Fundamentals of Microbiology (2+2)

Procedure

Slide culture preparation

• Aseptically, place a sheet of filter paper into bottom of Petri dish

• Place a sterile U-shaped glass rod on the filter paper

• Moisten the filter paper with sterile water ( about 5 ml)

• Place a sterile slide on the U -shaped rod

• Flame a scalpel to sterilize, cool 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 into one side

• Inoculate both top and bottom surfaces of the agar block with spores from the mold colony

• Place the inoculated block of agar in the center of the slide with one of the inoculated surfaces down

• Place a sterile cover glass on the upper inoculated surface of the agar cube

• Place the lid on the Petri dish and incubate at room temperature for 48 h. After 48 hours of incubation, examine the slide under low power objective for details of hyphae and 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. Add a drop of 95% ethanol to the hyphae on the cover glass

• After the evaporation of the alcohol, place the cover glass, mold side down, on the drop of lactophenol cotton blue stain on the slide

• Remove the slide from the Petri dish and add a drop of 95% ethanol to the hyphae and follow this up with a drop of lactophenol cotton blue stain.

• Cover the entire preparation with a clean cover glass

• Observe both the stained slides under the microscope