MEVL13 – Environmental Microbiology Lab
Experiment No. 2
Aim: To perform Gram staining of bacteria.
Date: _______________
Introduction
• Gram staining is the most common, important, and widely used differential staining technique in microbiology,
introduced by Danish bacteriologist Hans Christian Gram in 1884.
• It differentiates bacteria into Gram-positive and Gram-negative groups, which helps in the classification and
identification of microorganisms.
Principle
• When bacteria are stained with the primary stain Crystal Violet and fixed with the mordant (iodine), some bacteria
retain the primary stain while others are decolorized by alcohol.
• Gram-positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and low lipid content in
their cell wall.
• On decolorization, this thick cell wall dehydrates and shrinks, closing the pores in the wall and preventing the stain
from exiting the cell.
• Hence the decolorizer (ethanol) cannot remove the Crystal Violet–Iodine complex bound to the thick
peptidoglycan layer of Gram-positive bacteria, which appears BLUE or PURPLE in colour.
• In Gram-negative bacteria, the cell wall also takes up the Crystal Violet–Iodine complex, but due to the thin
peptidoglycan layer and thick outer lipid layer, the CV–Iodine complex gets washed off.
• When exposed to alcohol, the decolorizer dissolves the lipids in the outer membrane, allowing the crystal violet–
iodine complex to leach out of the cells.
• On staining again with safranin (counterstain), Gram-negative bacteria take up this stain and appear RED in colour.
Materials Required
1. Crystal Violet — the primary stain
2. Iodine — the mordant that holds the stain onto the microorganism
3. A decolorizer made of alcohol (95%)
4. Safranin — the counterstain
Procedure
1. Take a clean, grease-free slide.
2. Prepare a smear of the bacterial suspension on the slide with a loopful of sample.
3. Air dry and heat-fix the smear.
4. Pour Crystal Violet and keep for about 30 seconds to 1 minute; rinse with water.
5. Flood the slide with Gram's Iodine for 1 minute and wash with water.
6. Wash with 95% alcohol or acetone for about 10–20 seconds and rinse with water (decolorization step).
7. Add Safranin and keep for about 1 minute; wash with water.
8. Air dry, blot dry, and observe under the microscope.
Observations
Examine the stained smear under the microscope (oil immersion, 1000x) and record the Gram reaction, colour, and cell
morphology for each organism.
(Typical/expected reading shown below — replace the test sample row with your actual observation.)
Organism Gram reaction Colour observed Cell shape/arrangement
Staphylococcus aureus Cocci in clusters (grape-
Positive Purple / Blue
(known Gram +) like)
Rods, single or short
E. coli (known Gram −) Negative Red / Pink
chains
Test sample (unknown) __________ __________ __________
Result
On Gram staining and observation under the microscope, Staphylococcus aureus retained the Crystal Violet–Iodine
complex and appeared purple/blue in colour, occurring as cocci in grape-like clusters — confirming it as a Gram-
positive organism. E. coli lost the Crystal Violet–Iodine complex on decolorization and took up the counterstain
Safranin, appearing red/pink in colour as rod-shaped cells — confirming it as a Gram-negative organism.
Conclusion: The Gram staining technique successfully differentiated the test organisms based on the composition and
thickness of their cell walls. Gram-positive bacteria (thick peptidoglycan) retained the primary stain and appeared
purple/blue, while Gram-negative bacteria (thin peptidoglycan with an outer lipid membrane) were decolorized and
appeared red on counterstaining. This confirms the aim of the experiment was achieved.
Precautions
1. Fix the slide properly before staining.
2. Under-staining and over-staining should both be avoided.
3. Remove excess stain after each step.
4. Ensure proper washing between each staining step.
5. Do not over-decolorize, as this may cause Gram-positive organisms to appear falsely Gram-negative.
6. Use young (18–24 hr) cultures, since older cultures may give inconsistent Gram reactions.