SOP: Gram Staining of Bacteria

GIFSQ — Global Institute for Food Safety & Quality

Principle

The Gram staining technique differentiates bacteria into two groups on the basis of cell-wall behaviour: Gram-positive cells hold the blue primary dye through decolourization, while Gram-negative cells lose it and take up the red counterstain. The result is an aid, or tool, in classifying bacteria.

Apparatus required

• Glass slides
• Sterile loop
• Grease pencil (for marking the smear position)
• Bunsen burner
• Microscope with oil immersion objective

Reagents

a. Primary dye (crystal violet): solution A — dissolve 2 g of crystal violet (85% dye content) in 20 ml of ethyl alcohol (95%); solution B — dissolve 0.8 g of ammonium oxalate in 80 ml of distilled water. Before use, mix equal quantities of solutions A and B; if the mixture is too concentrated and difficult to decolourize, dilute solution A up to 10 times and mix the diluted solution with an equal quantity of solution B.

b. Mordant (Lugol’s iodine, Gram modification): dissolve 1 g of iodine and 2 g of potassium iodide in 300 ml of distilled water.

c. Counterstain (safranin): mix 10 ml of a 2.5% solution of safranin in 95% ethyl alcohol with 100 ml of distilled water.

Procedure

Clean the glass slides thoroughly and wipe them with a dry cloth; handle the slides only by grasping them at the edges

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Flame the slide surface to remove any grease, and mark with a grease pencil where the smear is to be made

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Heat a sterile loop to redness in a Bunsen flame, let it air-cool, transfer a loopful of bacterial suspension, spread it evenly over a small circular area, and let it air dry; reflame the loop to burn off the excess inoculum

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Fix the dried smear by passing the slide rapidly 5 to 6 times through the flame, film-side up — apply only even heat that the slide can tolerate on the back of one’s palm

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Apply the crystal violet solution and let it remain for 1 min; wash in tap water

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Apply the iodine solution (mordant), let it remain for 1 min, and wash in water

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Decolourize by adding alcohol dropwise on the tilted slide until all free colour (blue) has been removed; wash in water

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Flood the slide with safranin (counterstain) for 10 sec; wash in water and air dry

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Examine under the oil immersion objective of the microscope: violet cells that survive decolourization are Gram-positive; cells that lose the violet and turn red with safranin are Gram-negative

Precautions

• Do not over-decolourize — too much alcohol strips the dye from Gram-positive cells as well.
• Fix the smear with gentle heat only; scorching the slide distorts the cells.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.