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The following points highlight the two methods used for observation of fungi and actinomycetes. The methods are: 1. Slide Culture 2. Cover Slip Culture.
Method # 1. Slide Culture:
Principle:
This method is used for fungi and actinomycetes to observe the conidiophores, the conidiogenous cells, their mode of conidial formation (conidial ontogeny) and conidia under natural conditions without disturbing them.
Requirements:
1. Cultures of Aspergillus and Streptomyces.
2. Agar plates with 12-15 ml solidified medium.
3. Sterile Petri dishes with moist filter paper and a pair of glass rods.
4. Slides and 22×22 mm cover slips.
5. Forceps and inoculating needle.
6. Sterile blade/scalpel.
7. 95% alcohol.
Procedure:
1. Sterilise a clean slide by flaming it and place it across the glass-rods in the Petri dish with wet filter paper.
2. Cut a 10 mm agar blocks with a sterile scalpel, lift it and keep it in the centre of the slide in the Petri dish.
3. Inoculate a very small portion of Aspergillus / Streptomyces spores at the four corners of the agar block on the slide.
4. Flame the cover slip, cool and place it over the inoculated agar block, replace lid of Petri plate and incubate at 28°-30°C (room temperature) for 72-96 hours (3- 4 days).
5. Examine the slide under microscope where undisturbed conidiophores, conidial ontogeny and conidia can be observed of Aspergillus and Streptomyces. Add a drop of 95% alcohol and then stain with 1% cotton blue in lactophenol and mount in lactophenol.
Lactophenol:
Lactic acid20.0 g
Phenol 20.0 g
Glycerine 10.0 g
Method # 2. Cover Slip Culture:
Principle:
This method helps in observing the conidiophores, the conidiogenous cells and conidial ontogeny without disturbing the growth. It is useful for Actinomycetes also and helps for SEM studies.
Requirements:
1. Trichothecium, Streptomyces cultures.
2. Czapek’s and Glycerol asparagine agar plates.
3. Inoculating needles.
4. Forceps.
5. Cover slips (sterile) 4×4 mm.
6. Bunsen flame.
7. Cotton blue, lactophenol.
8. Microscope.
9. Glass marking pencil.
Procedure:
1. Pour melted cooled (45°C) agar media in plates and allow them to solidify and mark four quadrants at the bottom of the Petri dish with glass marking pencil.
2. Stab one cover slip in each quadrant at 45° angle.
3. Streak the inoculum on the agar adjacent to the cover slip.
4. Incubate at room temperature 28°- 30°C for 4-5 days until the organism is seen growing up the cover slip.
5. Remove the cover slip with a sterile forceps gently warm to fix the culture to cover slip (optional) place a drop of cotton blue on a slide place coverslip on it and observe under a microscope.
This gives a temporary mount. The cover slips (4×4 mm) can be cut into four equal pieces and used for cultures of fungi and actinomycetes. These cover slip cultures can be dehydrated (optional) coated with gold and scanned under a Scanning Electron Microscope (SEM).