SIMPLE STAINING

Definition of Simple Staining

Simple staining can define as one of the ordinaries yet popular method which is used to elucidate
the bacterial size, shape and arrangement to differentiate the group of bacteria. It stains the
bacterial cell uniformly and thus increases the visibility of an organism.
Simple staining sometimes interchangeable with the names like direct, positive or monochrome
staining. Now let us understand why simple staining is called by such alternative names.

Refers as Direct staining: Because it is a direct method that directly stains the bacterial cell with
a colourless background.
Refers as Positive staining: Because it makes the use of basic dyes which are positively charged
and binds with the negatively charged bacterial cell.
Also Refers as Monochrome staining: Because it adds contrast to the specimen by the use of a single
stain only.
Simple staining is a method of staining in which bacteria are stained by using a single
stain.
Simple staining is also called as monochrome staining or positive staining.
Examples of simple stain are Methylene blue, Safranin, Malachite green, Basic fuchsin
and crystal violet etc.
In simple staining procedure cell are uniformly stained.

Simple Stains

Simple stains can define as the basic dyes, which are the alcoholic or aqueous solution, diluted
up to 1-2%. These can easily release OH– and accepts H+ ion, and hence the simple stains are
positively charged. As the simple stains are positively charged, they usually refer to as “Positive
or Cationic dyes”.
It is commonly used to colour most of the bacteria. As the simple stain carry a positive charge,
that’s why they firmly adhere to a negative bacterial cell by which organism appears coloured
with a colourless background.

Examples of simple stain include safranin, methylene blue, crystal violet etc.
The basic stains have different exposure time 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

Principle

Its principle is based on the principle of producing a marked contrast between

the organism and around its surrounding, by the use of basic stain.
A basic dye consists of positive chromophore which strongly attracts to the negative cell
components and charged molecules like nucleic acids and proteins.

Procedure of Simple Staining

The method of simple staining involves three steps like:

1. Smear preparation
2. Heat fixing
3. Staining

Smear Preparation

Bacterial smear consists of a thin film of bacterial culture or inoculum. For the preparation of
smear, we need to perform the following steps like:

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

2. Add a drop of distilled water at the centre of the glass slide.
3. Then add inoculum from the bacterial culture with the help of 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.
Heat Fixing

There are many reasons to perform heat fixing, and it can not be skipped because:

 Heat fixing helps in the fixation of a specimen to the glass slide.

 Heat fixing helps the stain to penetrate into the smear.
After smear preparation, heat fixes the smear by passing the slides through the flame of Bunsen
burner for at least three times. Then, allow the slide to air dry.

Simple Staining of Bacteria

It is the last and the most crucial step which colours the bacterial cells and makes it visible,
through which one can identify the morphological characteristics of the bacteria. This stage
involves the following steps as follows:

1. Add stain to the heat fixed smear.

2. Allow the stain to stand for at least 1minute so that it can penetrate between the cells.
3. Wash off the glass slide carefully.
4. Blot dry the slide with absorbent paper but do not wipe the slide.
5. Examine the glass slide under the microscope from low to high power objective to get a
magnified view of the specimen. One can also add a drop of oil immersion over the stained area of
the glass slide and observe it under 100X objective.

Advantages

 Simple staining is a very simple method to perform which stains the organism by a single

reagent.
 It is a rapid method which reduces the performance time by taking only 3-5 minutes.
 Simple staining helps to examine or elucidate the bacterial shape, size and arrangement.
 It also helps us to differentiate the bacterial cells from the non-living structures.
 Simple staining can be useful in the preliminary study of the morphological characters of the
bacteria.
Disadvantages

 It does not give much information rather than the morphological characteristics of bacteria.
 Through simple staining, we cannot classify a particular type of organism.

Results

The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye used. If
methyene blue is used, some granules in the interior of the cells of some bacteria may appear more deeply
stained than the rest of the cell, which is due to presence of different chemical substances.

Left: Cocci in Cluster; Right: Bacilli (Image source: microrao.com)

Conclusion

Therefore, we can conclude that a simple staining method is the easiest way to colour the
microscopic object as it uses a single basic stain. The results of simple staining are based on the
type of basic stain that has been used.

The colour of a stain will decide the colour of a specimen that has to be identified. For example,
when the bacteria retain the colour of safranin, they appear pink-red, and same goes with the
other stains.

In simple staining, there is an attraction between the positive stain to the negative bacterial cell,
which results in the observation of coloured bacteria with a bright background.