Identification of fungi depends largely on their macroscopic features (colony characteristics, growth rate, color, texture, diffusible pigment, exudates, aerial and submerged hyphae) and microscopic features (arrangement of spores and sporing bodies). Arrangements of conidiophores and the way in which spores are produced (conidial ontogeny) help in the accurate identification of filamentous fungi.
This can be achieved by tease mount (a technique in which a part of growth is teased with needles, a drop of LPCB is added, covered with a coverslip and observed under the microscope). Although the most common technique used in mycology, identification is often difficult by tease mount method because of the dislodgement of conidia and spores from the conidiogenous cell.
To overcome this, slide cultures are put up which is considered best for preserving and observing the actual structure of a fungus. The method was first developed by Riddel in 1950 and currently, several modifications are in use.
Fungi are inoculated in small blocks of nutrition deficient agar medium (like cornmeal agar or 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.
Fungi when grown in nutrition deficient medium develop spores rapidly and adhere to the surface of the coverslip.
Identification is made by microscopically examining the undisturbed sporulating structures as they were arranged during growth on the agar block under the coverslip.
There are many variations on this method; however, the basic principle and procedure remain the same. Differences are based only on the set up of the procedure. Some use agar plates as a base whereas others may use wet tissue or molten agar.
Prepare a setup by either one of the following methods
- 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 (rod can be sterilized by flaming if held by forceps). Pour enough sterile water (about 4 ml) on filter paper to completely moisten it and place a sterile slide on the U-shaped rod.
- Alternatively, place several layers of paper towel into a sterile culture dish, add two applicator sticks or sterile glass rod, and position a sterile microscope slide on top.
- 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.
- 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. Note: The block should be smaller than the coverslip so that it fits under.
- Add the agar block to the surface of the sterile microscope slide.
- 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.
- Replace the lid of the culture and allow it to incubate at 30°C for 4-7 days.
- After the incubation period, remove the coverslip (working inside of a biological safety cabinet) and place it on a microscope slide containing a drop of lactophenol cotton or aniline blue.
- Observe microscopically for the characteristic shape and arrangement of spores.
The remaining agar block may be incubated further and used later (if the slide culture is unsatisfactory for the microscopic identification. The agar block is then removed and discarded, and a drop of lactophenol cotton or aniline blue is placed on the area of growth and a coverslip is positioned into place. Many laboratorians like to make two cultures on the same slide so that if characteristic microscopic features are not observed on examination of the first culture, the second will be available after an additional incubation period.
Although this method is ideal for making a definitive identification of an organism, it is the least practical methods among available methods. It should be reserved for those instances in which an identification cannot be made based on adhesive tape preparation or wet mount.
Slide cultures of slow-growing organisms suspected to be dimorphic pathogens such as H.capsulatum, B.dermititidis, C.immitis, P.brasiliensis or S. schenckii should not be made. Slide cultures must be observed only after a coverslip has been removed from the agar plug and not while it is in position on the top of the agar plug. The latter method of observation could be very dangerous, as it could cause a laboratory-acquired infection.
Advantages of slide culture:
- It is a cost-effective, rapid method for the identification of fungal isolates based on their morphological and/ microscopic characteristics.
- 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.
Disadvantages of slide culture:
As the agar medium used contains a limited nutrient source, fungal pathogens will survive for a short time period. Therefore, this method is not suited to fungi which take more than a few days to reach maturity, for example, members of the Xylariaceae and many fungi isolated as endophytes.
References and Further Reading
- A_revised_method_for_the_observation_of_conidiogenous_structures_in_fungi [accessed Dec 02 2019].
- Prakash, P.Yegneswaran.; Bhargava, K., 2016: A modified micro chamber agar spot slide culture technique for microscopic examination of filamentous fungi.
- Amrita Vishwa Vidyapeetham: Slide culture technique for fungi. [accessed Dec 03 2019].