SOP for Microbial Staining Procedures : Pharmaguideline

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SOP for Microbial Staining Procedures

Standard operating procedure to stain the bacterial cells, bacterial spores and fungus.


To lay down the procedures for microbial staining.


This is applicable to the microbiology laboratory.




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5.1 Do not overheat the smear (where ever applicable).
5.2 Staining reagent should not fall on the floor.
5.3 For oil immersion technique use only one drop of immersion oil/required quantity (do not over flood the slide with immersion oil).

5.4 Gram’s Staining Procedure

5.4.1 Principle This staining procedure differentiates bacteria into gram-positive and negative based on their ability to retain the primary dye (crystal violet) or lose the primary dye and accept the color of the counterstain (safranin). Gram-positive bacteria produces a blue/purple color with this staining procedure while gram-negative ones are red in color Crystal violet -- the primary dye Gram’s iodine -- the complexing agent Alcohol -- the decolorizer Safranin -- the counterstain

5.4.2 Procedure Air dry or heat fix the smear and flood the slide with crystal violet staining solution for 1 minute. Wash the smear in a gentle and indirect stream of water. Flood the smear with iodine (mordant) solution for 1 minute. Wash in water. Decolorize with alcohol by adding dropwise until all free blue color has been removed. Wash in water. Flood the slide with safranin (counterstain) for 1 minute. Wash in water and air dry. Examine under oil immersion objective

5.4.3 Results and Interpretation Gram-positive cells appear violet in color whereas Gram-negative cells appear in red color.

5.5 Spore Staining Procedure

5.5.1 Principle Endospore-forming is a distinguishing feature of the family Bacillaceae. Endospores resist adverse environmental conditions such as dryness, heat and poor nutrient supply. The position of the spore in the cell may be central, subterminal or terminal. It may be the same diameter as the cell, smaller, or larger causing a swelling of the cell. Bacterial endospores strongly resist the application of simple dyes, but once stained are quite resistant to decolorization. This character suggests one way to make the structure (endospore) visible. If simple stains are used, the body of the bacillus is deeply colored, whereas the spore is unstained and appears as clear area in the organism. By vigorous staining procedures the dye can be introduced into the spore. When thus stained, the spore tends to retain the dye after treatment with decolorizing agents. To make the distinction clear between the spore and the vegetative portion of the cell, a contrasting counterstain is usually applied in the ordinary fashion and the resulting picture shows the initial stain taken up by the spore and the second stain appear in the cytoplasm. Thus it makes a very convenient method of distinguishing the endospore from vegetative cells.

5.5.2 Procedure (Schaeffer-Fulton method) Heat fix a bacterial smear with minimal flaming. Heat the underside of the slide with Bunsen burner or on a hot plate until steam rises (without boiling). Flood the slide with malachite green and leave it for 3 minutes, while the water continue to boil. Wash in water. Counterstain with safranin for 30 seconds. Examine under oil immersion objective.

5.5.3 Results and Interpretation Red colored cells containing green spores can be seen.

5.6 Fungal staining

5.6.1 Principle

The lactophenol cotton blue wet mount preparation is the most widely used method of staining and observing fungi. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls.

5.6.2 Procedure Place a drop of 70% alcohol on a microscope slide. Immerse the specimen/ material in the drop of alcohol. Add one, or at most two drops of the lactophenol/cotton blue mountant/stain before the alcohol dries out. Place the coverslip on the edge of the drop lower gently, avoiding air bubbles.

5.6.3 Results and Interpretation In case of Aspergillus niger compare the characteristic of fungal spore as follow A.niger produce colonies that are composed of white or yellow felt that is covered by dark asexually produced fungal spores. Mycelial, or threadlike, hyphae are divided by a septum and transparent.


6.1 SOP – Standard operating procedure
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