Gram Staining protocol-gram staining principle, procedure,result,precautions
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
- Distilled water drop
- 95% ethyl alcohol
B. Others Materials
- Staining tray
- Inoculation loop
- Glass rods
- Blotting paper