SLIDE CULTURE TECHNIQUE

SLIDE CULTURE TECHNIQUE

AIM

       To identify the Fungi morphology without any disturbance by Slide culture technique.

PRINCIPLE   

   Fungi are the group of Eukaryotic microorganisms and they are identified mostly by close examination of its morphology and the characteristics it possess. Identification of Fungi is often difficult by tease mount method because of the dislodgement of conidia and spores from the Conidiogenous cell. To overcome this, Slide cultures technique is considered best for preserving and observing the actual structure of a fungus without any disturbances. The method was first developed by Riddel in 1950 and currently, several modifications are in use.

     In Slide cultures technique, fungi are inoculated in small blocks of Nutrient deficient agar medium (Cornmeal agar or Potato dextrose agar) (Fungi when grown in nutrition deficient medium develop spores rapidly and adhere to the surface of the coverslip), 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 (LPCB), may be added and observed for microscopic structures. 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. 

MATERIALS REQUIRED

• Fungal culture
• Cornmeal agar or Potato dextrose agar
• Petridish
• Scalpel or Spatula
• Glass slide
• Cover slip
• Lactophenol cottonblue (LPCB) stain

 PROCEDURE

a)     Prepare the Cornmeal agar or Potato dextrose agar medium and cut the block of agar (7 × 7 mm) by inserting the scalpel and carefully transfer this block aseptically to the centre of the Glass slide.

b)   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.

c)     Aseptically, place a sterile Cover slip on the upper surface of the agar cube.

d)    Place the Glass slide with Fungi inoculated agar block on the Petridish with wet cotton on both sides and incubate at room temperature for 48 hours.

e)     After 48 hours, gently remove the Cover slip from the fungi inoculated Agar block and place it on a microscope slide containing a drop of Lactophenol cotton blue.

f)      Observe microscopically for the characteristic shape and arrangement of spores.

   

Figure – 1: Slide culture technique

OBSERVATION AND RESULTS

          The morphology of the fungi was observed clearly without any disturbance under microscope in 45 X objective.