Gram Staining: Principle, Procedure, Interpretation

 Gram Staining is the common, important, and most used differential staining techniques in microbiology,
which was introduced by Danish Bacteriologist Hans Christian Gram in 1884.

 This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the
classification and differentiations of microorganisms.

 The difference in cell wall composition makes the difference.

 Bacteria are stained with gentian violet and then treated with Gram's solution; after being decolorized with
alcohol and treated with safranine and washed in water, those that retain the gentian violet are Gram-
positive and those that do not retain it are Gram-negative.
Gram positive Bacteria
• Gram positive bacteria have a thick cell wall of peptidoglycan and other polymers.
• Peptidoglycan consists of interleaving filaments made up of alternating acetylmuramic acid and
acetylglucosamine monomers.
• There is a “membrane teichoic acid” between the cell wall and cell membrane.
Gram Negative Bacteria
• Gram negative bacteria have an outer membrane of phospholipids and bacterial Lipopolysaccharides
outside of their thin peptidoglycan layer.
• The space between the outer membrane and the peptidoglycan layer is called the periplasmic space.
• The outer membrane protects Gram negative bacteria against penicillin and lysozymes.
Parameter Gram-positive bacteria Gram-negative bacteria
Cell Wall A single-layered, smooth cell wall A double-layered, wavy cell-wall
The thickness of the cell wall is 20 to 80 The thickness of the cell wall is 8 to 10
Cell Wall thickness
nanometres nanometres
Peptidoglycan Layer It is a thick layer/ also can be multi-layered. It is a thin layer/ often single-layered.
Teichoic acids Teichoic acids are present. Teichoic acids are not present.
Lipopolysaccharide Lipopolysaccharide is not present. Lipopolysaccharide is present.
Outer membrane The outer membrane is not present. The outer membrane is mostly present.
Lipid content The Lipid content is very low. The Lipid content is 20% to 30%.
Resistance to Antibiotic These are very susceptible to antibiotics. These are very resistant to antibiotics.

•The cell wall of gram-positive

 The Gram Reaction is dependent on permeability of the bacterial cell wall and cytoplasmic membrane, to
the dye-iodine complex.
 In Gram positive bacteria, the crystal violet dye –iodine complex combines to form a larger molecule
which precipitates within the cell.
 Also the alcohol/acetone mixture which act as decolorizing agent, cause dehydration of the multi-layared
peptidoglycan of the cell wall.
 This causes decreasing of the space between the molecules causing the cell wall to trap the crystal violet
iodine complex within the cell.
 Hence the Gram positive bacteria do not get decolorized and retain primary dye appearing violet.
 Also, Gram positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly.
 In the case of Gram negative bacteria, the alcohol, being a lipid solvent, dissolves the outer
lipopolysaccharide membrane of the cell wall and also damage the cytoplasmic membrane to which the
peptidoglycan is attached.
 As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the
process of decolourisation.
 Hence when a counter stain is added, they take up the colour of the stain and appear pink.
 Basic requirements for staining
•Clean grease-free slide.
•f

• Bacteria to be stained.
•Inoculating loops- to transfer bacterial suspension to slide.
•Bunsen burner – to sterilise inoculating loops before and after smear preparation.
•Pencil marker – to mark (particularly central portion of slide) where bacterial smear is applied.

•STAINING REAGENTS:
•1. Crystal violet – Primary stain
• 2. Gram’s iodine- mordant/fixative
•3. Acetone (95%)- decoloriser
•4. Safranine/dilute carbol fuchsin – counterstain
 Procedure of Gram Staining

1. Take a clean, grease free slide.

2. Prepare the smear of suspension on the clean slide with a loopful of sample.

3. Air dry and heat fix

4. Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.

5. Flood the gram’s iodine for 1 minute and wash with water.

6. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.

7. Add safranin for about 1 minute and wash with water.

8. Air dry, Blot dry and Observe under Microscope.

9. Interpretation
 Smear preparation: Basic initial steps before staining
•Putting of bacterial suspension (bacteria in liquid) to be stained on the central portion of slide in a circular
fashion,
• air-dried
•heat-fixed
•the resultant preparation called bacterial smear- appears dull white.
 REQUIREMENTS – STAINING REAGENTS

Crystal violet - all bacteria take crystal violet- so all appears violet. 2. Iodine – Crystal Violet-
iodine(CV-I) complex is formed. 3. Acetone- bacteria with high lipid content loose CV-I
complex(appear colouless) but bacteria with less lipid content retains CV-I complex ( appear
violet). 4. Safranine/ dilute carbol fuchsin – only colouless bacteria takes – appear pink.

Crystal violet – 1 min - wash. • Iodine – 1 min – wash. • Acetone add drop by drop and watch
out colour comes out – wash immediately. • Safarnine/dilute carbol fuchsin – 1 min- wash. •
Allow to dry – examine under microscope. Note: Results should be confirmed only with 100x.
SMEAR FIXATION: • 1) Heat fixation • a) Pass air-dried smears
through a flame two or three times. Do not overheat. • b)
Allow slide to cool before staining. • 2) Methanol fixation • a)
Place air-dried smears in a coplin jar with methanol for one
minute. Alternatively, flood smear with methanol for 1 minute.
• b) Drain slides and allow to dry before staining.
16. Method of Picking material from Agar plates Wrong Right
17. Prefer to pick up colonies with loop and smear on Clean
glass slide
•Observe the Following Under Oil Immersion lens
•19. • Bacteria that manage to keep the original
purple dye have only got a cell wall - they are
called Gram positive. • Bacteria that lose the
original purple dye and can therefore take up the
second red dye have got both a cell wall and a cell
membrane - they are called Gram negative. Colors
makes the Difference in Gram staining
•20. Colour: Purple colored bacteria – Gram
positive Pink colored bacteria – Gram negative
Shape: Spherical – cocci Rod – bacilli Arrangement
Cocci in clusters – staphylococci Cocci in chains -
streptococci