INTRODUCTION:
Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Gram staining method was named after the Danish bacteriologist Hans Christain Grams (1853-1938) who originally devised it in 1882 to discriminate between pneumococci and klebsiella pneumoniae bacteria in the lungs tissue.it is a differential media which allows most bacteria to be divided into two groups, Gram-positive bacteria and Gram-negative bacteria. The technique is based on the fact that the Gram positive cell wall has a stronger attraction for crystal violet when Gram's iodine is applied than does the Gram negative cell wall. Gram's iodine is known as a mordant. It is able to form a complex with the crystal violet that is attached more tightly to the Gram-positive cell wall than to the Gram-negative cell wall. This complex can easily be washed away from the Gram-negative cell wall with ethyl alcohol. Gram-positive bacteria, however, are able to retain the crystal violet and therefore will remain purple after DECOLORIZING with alcohol. Since Gram-negative bacteria will be colorless after decolorizing with alcohol, COUNTERSTAINING with safranin will make them appear pink. The procedure was discovered by Christian Gram in 1884. The chemical basis was not understood by Gram and is still not fully understood today. It is known, however, that the two groups of bacteria have very different cell walls and that the type of cell wall dictates the way a bacterium responds to the Gram stain.The Gram stain is probably the most commonly used staining procedure in microbiology. It is extremely useful in identifying bacteria. It is important that you understand the color changes that occur at each step in the Gram staining.
REASONS OR IMPORTANCES OF GRAM STAINING:
- Differentiation of bacteria into gram-positive and gram-negative is the first step towards classification of bacteria.
- it is also the first step towards identification of bacteria in cultures.
- Choice of culture media for innoculation can be made empirically based on gram stain report
- Useful in estimation of total count of bacteria.
MATERIALS:
- Crystal violet (primary stain)
- Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall)
- Decolorizer (e.g. ethanol)
- Safranin (secondary stain)
- Water (preferably in a squirt bottle).
- Microscopic slides .
PROCEDURE:
- Place a loopful of sterile distilled water onto a microscope slide.
- Touch an isolated colony with inoculating loop and swirl it in the drop of water on the slide.
- Let the smear air dry at room temperature
- Heat fix the smear by waving the slide over a flame, being careful not to over heat
- Flood the slide with crystal violet, and let stand for one minute.
- Wash the slide briefly with cold water.
- Flood the slide with gram's iodine; let stand for one minute; wash off with water.
- Decolorize until the solvent flows colorlessly from the slide.
- Flood the slide with safranine; let stand for 30 seconds; wash off with water.
- Blot the slide dry with bilbous paper.
- Examine the slide under the microscope for gram reaction (100x oil-immersion objective) . NOTE: Only perform gram stains on fresh cultures (24hr incubation). Using older
cultures often yields unexpected results. Be sure to stop the application of
decolorizer immediately after it runs clear to avoid over-decolorizing. Always
run positive and negative controls from known cultures.
