Characterisation of Microorganism – Grams Staining

Sampling of probiotic bacteria from food samples and isolation on specialised enriched media


Simple staining depends on the fact that bacteria differ chemically from their surroundings and thus can be stained to contrast with their environment. Bacteria also differ from one another chemically and physically and may react differently to a given staining procedure. This is the principle of differential staining. Differential staining can distinguish between types of bacteria. The Gram stain (named after Christian Gram, Danish scientist and physician, 1853–1938) is the most useful and widely employed differential stain in bacteriology. It divides bacteria into two groups— gram negative and gram positive. The first step in the procedure involves staining with the basic dye crystal violet. This is the primary stain. It is followed by treatment with an iodine solution, which functions as a mordant; that is, it increases the interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more strongly stained. The smear is then decolorized by washing with an agent such as 70% ethanol or isopropanol-acetone. Gram-positive bacteria retain the crystal violet-iodine complex when washed with the decolorizer, whereas gram-negative bacteria lose their crystal violet-iodine complex and become colorless. Finally, the smear is counterstained with a basic dye, different in color than crystal violet. This counterstain is usually safranin. The safranin will stain the colorless, gram-negative bacteria pink but does not alter the dark purple color of the gram-positive bacteria.


  1. Bacterial culture
  2. Crystal violet
  3. Gram’s iodine
  4. 70% alcohol
  5. Carbol fuschin (1:10)
  6. Inoculating loop
  7. Immersion oil
  8. Bunsen burner
  9. Bright field microscope
  10. Clean glass slides


  1. Prepare heat-fixed smears of the given bacterial culture.
  2. Place the slides on the staining rack.
  3. Flood the smears with crystal violet and let stand for 1 minute.
  4. Rinse with water for 5 seconds.
  5. Cover with Gram’s iodine mordant and let stand for 2 minutes.
  6. Rinse with water for 5 seconds.
  7. Decolorize with 70% ethanol for 15 seconds. Do not decolorize too long. Add the decolorizer drop by drop until the crystal violet fails to wash from the slide.
  8. Rinse with water for 5 seconds.
  9. Counterstain with safranin or carbol fuschin (1:10 diluted) for about 30 seconds.
  10. Rinse with water for 5 seconds.
  11. Blot dry with tissue paper and examine under oil immersion. Gram-positive organisms stain blue to purple; gram-negative organisms stain pink to red. There is no need to place a coverslip on the stained smear.
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