SLIDE CULTURE TECHNIQUE

 

 

Morphological observation of Fungi – Slide  culture

 

AIM: To cultivate and to study the fungi morphology on the slide by following slide culture technique.

PRINCIPLE: Fungi are inoculated in small blocks of nutrition deficient agar medium like potato dextrose agar, covered with a coverslip and incubated. After incubation, the coverslip is removed from the agar block and placed on another slide to which a dye, such as lactophenol cotton blue, may be added and observed for microscopic structures. Identification is made by microscopically examining the undisturbed sporulating structures as they were arranged during growth on the agar block under the coverslip.

MATERIALS REQUIRED: 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, Scalpel, Inoculating needle, Sterile distilled water, 95% ethanol, Forceps

PROCEDURE:

1.      Cut an agar block of desired dimensions (using sterile scalpel) from a solid medium and just flip the block up onto the surface of the same agar plate. Proceed to step 4 directly in such cases.

2.      Cut a small block (range: 5×5 mm-1x1cm) of a suitable agar medium that has been previously poured into a culture dish to a depth of approximately 2 mm by using sterile scalpel blade or with a sterile test tube. 

3.      Add the agar block to the surface of the sterile microscope slide.

4.      With a right-angle wire, inoculate the four quadrants of the agar block with the organism and apply a sterile coverslip onto the surface of it.

5.      Replace the lid of the culture and allow it to incubate at 30°C for 4-7 days.

6.      After the incubation period, remove the coverslip and place it on a microscope slide containing a drop of lactophenol cotton or aniline blue.

7.      Observe microscopically for the characteristic shape and arrangement of spores.

 

 

RESULT: As fungi grow directly on the slide on a thin film of agar, there is no need to remove a portion of the fungus from a culture plate and transfer it to the slide. This reduces the chance of damage to fragile reproductive structures or spore-bearing structures of fungi.