Gram Staining Procedure Of Bacteria (Step By Step)

Gram staining: this technique was development by Danish physician, Hans Christian Gram, in 1884; which is very useful in taxonomic grouping of bacteria. The basic distinction between gram+ and gram-bacteria is in the difference of cell wall structure, along with some other characteristics. The crystal violet, iodine, ethyl alcohol (95%) and safranin are used for staining. First, the cell are stained

with crystal violet, then with gram iodine and finely washed in 95%ethanol which differentiate two  i.e., Gram+ and the other one which loses stain i.e., Gram-. The slides are washed with distilled water, followed by counter-stain with safranin for 30sec. and finely the slide are again washed with distilled water. Gram+ bacteria remain dark violet or purple coloured, but gram- bacteria become red (pink). This grouping does not depend on the shape of bacteria e.g., the bacillus shaped  bacteria like Bacillus subtails is gram+, while the other rod-shaped bacteria like coxiella burnetii is gram-. The coccus-shaped bacteria such as Escherichia coli is gram-, but sporosarcina is gram+. The detailed flow chart of the gram staining procedure is giving bellow:

groups : one which retains stain becomes dark-violet or purple coloured

Process

Take a clean grease free slide

Prepare a smear of bacteria on the slide

Air dry and then heat fix the smear over flame

Apply few drops of crystal violet stain on slide and keep it for 2-60sec.

Rinse the slide in distilled water

Apply few drops of gram’s iodine on the slide and keep it for 20-30sec.

Rinse the slide in distilled water again

Wash the slide with 95% ethyl alcohol for 10-20 sec.

Stains used for gram-staining:

  1.  Crystal violet

Solution A

  •         Crystal violet (90% dry content)……………………… 2gm
  •        Ethyl alcohol (95%)………………………………… 20ml

Solution B

  •          Ammonium oxalate…………………………………. o.8 gm
  •          Distilled water……………………………………… 80 ml

Mix solution A & B.

  1.  Iodine solution
  •        Iodine ……………………………………………….. 1 gm
  •          Potassium iodite……………………………………. 2 gm
  •        Distilled water………………………………………. 300 ml
  1.  Safranin
  •        Safranin (2.5% soln, in 95% ethanol)……………….10 ml
  •      Distilled water……………………………………….100 ml

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