Slide culture Dalia Kamal Eldien Practical NO(4)

Slide culture Special techniques used in mycological lab include Slide culture and Scotch tape mount 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 fungi features that are key to identification, notably the spore-bearing structures, to be damaged.

Method 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. Pour enough sterile water  on  filter paper to completely moisten it. With forceps, place a sterile slide over 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 .

Method 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. Aseptically, place a sterile cover glass on the upper surface of the agar cube.

Method 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.

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

Cut the block of agar

Pick up the block of agar

U-shaped glass rod

Wet the filter paper

Culture the tested fungi

Cover by cover slip

Growth of fungi after incubation

Examine under microscope

Scotch tape 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 Cut a strip of Scotch transparent tape and place ends between thumb and index finger, gummed side out. Open plate with opposite hand and press tape against the colony to identify.

Place a drop of lactophenol on a labeled slide, Press tape against slide with lactophenol. Smooth the tape back on the slide by opening fingers and using guaze. Place another drop of lactophenol on top of the tape. Place a large 20x40 mm coverslip on top of slide. Examine the slide under the microscope.

Scotch tape

Scotch tape preparation

Examine under the microscope