape and place ends between thumb and index finger, gummed side out.
(2) Making a loop by closing fingers, open plate with opposite hand and press tape against the colony to identify.
(3) Place a drop of lactophenol on a labeled slide.
(4) Press tape against slide with lactophenol.
(5) Smooth the tape back on the slide by opening fingers and using gauze.
(6) Place another drop of lactophenol on top of the tape.
(7) Place a large 20x40 mm coverslip on top of slide.
(8) Examine the slide under the microscope.

27. Slide Culture A- Slide Culture Preparation
Aseptically, with a pair of forceps, place a sheet of sterile filter paper in a Petri dish.
Place a sterile U-shaped glass rod on the filter paper. (Rod can be sterilized by flaming, if held by forceps.)
Pour enough sterile water  (about 4 ml) on  filter paper to completely moisten it.
With forceps, place a sterile slide on the U-shaped rod.
Gently flame a scalpel to sterilize, and cut a 5 mm square  block  of  the medium  from  the  plate  of Sabouraud’s agar or Emmons’ medium.
Pick up the block of agar by inserting the scalpel and carefully transfer this block aseptically to the center of the slide.

28. Inoculate four sides  of the agar square with spores or mycelial fragments of the fungus to be examined. Be sure to flame and cool the loop prior to picking up spores.
Aseptically, place a sterile cover glass on the upper surface of the agar cube.
Place the cover on the Petri dish and incubate at room temperature for 48 hours.
After  48  hours,  examine  the  slide  under  low power. If growth has occurred there will be growth of hyphae  and production of  spores.  If  growth  is  inadequate  and spores are not evident, allow the mold to grow for another  24–48  hours  before  making  the  stained slides.