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Simple Staining Procedure in Microbiology

In this simple staining procedure in microbiology post we have briefly explained about simple staining of bacteria principle, objectives, requirements, procedure, uses and limitations.

Simple Staining of Bacteria

The basic dyes, which are alcoholic or aqueous solutions, are referred to as simple stains (diluted up to 1-2 percent).

The simple staining of bacteria are positively charged because they can quickly release OH and receive H+ ions. Simple stains are known as positive or cationic dyes because they are positively charged.

Most microorganisms are usually coloured with simple staining of bacteria. Because the simple stain has a positive charge, it sticks to a negative bacterial cell and colours it while leaving the background colourless. Safranin, methylene blue, crystal violet, and other basic stains are examples.

Principle

Its principle is based on producing a marked contrast between the organism and its surroundings by using basic stain.

A basic dye consists of a positive chromophore, which strongly attracts the negative cell components and charged molecules like nucleic acids and proteins.

Thus, a simple staining of bacteria technique results in a coloured bacterial cell against a colourless background.

Requirements

Materials

1. Bunsen burner

2. Inoculating loop

3. Microscope

4. Distilled water

5. Tissue paper

6. Toothpicks

7. Microscopic Slides

Chemicals

1. Methylene blue Stain

2. Crystal violet Stain

3. Carbol fuchsin Stain

Simple Staining of Bacteria Procedure

1. Take 1–2 drops of sterilized water on clean glass slide. Pick culture of bacteria with the help of inoculation loop under aseptic conditions, and make suspension with water.

2. Prepare smear of bacteria by spreading it over slide. Air-dry the smear and heat fix. Spread 2–4 drops of staining reagent carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue, 1 to 2 minutes. Pour off the extra stain and was slide in running water.

3. Keep the slide in inclined position, and remove water droplets carefully using blotting paper.

4. View the slide under low (100X), high (400X), and oil immersion (1000) magnification of compound microscope.

5. Observation: Blue-colored cocci cell arranged in the form of chain and blue-colored rod-shape cells are visible under oil immersion microscope. Draw the diagram of it, and note down the observations.

Results

Simple stain

Simple stain of Bacillus cereus, a bacterial rod

Bacilli and diplobacilli: Rod-shaped bacteria, purple, Spirilla: spiral-shaped bacteria, purple, Cocci: spherical-shaped bacteria, purple.

Advantages

1.Simple staining of bacteria is a very simple method to perform, which stains the organism by using a single reagent.

2. It is a rapid method that reduces the performance time by taking only 3-5 minutes. Simple staining of bacteria helps to examine or elucidate the bacterial shape, size and arrangement.

3. It also helps us to differentiate the bacterial cells from the non-living structures. Simple staining of bacteria can be useful in the preliminary study of the bacteria’s morphological characteristics.

Limitations

1. It does not give much information about the cell apart from the bacteria’s morphological characteristics. Through simple staining of bacteria, we cannot classify a particular type of organism.

Further Readings

Reference