SOP For Lactophenol Cotton Blue Staining for Identification of Fungi
1.0 Objective
1.1 To establish a procedure for performing the Lactophenol Cotton Blue (LPCB) staining technique for microscopic identification of fungal isolates.
2.0 Scope
2.1 This SOP is applicable to the identification of fungal colonies isolated in the Microbiology Laboratory.
3.0 Responsibility
3.1 Microbiologist or above – Microbiology Laboratory:
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Preparation and execution of the staining procedure.
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Microscopic examination and interpretation of results.
3.2 Head – Microbiology Section / Nominee:
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Review and approval of the SOP.
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Ensuring compliance with the procedure.
4.0 Accountability
4.1 Head – Quality Control / Nominee:
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Overall compliance and implementation of this SOP.
5.0 Procedure
5.1 Preparation of Slide
5.1.1 Take a clean, dry glass slide.
5.1.2 Place two to three drops of Lactophenol Cotton Blue (LPCB) stain at the center of the slide.
5.2 Preparation of Fungal Mount
5.2.1 Using a sterile inoculating needle (flamed and cooled), gently pick a small portion (tuft) of the fungal colony along with spores from the culture plate.
5.2.2 Transfer the fungal material onto the drop of LPCB stain on the slide.
5.2.3 Using two sterile flamed and cooled needles, carefully tease and separate the fungal tuft into smaller fragments to obtain a thin, even preparation.
5.3 Mounting
5.3.1 Carefully place a clean coverslip over the stained preparation.
5.3.2 Avoid trapping air bubbles while placing the coverslip.
5.4 Microscopic Examination
5.4.1 Examine the prepared slide under the microscope.
5.4.2 Initially observe under low power (10X objective) to locate the fungal structures.
5.4.3 Further examine under high power (40X objective) to study detailed morphological characteristics such as hyphae, spores, conidia, and spore arrangement.