Lactophenol cotton blue stain is used to identify fungi grown on culture from any clinical material.
Principle of Lactophenol cotton blue stain
- The stain is used to stain fungus grown on ‘slide culture technique’ or culture media, and the preparation is examined under a bright field microscope.
- The fungus is identified using colony morphology on culture medium and microscopic morphology using the ‘slide culture technique.’
Type of Sample used for Lactophenol cotton blue stain
Any clinical specimen, tissue biopsy and/or bodily fluids collected for fungal etiology and investigation. Culture preparation showing sporulation
- Micro slides
- Filamentous fungal isolate
- “4 mm” Nichrome wire loop
- Cover slip
Lactophenol cotton blue stain: Ingredients:
- Phenol crystals 20 g
- Lactic acid 20 mL
- Glycerol 40 mL
- Cotton blue 0.05 g
- Distilled water 20 mL
The ingredients are dissolved by heating them in a water bath. Then 0.05 g of Cotton Blue is added.
Equipment used in Lactophenol cotton blue Staining
- Biological safety cabinet (Class II)
- Light Microscope – 100 × magnification
Lactophenol cotton blue staining Procedure
- The fungus grown on culture medium is inoculated onto four corners of a piece of SDA medium cut into 3×3 cm size, and a clean cover slip is placed over the medium.
- The inoculated bit is positioned on top of a microscopic glass slide. After keeping the slide culture in a moist chamber, the preparation is incubated at 25° C in an incubator.
- The preparation is observed every day for the production of spores by the fungus.
- After the spores have formed, a clean microscopic glass slide is prepared and a drop of lactophenol cotton blue is added.
- The slide culture cover slip is then removed from the agar, placed on the slide, and examined with a 40X objective under the microscope.
Interpretation of Results
- The stain is taken up by the fungus, which turns blue.
- Microscopic morphology is well appreciated.
- The fungus grown from a clinical specimen is identified and reported using colony morphology and microscopic morphology.
- Observe all precautions as for handling a biological fluid.
- Wear facemask and disposable gloves while transferring and handling the specimen.
- Process the specimen under the biosafety hood only.
Fungi. In Mackie & McCartney Practical Medical Microbiology. Year 1990, 14th edition Edited by Collee, Dugid, Fraser, Marmion. Pg. 600-50.