भारतीय विज्ञान शिक्षा एवं अनुसंधान संस्थान तिरुपति

INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH TIRUPATI

DEPARTMENT OF BIOLOGY
BIO-112 Basic Biology course

Experiment 5- Gram Staining experiment

Aim: To perform Gram staining experiment using Escherichia coli and Bacillus subtilis cultures.

Materials required: Components of Gram staining kit- Crystal violet, Grams Iodine, Decolorizer, Safranin,
Glass slides, 70 % ethanol, Tissue rolls, inoculating loop, bacterial cultures and spirit lamp.

Principle:

The cell wall is the layer, usually fairly rigid, that lies just outside the plasma membrane. It is one of the most
important procaryotic structures for several reasons: it helps determine the shape of the cell; it helps
protect the cell from osmotic lysis; it can protect the cell from toxic substances; and in pathogens, it can
contribute to pathogenicity. Cell walls are so important that relatively few procaryotes lack them. Those that
do have other features that fulfill cell wall function. The bacterial cell wall also is the site of action of several
antibiotics. Therefore, it is important to understand its structure.
After Christian Gram developed the Gram stain in 1884, it soon became evident that most bacteria could be
divided into two major groups based on their response to the Gram-stain procedure. Gram-positive bacteria
stained purple, whereas gram-negative bacteria were colored pink or red by the technique. The true
structural difference between these two groups did not become clear until the advent of the transmission
electron microscope.

Difference between Gram positive and Gram negative bacteria in terms of

Peptidoglycan layer thickness, periplasmic space, teichoic acid presence or absence, lipopolysaccharide
layer.

Composition of the peptidoglycan layer and cross-linking agent, significance of the unusual D- stereo-isomer
amino acids as part of the peptidoglycan layer, roll of cell wall in osmotic protection.

Mode of action of lysozyme and antibiotics like penicillin act on the cell wall.

Main principle of Gram staining:The difference between gram-positive and gram-negative bacteria is
thought to be due to the physical nature of their cell walls. If the cell wall is removed from gram-positive
bacteria, they stain gram negative. Furthermore, genetically wall-less bacteria such as the mycoplasmas also
stain gram negative. During the procedure, bacteria are first stained with crystal violet and next treated with
iodine to promote dye retention. When bacteria are treated with ethanol in the decolorization step, the
alcohol is thought to shrink the pores of the thick peptidoglycan found in gram- positive bacteria, causing the
peptidoglycan to act as a permeability barrier that prevents loss of crystal violet. Thus the dye-iodine
complex is retained during the decolorization step and the bacteria remain purple. In contrast, gram-
negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol
treatment also may extract enough lipid from the outer membrane to increase the cell wall’s porosity
further. For these reasons, alcohol more readily removes the crystal violet-iodine complex from gram-
negative bacteria. Thus gram-negative bacteria are easily stained red or pink by the counterstain safranin.
 भारतीय विज्ञान शिक्षा एवं अनुसंधान संस्थान तिरुपति
INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH TIRUPATI
DEPARTMENT OF BIOLOGY
BIO-112 Basic Biology course

Procedure:

1. Prepare a thin smear on a clear, dry glass slide.

2. Allow it to air dry and fix it by gentle heat.

3. Flood with Grams Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of
known gram-negative organisms, use less crystal violet).

4. Wash with tap water.

5. Flood the smear with Grams Iodine (S013). Allow it to remain for 1 minute.

6. Decolourize with Gram's Decolourizer (S032) until the blue dye no longer flows from the smear. (Acetone
may be used as a decolourizing agent with caution, since this solvent very rapidly decolourises the smear).

7. Wash with tap water.

8. Counterstain with 0.5% w/v Safranin (S027) for 20 seconds and rinses off with water.

9. Wash with tap water.

10. Wipe the lower surface (opposite surface of the smear prepared portion of the glass slide) of the glass
slide and place it on the stage of the compound microscope and focus the sample.

NOTE: PLEASE ENSURE THE OBJECTIVE LENS DO NOT TOUCH THE SAMPLE PREPARED ON THE GLASS SLIDE

Observation and Inference:

Observation in terms of color of Gram positive and Gram negative stained sample.

Shape of the bacteria- cocci or bacilli form?

Result: Gram staining results for E.coli and B. subtilis samples.

Reference and sources:

1.Prescott Microbiology Details of the aspects mentioned in the principle section of the handout will be
given in detail (Pages 46 to 52 of the book).
भारतीय विज्ञान शिक्षा एवं अनुसंधान संस्थान तिरुपति
INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH TIRUPATI
DEPARTMENT OF BIOLOGY
BIO-112 Basic Biology course

Image courtesy of Prescott’s principle of Microbiology

भारतीय विज्ञान शिक्षा एवं अनुसंधान संस्थान तिरुपति
INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH TIRUPATI
DEPARTMENT OF BIOLOGY
BIO-112 Basic Biology course
भारतीय विज्ञान शिक्षा एवं अनुसंधान संस्थान तिरुपति
INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH TIRUPATI
DEPARTMENT OF BIOLOGY
BIO-112 Basic Biology course