Gram Staining.pptx

Gram staining process

1. Study of bacteria by using gram staining technique

2. The Gram staining method is named after the Danish bacteriologist Hans Christian Joachim Gram who used it to discriminate between Pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.

3. Bacterial species with walls containing small amounts of peptidoglycan and characteristically, lipopolysaccharide , are Gram-negative whereas bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive. Peptidoglycans are mainly a polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic acid. The Gram-negative bacteria also have an additional outer membrane which contains lipids, which is separated from the cell wall by means of a periplasmic space

4. Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram -negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by the Safranin . Gram staining is thus based on the ability of bacterial cell wall to retain the crystal violet dye during solvent treatment by peptidoglycan in the cell wall.

5. Gram staining consists of four components   Primary stain (Crystal violet, methyl violet or Gentian violet) Mordant (Gram's Iodine ) Decolorizer (acetone/alcohol) Counterstain ( safranin )

6. Material Required Curd solution for bacteria (for gram positive bacteria, Lactobacillus ), Half cooked rice solution (rich in gram negative bacteria, Escherichia coli ), distilled water (preferably in a wash bottle), Crystal Violet (primary stain: 1g crystal violet in 100 ml of distilled water), Gram's Iodine (mordant: 1g iodine and 2g Potassium iodide (KI) in 300 ml of distilled water), Decolorizer (e.g. acetone or 95% ethanol), Safranin (secondary stain: 1g safranin in 100 ml of distilled water), Glass slides, Collecting Loop, Matchbox, Bunsen Burner, Slides, Diamond marker, Coplin jars, Slide holder, blotting paper, microscope.

7. Procedure :   Mark the clean slides for different bacterial samples (curd and half cooked rice solution ). Put a small drop of the bacterial sample on one edge of the slide and make a smear with another slide at 45o angle to the drop . For fixing the bacteria, heat the slide by passing the slide with smear briefly through the spirit lamp without exposing the dried film directly to the flame . Add crystal violet solution on the slide so as to flood it for 60 sec . Remove the excess stain by washing the slide with distilled water .  

8. Add Gram's Iodine solution to cover the smear on the slide for 30 sec  Wash the smear with distilled water and drain. Again wash the smear with acetone for decolorization very briefly (2-3 sec or one dip). Wash the smear immediately with distilled water to stop the decolorization .  Counter stain the smear with safranin for 60 sec. Wash the smear on slide again with distilled water and drain. Allow the smear to air dry and observe under a microscope (40X)

9. Applications of Gram staining :   Differentiation of bacteria into Gram positive and Gram negative is the first step towards classification of bacteria . It can be used as identification of bacteria in cultures . Observation of bacteria in clinical specimens provides a vital clue in the diagnosis of infectious diseases . Useful in estimation of total count of bacteria . Empirical choice of antibiotics can be made on the basis of Gram stain’s report . Choice of culture media for inoculation can be made empirically based on Gram’s stain report.

10. Precautions : Smear should be even and thin . The entire slide should not be covered with the sample. This will make handling difficult and areas may be missed during decolorization . Overheating should be avoided as it may alter cell morphology or cause organisms to decolorize more quickly . 95% ethanol will decolorize slower than acetone/alcohol, than does acetone. Therefore, timing for decolorization should be set accordingly and taken care of .   The decolorization step must be performed carefully, otherwise over- decolorization may occur. This step is critical and must be timed correctly otherwise the crystal violet stain will be removed from the Gram-positive cells also.