Microscopic Examination of fungi notes for microbiology students.pptx

Microscopic examination of fungi involves using a microscope to observe fungal structures, such as spores, hyphae, and reproductive organs. This technique aids in identifying fungal species, diagnosing infections, and studying fungal morphology and behavior. Staining and mounting techniques enhance visibility and detail during examination.

1. Microscopic examination of fungi by different techniques Public Heath Microbiology BY- SANJU SAH St. Xavier’s College, Maitighar , Kathmandu

2. Techniques Wet Mount Method Lacto Phenol Cotton Blue Teased Mount (LPCB-TM) Scotch Tape Method Slide Culture Technique. Cover Slip Technique

3. 2.Lactophenol cotton blue teased mount method This method is used study the morphology of hyphae,spores and other structures. Fragment of fungal culture is teased out using two teasing needle and placed on a glass slide in a drop of LCB stain. Then the preparation is covered with cover slip and observed under microscope Advantages: • It is the most widely used method for staining because it is easy and fast. • This stain is useful for making permanent mount of the fungus which is in culture. Disadvantage The whole morphology cannot be observed.

4. 3. Scotch tape method On a clean glass slide place a drop of Lactophenol cotton blue, Touch a adhesive side of tape of transparent scotch tape on the surface of colony at a point intermediate between its centre and periphery. Then fix theadhesive side of the tape over an area on the glass slide containing lactophenol cotton blue. The slide is observed under microscope

5. Disadvantages: Only the superficial structures of the fungi tend to stick to the tape. This technique is rarely used because of the technical inconvenience.

6. 4.Slide culture technique This technique is the best technique for the microscopic examination of mold cultures. But it takes time, some fungi take months to sporulate. The time and the nature of the block of agar are determinant factors. This technique allows the intact morphology of the fungus to be seen under the microscope.

7. Procedure 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 (about 4 ml) 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 dextrose agar. Pick up the block of agar by inserting the scalpel and carefully transfer this block aseptically to the centre of the slide.

8. 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. 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.

9. Advantages of slide culture: It is a rapid method of preparing fungal colonies for examination and identification. 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.

10. 5.Cover Slip Technique This simplified technique allows examination at different stages of development of the fungus. Cover slip is penetrated on agar plate making and angle of 45 º When fungus grows the aerial mycelium attach on coverslip and it is removed from agar plate. Then it is observed under microscope with lactophenol cotton blue. Diadvantage The aerial part of the fungi can only be observed by this method.

11. 1 . Wet mount method Potassium Hydroxide (KOH) mount is a very useful test for the laboratory diagnosis of fungal infection of tissues especially skin, hair and nails. KOH separates the fungal elements from intact cells as it digests the protein debris and dissolves cement substances that holds the keratinized cells together and makes it easy to visualize under the microscope. Another wet mouth method is done by using stain like Lactophenol Cotton Blue.

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