Gram Staining Protocol

In this practical we are going to discuss about Gram Staining protocol with gram staining principle, procedure  in a very easy way.


To perform the Gram staining technique to differentiate and classify the two major groups : Gram positive bacteria and Gram negative bacteria.


In 1884 the Danish bacteriologist Hans Christian Gram developed a staining technique that separates bacteria into two groups : that are gram positive and gram negative. The procedure is based on the ability of microorganism to retain the purple color crystal-violet during decolonization with alcohol.Gram negative bacteria are decolorized by the alcohol,losing the purple color of crystal violet.Gram positive bacteria are not decolourized and remain purple.After decolonization, safranin, a red counter stain,is used to impart a pink color to the decolonized gram-negative organisms.

In gram staining protocol, crystal violet,the primary stain,causes both gram positive and gram negative organisms to become purple after 20 seconds of staining.When Gram’s Iodine, the mordant, is applied to the cells for 1 minute,the color of gram positive and gram negative bacteria remains the same;purple color.The function of the mordant here is to combine with crystal violet to form a relatively insoluble compound in the gram positive bacteria remain purple.In the final step a counter-stain, safranin ,adds a pink color to the de-colorized gram negative bacteria without affecting the color of the purple gram positive bacteria.

This techniques seems quite simple,performing it with a high degree of reliability is a goal that requires some practice and experience.Here are some suggestions that can be helpful;

  • First, don’t make your smear too thick. Only a very small amount of culture is needed; a visual detection of the culture on an inoculation loop already indicates that too much is taken
  • Second, when working with unknowns keep in mind that cultures of gram positive bacteria tends to de-colorized more rapidly than young ones.
  • Leaving the decolorizer on for longer than 8 seconds will cause excess decolorization in the gram-positive cells, and proper staining will not occur.
  • While working with Bacillus,causing them to appear gram positive, another point to remember that, one should use cultures that are totally fresh.Because Bacillus shows gram variability over aging of cultures i.e. sometimes it may be gram positive and sometimes gram negative in nature.


The basic principle of Gram’s Staining is to determine by the properties of certain bacterial cell walls to retain the crystal violet dye. The cell walls for gram-positive microorganisms have a higher lipid content than gram-negative cells. First, crystal violet ions penetrate the cell wall of both types of cells. Then, iodine solution is added to form CV-I complex (where Iodine works as denaturing agent and lipid solubilizer), that makes the dye difficult to remove.That’s why this step is referred as “fixing” of the dye.
After treating with iodine, the cells are treated with decolorizer, a mixture of ethanol and acetone, which dissolves the lipid layer from the gram-negative cells, and dehydrating the thicker gram-positive cell wall. As a result, the stain leaches from gram-negative cells and is sealed in gram-positive cells. With expedient removal of the decolorizer, cells will remain stained.Then the safranin uses as counter stain. The addition of a safranin counter-stain to dye the gram-negative cells with a pink color for easier observation under a microscope. Thus, gram-positive cells will be stained purple and gram-negative cells will be stained pink on the basis of gram staining techniques .


A. Gram staining reagents

  • Crystal violet
  • Grams iodine solution
  • Acetone
  • Distilled water drop
  • 95% ethyl alcohol
  • Safranin

B. Others Materials

  • Staining tray
  • Inoculation loop
  • Glass rods
  • Blotting paper
  • Burner
  • Microscope

Preparation of solution

A. Gram Crystal Violet Solution

  • Dissolve 20 g of crystal violet in 100 ml of ethanol to make a crystal violet stock solution.
  • Similarly, dissolve 1 g of ammonium oxalate in 100 ml of water to make an oxalate stock solution.
  • The working solution is obtained by mixing 1 ml of the crystal violet stock solution with 10 ml of water and 40 ml of the oxalate stock solution. Store the working solution in a drop bottle.

