Cell Staining: The Simple Stain

Cell Staining
Cell staining is a technique that can be used to better visualize cells and cell components under a
microscope. By using different stains, one can preferentially stain certain cell components, such as a
nucleus or a cell wall, or the entire cell.
Why do we have to stain bacteria?
It is very difficult to observe microorganisms by our naked eyes because they are very minute and
transparent as well as colorless when they are suspended in aqueous medium. The refractive index of
microorganism is not very different from the medium in which they grow, due to which they cannot be
observed in unstained preparation. Staining helps to observe the organism clearly.
The Simple Stain
The use of a single stain to colour a bacterial organism is commonly referred as simple staining. All types
of bacteria appear as the color of that stain when viewed under the microscope. E.g crystal violet,
safranin, and methylene blue.
Principle:
In order to observe most bacterial cells using bright field microscopy the cells must be dark enough to
see, that is they must have contrast to the light. To create contrast a simple stain can be used. Simple
stains use basic dyes which are positively charged. These positive dyes interact with the slightly
negatively charged bacterial cell wall thus lending the color of the dye to the cell wall.
Requirements:
1. Fresh culture sample 24-hour agar culture of Staphylococcus aureus/24- hour agar culture of
Escherichia coli
2. Stains (Methylene blue or Safranin or crystal violet)
3. Bunsn burner
4. Inoculating loop
5. Microscope
6. Distilled water
7. Tissue paper
8. Toothpicks
9. Microscopic slides
10. Permanent marking pen
Procedure
1. Take a clean, grease free glass slide. In order to make the slide grease free, wash it, rinse with
alcohol and then clean with filter paper to make it dry.
2. Prepare separate bacterial smears of the given sample organism.
3. Smears must be heat fixed prior to staining.
4. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the
appropriate exposure time for each: carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60
seconds; methylene blue 1 to 2 minutes.
5. Gently wash the smear with distilled water to remove excess stain. During this step, hold the slide
parallel to the stream of water; in this way you can reduce the loss of organisms from the
preparation.
6. Using bibulous paper, blot dry, but do not wipe the slide. Or let it for air dry sometime
7. Examine the stained slides under 10X, 40X and then under oil immersion respectively.
8. Observe the morphology of the organisms with reference to their shapes (bacilli, cocci, spirilla) and
arrangements (chains, clusters, pairs).
9. Record your observation
Observation:
A) Coccus B) Bacillus
Result Interpretation of Simple Staining

Bacilli and diplobacilli: Rod-shaped bacteria, purple
Spirilla: spiral-shaped bacteria, purple
Cocci: spherical-shaped, bacteria, purple
The Gram Stain

Gram staining method, the most important procedure in Microbiology, was developed by Danish
physician Hans Christian Gram in 1884. This differential staining procedure separates most bacteria into
two groups on the basis of cell wall composition;
1. Gram-positive bacteria (thick layer of peptidoglycan)- stains purple
2. Gram-negative bacteria (thin layer of peptidoglycan and high lipid content) –stains red/pink
Principle of Gram Staining
When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the
bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram
positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is
low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in
the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-
Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears
blue or purple in colour.
In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer
of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off.
When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the
crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take
the stain and appears red in color.
Reagents Used in Gram Staining
 Crystal Violet, the primary stain

 Iodine, the mordant
 A decolorizer made of acetone and alcohol (95%)
 Safranin, the counterstain
Procedure/Protool of Gram Staining
Gram staining techniques involve the following steps:
1. Fixation/Smear Preparation
Fix material on a slide with methanol or heat. If the slide is heat fixed, allow it to cool to the touch before
applying the stain.
2. Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple
3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine

(CV-I) complex; all cells continue to appear blue.
4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative

cells.The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-
rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-
walled, gram-positive bacteria. The gram-negative bacteria appear colorless and gram-positive
bacteria remain blue.
5. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-
negative cells red/pink; the gram-positive bacteria remain blue.
6. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and
then blot dry with absorbent paper.
Result Interpretation
Gram Positive: Blue/Purple Color
Gram Negative: Red Color
Some Important Questions
Why do we have to stain bacteria?
It is very difficult to observe microorganisms by our naked eyes because they are very minute and
transparent as well as colorless when they are suspended in aqueous medium. The refractive index of
microorganism is not very different from the medium in which they grow, due to which they cannot be
observed in unstained preparation. Staining helps to observe the organism clearly.
Why do fixation?
The main purpose of this step is to adhere the bacterial cells to the microscope slide (it also denatures the
proteins and kills them too). If you forget to do this step, then the cells will be 'washed' off in all the
subsequent steps of your staining process. You will literally have no cells on your slide to stain!
What is an example of mordant?

Iodine solution
What is the purpose of adding decolorizing agent?
A decolorising agent removes the dye from a stained cell, e.g., alcohol, acetone or ether.
What is the purpose of adding mordant?
A Mordant increases the affinity or attraction between the cell and the dye, e.g. Iodine

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Cell Staining: The Simple Stain

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Cell Staining

Why do we have to stain bacteria?

The Simple Stain

Result Interpretation of Simple Staining

The Gram Stain

Principle of Gram Staining

Reagents Used in Gram Staining

 Crystal Violet, the primary stain

3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine

4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative

What is an example of mordant?

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