Procedure for accessing flash
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7-10 day old fungal culture
Preparation of Sabouraud agar (pH-5.6)
Sabouraud agar supplemented with aureomycin*
*Aseptically add aureomycin ,10µg per ml, to the sterile, molten and cooled medium
- Sterile Petri dish
- Filter paper (9cm diameter)
- U-shaped glass rod
- Microscope slides and coverslips (Sterile)
- Sabouraud’s plate with mixed culture of fungi
- Sterile Sabouraud’s agar plate
- Lactophenol cotton blue stain
- Glass capillary tube
- Inoculating needle
- Sterile distilled water
- 95% ethanol
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 centre of the slide.
- 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.
B) Application of Stain
- Place a drop of lactophenol cotton blue stain on a clean microscope slide.
- Remove the cover glass from the slide culture and discard the block of agar.
- Add a drop of 95% ethanol to the hyphae on the cover glass. As soon as most of the alcohol has evaporated place the cover glass, mold side down, on the drop of lactophenol cotton blue stain on the slide. Examine the slide under microscope
Advantages of slide culture:
- It is a rapid method of preparing fungal colonies for examination and identification.
- Permits fungi to be studied virtually in situ with as little disturbance as possible
- Fungi are identified mostly by close examination of its morphology and the characteristics it possess. In slide cultures, we are growing the fungi directly on the slide on a thin film of agar. By doing this, there is no need to remove a portion of the fungus from a culture plate and transfer it to the slide. So there is less chance for the features that are key to identification, notably the spore-bearing structures, to be damaged.
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