B. Methylene Blue Solution

  • Dissolve 1 g of methylene blue, 90% dye content, in 100 ml of ethanol, this is solution A.
  • Mix 0.03 g of KOH in 300 ml of water, this is solution B.
  • Mixing solutions A and B yields the working solution.

C. Gram Iodine Solution

  • Dissolve 1 g of iodine, 2 g of potassium iodide and 3 g of sodium bicarbonate in 300 ml of water.

D. Gram Decolorizer Solution

  • Mix equal volumes of 95% ethanol and acetone.

E. Gram Safranin Solution

  • Dissolve 2.5 g of Safranin O in 100 ml of 95% ethanol to make a stock solution.
  • Working solution is obtained by diluting one part of the stock solution with five parts of water.


Smear Preparation

  • Transfer a drop of the suspended culture to be examined on a slide with an inoculation loop. If the culture is to be taken from a petri dish or a slant culture tube, first add a drop of water on the slide and aseptically transfer a minute amount of a colony from the petri dish.
  • Spread the culture with an inoculation loop, creating an even thin film over a circle of 1.5 cm in diameter (approximately dime sized). Thus, a typical slide can simultaneously accommodate 3 to 4 small smears if more than one culture is to be examined.
  • Hold the slide with a cloth pin. Air-dry the culture and fix it or over a gentle flame while moving the slide in a circular fashion to avoid localized overheating.
  • The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture. Heat can be applied to facilitate drying the smear, however, ring patterns can form if heating is not uniform (e.g. taking the slide in and out of the flame).

Gram Staining

  • Add about 5 drops of crystal violet stain over the fixed culture. Let stand for 60 seconds. A clothes pin is used to hold the slide during the staining procedure to avoid staining one’s hand.
  • Pour off the stain and gently rinse the excess stain with a stream of dH2O. Note: The objective of this step is to wash off the stain, not the fixed culture.
  • Add about 5 drops of the iodine solution on the smear, enough to cover the fixed culture. Let stand for 60 seconds.
  • Pour off the iodine solution and rinse the slides with running water. Shake off excess water from the surface.
  • Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with water after 30 seconds. Stop when the solvent is no longer colored as it flows over the slide.
  • Leaving the decolorizer on for longer than 10 seconds will cause excess decolorization in the gram-positive cells, and proper staining will not occur.
  • Counter-stain with 5 drops of the Safranin solution for 60 seconds.
  • Wash off the red Safranin solution with water. Blot with bibulous paper to remove any excess water. Alternatively, the slide may be shaken to remove most of the water and air-dried.
  • Liberally wash off any spilled stain immediately with water to avoid leaving permanent marks on the sink, lab bench or glassware.
  • Examine the finished slide under a microscope.

Observation & Results


In a mixed culture some bacteria appear purple and some pink under microscope after gram staining procedure. It may also possible that in a single bacterial culture all bacteria will appear pink or purple after proper gram staining protocol is followed

Sample appear purple in color because crystal violet binds with thick peptidoglycan layers and the pore of cell-wall were closed due to denaturing properties of alcohol. After addition of alcohol crystal violet retained inside the cell and showed purple or violet color. So we conclude that sample that appears pink is gram positive in nature.

Other sample appears as red in color because the gram negative bacterial cell wall has large amount of lipid layers which dissolved in alcohol. So the primary stain is washed out from the cell wall and after addition of counters stain safranin it was appeared as red or pink. So it is confirmed that it it is gram negative.


  • Always use fresh and young cultures ( less than 24 hours old ) to avoid misleading results. Since old cultures of gram positive bacteria tends to decolourize more rapidly; thus might appear to be gram negative.
  • Excessive heat should be avoided during heat fixing
  • Over decolourization of the smear should be avoided
  • Smear should be thin and uniform.


  • “Gram stain demonstration slide, 1,000x 2” by Marc Perkins – OCC Biology Department is licensed with CC BY-NC 2.0. To view a copy of this license, visit

Read  – DNA Estimation By DPA method

See – Gram Staining Practical pdf

Leave a Comment

Your email address will not be published. Required fields are marked *