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Gram Staining

Aim: Identification of given microorganisms sample, either Gram positive or Gram negative based on Gram staining. Introduction: The Gram stain devised by Christian Gram in 1882 has become one of the most important diagnostic procedures in microbiology. This stain differentiates two main categories of bacteria according to the structure of the cell wall. Bacteria with a predominantly peptidoglycan cell wall stain blue/purple with this technique and since they retain the primary stain are called Gram positive (Gram +). Cells with only a minor peptidoglycan component and possessing a lipopolysaccharide cell wall give up the primary stain during destaining and therefore exhibit the red counter stain. These cells are called Gram negative (Gram-). A Gram stain is usually performed on a smear preparation that has been heat fixed. One function of fixation is to secure (fix) the cells to the slide. Examples of Gram positive Bacteria Corynebacterium Lactobacillus Bacillus Streptococcus Staphylococcus Clostridium Examples of Gram negative Bacteria Escherichia coli Klebsiella Salmonella Shigella Pseudomonas Campylobacter Vibrio cholerae Yersinia Principle Both Gram-positive (Gm+) and Gram-negative (Gm) organisms form a complex of crystal violet and iodine within the bacterial cell during the Gram-

staining procedure. Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain. In contrast, cell wall permeability of Gm- organisms is increased by ethyl alcohol washing because it removes the outer membrane from the Gramnegative cell wall. This allows the removal of the crystal violet-iodine complex from within the cell. The decolorized Gm- cell can then be rendered visible with a suitable counterstain, in this case Safranin, which stains them pink. Pink which adheres to the Gm+ bacteria is masked by the purple of the crystal violet. Requirements 1. Overnight culture of the bacterial suspension 2. Glass slide and coverslips 3. Bunsen burner 4. Inoculation loop 5. Tissue paper 6. Distilled water
7. Gram staining kit: Grams reagent (crystal violet), Mordant (Iodine),

Decolorizer (95% Ethanol or Acetone), Counterstain (Safranin) 8. Microscope Procedure Prepare a light suspension of cells from very young cultures grown on appropriate agar medium. If the suspension prepared is too turbid, dilute with distilled water. 1. 2.

Add one drop to a clean glass slide and spread the drop with a loop over the surface of the slide. Allow to air-dry. Carefully fixate the cells by moving the slide into a flame (bacteria upwards). Flood the slide for 30sec -1minute with Gram's reagent (crystal violet).


Wash the slide under slow running tap water until crystal violet diappears. Flood the slide with Mordant (Iodine) solution for 1-1.5minute. Place slide diagonally in glass box and rinse of Iodine solution with water. Add excess amount of fresh safranin and wait for 35 seconds. Rinse slide with water. Allow the slide to air-dry.

5. 6.



10. Examine the preparations under the bright field microscope. 11. Gram-positive cells appear purple and Gram-negative cells pink.

(The Iris of the microscope condenser should be opened as wide as possible. With a closed condenser colours can hardly be discriminated.). Observation: ---Results: ------

Flow chart Bacterial smear preparation

Heat fixation

Stain with cryastal violet 1minute

Wash with water until crystal violet disappears

Flood with Iodine 1.5minute

Wash with water

Add safranin 35sec

Wash with water

Observe under Microscope

BY Aravind Setti Dept of Genetics Osmania University