Simple Staining

 

Simple Staining of a Given Bacterial Sample

Objectives:

• To perform simple staining on a bacterial smear.
• To observe bacterial morphology (shape and arrangement) under a microscope.
• To differentiate bacteria from the background using a single stain.

Theory:

A stain is a chemical substance used to color cells or cell components to make them visible under a microscope. A stain consists of three main components:

• Benzene (solvent or base structure)
• Chromophore (color-producing part)
• Auxochrome (ionizing group that binds the stain to the cell)

The chromophore gives the color, while the auxochrome helps the stain attach to the bacterial cell by forming ionic or electrostatic bonds.

Types of Stains (based on the nature of the chromogen charge):

1. Basic stains – Have positively charged chromogen (e.g., methylene blue, crystal violet, safranin). These bind to the negatively charged bacterial cell wall, making the cells appear colored. Basic stains are most commonly used in microbiology.
2. Acidic stains – Have negatively charged chromogen (e.g., eosin, picric acid). These repel the bacterial cell and stain the background, not the cell (used in negative staining).
3. Neutral stains – Contain both positive and negative chromogens, and are used mainly in staining blood cells (e.g., Leishman stain, Wright’s stain).

Simple staining uses a single basic dye (such as methylene blue, crystal violet, or safranin) to stain bacterial cells. The basic dye has a positively charged chromogen that binds to the negatively charged bacterial cell surface, causing the cells to appear colored under a microscope. Since only one stain is used, all cells take up the same color, and this method is primarily used to study:

• Bacterial size
• Shape (coccus, bacillus, spirillum)
• Arrangement (single, pair, chain, cluster)

Requirements:

S.N.	Materials/Equipment
1	Clean glass slide
2	Inoculating loop
3	Bunsen burner
4	Methylene blue (or any basic stain)
5	Bacterial culture (sample)
6	Distilled water
7	Blotting paper
8	Microscope

 

Procedure:

o   Take a clean glass slide.

o   Add a drop of sterile distilled water if using solid culture.

o   Using a sterilized inoculating loop, transfer a small amount of bacterial culture onto the slide and mix it with water to form a thin smear.

o   Air dry the smear.

o   Pass the dried slide (smear side up) through a flame 2–3 times to fix the bacteria.

o   Flood the smear with methylene blue and allow it to stand for 1 minute.

o   Gently rinse the slide with distilled water to remove excess stain.

o   Blot the slide gently with blotting paper.

o   Observe under low power (10X) and then under high power (100X oil immersion) for better visualization.

Observation

S.N	Stain Used	Sample	Shape Observed	Arrangement
1	Methylene Blue	B	Bacillus (Rod)	Single/Chains
2	Methylene Blue	S	Coccus (Spherical)	Clusters/Pairs

Figure: Cocci in a cluster observed

Result & Discussion:

The bacterial cells appeared blue due to methylene blue staining. Bacterial cells have a net negative charge on their surface, primarily due to the presence of teichoic acids in Gram-positive bacteria and lipopolysaccharides in Gram-negative bacteria. This negative charge allows for effective interaction with basic dyes, which have positively charged chromophores.

In this experiment, methylene blue, a basic dye, was used. The positively charged auxochrome of the dye binds to the negatively charged bacterial surface through electrostatic attraction. As a result, the bacterial cells absorb the dye and appear blue under the microscope, while the background remains unstained. This method helps visualize the morphology (shape and size) and arrangement of bacteria.

Conclusion:

Simple staining was successfully performed using methylene blue.

Precautions:

• Use a clean slide to prepare good smear.
• Do not overheat during heat fixing as it may distort the cells.
• Avoid overstaining or overwashing to preserve clarity.
• Always sterilize the loop before and after use.
• Handle stains carefully; some are toxic or may stain skin.

References:

1. Cappuccino, J.G., & Welsh, C. (2017). Microbiology: A Laboratory Manual. 11th Ed. Pearson.
2. Pelczar, M.J., Chan, E.C.S., & Krieg, N.R. (2007). Microbiology: Concepts and Applications. McGraw-Hill.