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Microbiology Laboratory
Lab 6: Simple Staining- Principle, Procedure and Result Interpretation
Simple staining is one of the conventional staining techniques. From the name, it is quite clear that
it is a very simple or direct staining method that uses a single stain only. The microorganisms are
invisible to the naked eye, and to make them visible, staining is performed that gives divergence to
a microscopic image. Direct staining makes the use of basic dyes like methylene blue, safranin,

crystal violet, malachite green etc. called "simple or direct stains".

The simple stain can be used as a quick and easy way to determine the cell shape, size, and
arrangement of bacteria. True to its name, the simple stain is a very simple staining procedure
involving a single stain solution. Any basic dye, such as methylene blue, safranin, or crystal violet,
can be used to color the bacterial cells. These stains will readily give up a hydroxide ion or accept a
hydrogen ion, which leaves the stain positively charged. Since most bacterial cells and cytoplasm
surface is negatively charged, these positively charged stains adhere readily to the cell surface. After
staining, bacterial cell morphology (shape and arrangement) can be appreciated.

Simple staining is defined as one of the ordinaries yet the popular method used to elucidate the
bacterial size, shape and arrangement to differentiate the various bacteria groups. It stains the
bacterial cell uniformly and thus increases the visibility of an organism. The term simple
staining sometimes interchangeable with the terms like direct, positive or monochrome
staining. Also, Simple stains can be defined as the basic dyes, which are the alcoholic or aqueous
solutions (diluted up to 1-2%). These can easily release OH and accepts H ion, due to which the
simple stains are positively charged. As the simple stains are positively charged, they usually termed
as positive or cationic dyes. It is commonly used to color most bacteria. As the simple stain carry a

positive charge, it firmly adheres to a negative bacterial cell and makes the organism colored by
leaving a background colorless. Examples of simple stains include safranin, methylene blue, crystal
violet etc.

The basic stains have different exposure times to penetrate and stain the bacterial cell.

Basic stains Exposure time to stain the bacteria

Methylene blue 1-2 minutes

Crystal violet 20-60 seconds

Carbol fuschin 15-30 seconds
Safranin 30-60 seconds
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Principle of Simple staining

In simple staining, the bacterial smear is stained with a single reagent, which produces a distinctive
contrast between the organism and its background. 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, Basic stains with a positively charged chromogen are
preferred because bacterial nucleic acids and certain cell wall components carry a negative charge
that strongly attracts and binds to the cationic chromogen Thus, a simple staining technique result in
a colored bacterial cell against a colorless background. The purpose of simple staining is to elucidate

the morphology and arrangement of bacterial cells. The most commonly used basic stains are
methylene blue, crystal violet, and carbol fuchsin

BASIC STAIN NEGATIVELY

WITH POSITIVE CHARGED
CHROMOPHORE BACTERIAL CELL

BINDNG

BACTERIAL CELL

BACKGROUND

ATTRACTION BETWEEN
BASIC STAIN AND
NEGATIVELY CHARGED
BACTERIAL CELL

PRINCIPLE OF SIMPLE STAINING

Reagents and Equipment's for Simple Staining

Methylene blue, crystal violet, and carbon fuchsin, Micro incinerator or Bunsen burner, inoculating
loop, staining tray, microscope, lens paper, bibulous (highly absorbent) paper, and glass slides.
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Procedure of Simple Staining

It involves the following three steps:

1.Smear preparation
2. Heat fixing
3. Staining

Smear Preparation

A bacterial smear appears as a thin film of bacterial culture. For the smear preparation, we
need to perform the following steps:

1. Take a clean, grease-free glass slide.

2. Add a drop of distilled water to the center of the glass slide.
3. Then, add inoculum from the bacterial culture with a sterilized inoculating loop on the glass
slide.
4. After that, mix the inoculum with a drop of distilled water to make a thin film by uniformly
rotating the inoculating loop until a thin bacterial film is formed.

SMEAR PREPARATION
1. Take clean glass slide
Distilled
water 2. Add a drop of
distilled water

3. Smear preparation

Heat Fixing

After smear preparation, move the prepared slides over the Bunsen burners flame at least three
times. Then, allow the slide to air dry. There are many reasons to perform heat fixing, and it
cannot be skipped because:
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HEAT FIXIING
Heat fixing helps in the
fixation of a specimen to the
glass slide.
Heat fixing

Heat fixing helps the stain to

penetrate the smear.

Air drying

Staining of Bacteria

It is the last and the most crucial step, in which one can identify the morphological characteristics
of the bacteria through microscopic examination, once the cells get stained. This stage involves
the following steps, which are as follows:

1. Add stain to the heat fixed smear.

2. Allow the stain to stand for at least 1
minute so that it can penetrate between the cells.
3. Wash off the glass slide carefully.
4. Blot dry the slide with absorbent paper (do not wipe the slide).
5. Examine the glass slide under the microscope from low to high power to get a magnified view of
the specimen. One can also add a drop of oil

SIMPLE STAINING
Air dried
slide

Flood with stain

Washing

Air
drying

Observation under
microscope
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6. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.

7. Place the stained smear on the microscope stage smear side up and focus the smear using the 10X

objective

8. Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to

be studied,apply immersion oil directly to the smear, and focus the smear under oil with the 100X

objective.

Liquid AirDry
Step 1.
Media
Heat Fix
Smear OR
ArDr

Solid
Media

Methylene lodine
blue
Step 2. 1min
F1min
Blot dry and view
Stain slide under 100x oil
immersion