TECHNIQUES USED FOR MOULD IDENTIFICATION
Colony Characteristics
• To evaluate colony characteristics of filamentous fungi, it is necessary to subculture the fungus to the same media that the original colony descriptions are based upon.
• Visual examination of the colony will rapidly reveal important data concerning color, texture, diffusible pigments, exudates, growth zones, aerial and submerged hyphae, growth rate, colony topography, and macroscopic structures such as ascocarps, pycnidia, sclerotia, sporodochia, and synnemata.
Lactophenol Mounts
Prepare a mount of the fungus in lactophenol within the biological safety cabinet.
Lactophenol Cotton Blue Mounting Media
a. Formulation:
Phenol (concentrated) : 20.0 ml
Lactic acid : 20.0 ml
Glycerol : 40.0 ml
Cotton blue : 0.05 gm
Distilled water : 20.0 ml
b. Preparation:
i. Mix phenol, lactic acid, glycerol and water in a 250 ml screw cap bottle.
ii. For lacto-phenol cotton blue, dissolve 0.05 gm of cotton blue in 20 ml of water. Then add phenol, lactic acid and glycerol. Store in a 250 ml screw cap bottle.
c. Quality control:
Appearance : Deep blue liquid, slightly viscous
Usage : A mounting medium for microscopic examination.
Caution : Avoid exposure to phenol vapors during use.
TECHNIQUES OF MICROSCOPIC EXAMINATION
Principle
The simplest technique consists in placing a fragment of the colony between a slide and cover slip. This approach is rapid and often adequate for identification. Delicate fungal structures, however, are often broken by this process, rendering identification difficult.
The adhesive tape technique assists in maintaining the integrity of fungal structures by fixing them on the adhesive surface of a piece of transparent (Not frosted) tape.
The slide culture technique consists in growing a micro colony of the fungus on a slide or on a cover slip. The developing fungal colony adheres to the surface of the glass and facilitates the observation of intact conidial structures.
Methods
Direct Examination of a Portion of the Colony
• With the aid of sterile needle, detach a small fragment of the colony to be identified and place it in a drop of lactophenol cotton blue
• Using two needles gently spread apart the material to be examined
• Cover the preparation with a cover slip and press gently to flatten the preparation
• Examine under microscope with low magnification and then under higher magnification.
Adhesive Tape Technique
• Cut a piece of adhesive cellulose tape and fold it back on itself with the adhesive side turned outward. To hold on to the ends of the loop, forceps may be useful
• Press the adhesive side of the tape onto the surface of the colony and pull it away. The aerial hyphae of the colony will remain glued on to the tape surface
• Place the tape in a drop of lacto phenol-cotton blue previously placed at the center of a glass slide
• Examine microscopically (Fig. 7.1).
Slide Culture Technique (Ridell’s Method)/Title Case
• Place a bent, U-shaped glass rod in the bottom of a sterile petri plate or sterile wet cotton ball in two corners of petriplate. Place a clean, sterile slide on the glass rod and then place a block of medium at the center of this slide
• Inoculate the sides of the medium block with small pieces of the culture to be identified.
• Place a cover slip sterilized by rapid passage over a flame onto the block of medium
• Pipette around 5 ml of sterile water onto the bottom of the petri plate to ensure maintenance of humidity, and then close the petri plate
• Incubate at an optimal temperature, generally 25°C
• The slide may be taken out of the petri plate and examined under the microscope at regular intervals (once in 7 days) to determine whether sporulation has occurred
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Fig. 7.1: Adhesive tape technique
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• When the culture has sporulated, lift the cover slip from the medium block and fix it by passing it rapidly in close proximity to a burner flame. Mount it by placing it over a drop of cotton blue or other mounting fluid on a glass slide (Figs 7.2A to C)
• It is also possible to mount the portion of the culture which remains attached to the slide after disposing of the block of medium
• These preparations may be sealed by applying a layer of nail polish around the perimeter of the cover slip. Such preparations, if well sealed, may remain in good condition for 1-5 years.
Simplified Slide Culture Technique (Harris Method, Modified)
• Beginning with a petri plate of growth medium, cut two small blocks of medium from one side of the plate and place them on the surface of the medium near the opposite side
• Inoculate the sides of the two blocks with small pieces of the culture to be studied
• Place a cover slip sterilized by rapid passage through a flame onto each of the two blocks, close the petri plate, and incubate
• When appropriate, fix and mount the cover slips as described above.
REMARKS
When cover slips are being mounted, do not use more than a small quantity of mounting fluid between slide and cover slip. When the thickness of the preparation is minimized, most
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| Figs 7.2A to C: Classic slide culture technique (Ridell’s Method) A. Inoculation of fungal colony B. Dematiaceous fungus grown well after 5 days C. Well grown hyaline fungus ready to be observed for the morphological form |
of the fungal structures will be in the same plane, making examination easier. This detail is especially important for photography. In addition, an excess of liquid will prevent proper sealing of the cover slip in any attempt to make a permanent mount of the preparation. More enduring mounts can be produced by placing a small drop of lactophenol cotton blue on the cover slip bearing the fungal growth and covering it with a second cover slip of smaller size. This whole preparation is then placed with the smaller cover slip facing downward onto a drop of balsam or other permanent mounting material in the center of a slide.
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