Bacteria can be classified into two different categories i.e. gram-positive bacteria and gram-negative bacteria. To differentiate the type of bacteria present in any collected sample there is a technique known as GRAM STAINING OR GRAM STAIN. It is sometimes also known as gram’s method. This technique is a different step process which can easily distinguish and classify between different types of bacteria. This was named after the great scientist Hans Christian Gram.
Gram Staining method differentiates bacteria on the basis of their physical and chemical structure of cell wall. They are stained with different reagents and are observed in different colors when seen under the microscope. It is due to the fact that gram positive bacteria have a thick layer of peptidoglycan which allows it to retain the primary stain which is crystal violet and thus they appear purple whereas on the other hand, gram-positive bacteria have thin peptidoglycan cell wall and thus only retain the secondary or counter stain which is Safranin and thus they appear slightly pinkish when observed under microscope. Gram staining is the basic technique which is widely used in the microbiology labs to distinguish between both the types of bacteria. It provides a great help to microbiologists to perform their clinical tasks. If any infection is suspected in the patient then after the collection of sample, the gram staining is done and the type of bacteria is observed.
Gram staining is completed in the various steps –

  1. Fixation of clinical materials i.e. the sample collected are being fixed on the glass slide by first making a smear using water and then that smear is either heat fixed or methanol fixed. It is a first and a very important step. Methanol fixation was later discovered keeping in mind its advantage of not destroying the morphology of host cell, as well as bacteria present in that. It is majorly used for the testing of blood samples collected from patients.
  2. Application of primary stain i.e. crystal violet. Primary stain means the first stain which is applied onto the fixed smear which stains all the calls purple or blue.
  3. To distinguish the slide is then washed off in a gentle and indirect stream of water for 2 seconds which removes the extra stain that is not absorbed by the cells.
  4. The next step involves the application of a mordant i.e. Iodine solution. This forms a complex with crystal violet due to which all the cells start appearing blue.
  5. Again the slide is washed for 2 seconds to remove extra stain.
  6. Addition of a decolorizing agent is the next step which will ultimately remove the excess stain which bacteria has not absorbed. The decolorizer contains the combination of acetone and alcohol. In this step, the gram positive bacteria continue appearing violet or blue in color whereas gram negative bacteria start appearing colorless.
  7. Application of counter stain or secondary stain i.e. safranin is the next step. It should let remain on the slide for 30 second to 1 minute. This will stain all the colorless gram negative bacteria pink and gram positive bacteria remains blue in color.
  8. Again the slide is washed off in a gentle stream of water.
  9. The prepared stained slide is then observed under a microscope using immersion oil (for observing under 100x).