Simple Staining Techniques in Microbiology

In this simple staining techniques in microbiology post we have briefly explained about simple stain, definition, simple staining of bacteria principle, procedure, steps, common terms and some common simple stains.

Simple Staining Techniques in Microbiology

Staining is a microscopy technique used to increase contrast in a microscopic image. Stains and dyes are frequently used in biological tissues for viewing, which is often done with the help of various microscopes. Stains can be used to define and examine bulk tissues (such as muscle fibres or connective tissue), cell populations (such as different blood cells), or organelles within individual cells.

Bacteria have nearly the same refractive index as water, so they are opaque or nearly invisible to the naked eye when observed under a microscope. To make the cells and their internal structures more visible under the light microscope, various staining methods are used. Microscopes are useless unless the specimens for viewing are properly prepared. Microorganisms must be fixed and stained in order to be visible, highlight specific morphological features, and be preserved for future use.

Simple Staining

One of the traditional staining techniques is simple staining of bacteria. It is clear from the name that it is a very simple or direct staining method that uses only one stain. To make the microorganisms visible to the naked eye, staining is used, which causes a divergence in a microscopic image. Direct staining employs basic dyes such as methylene blue, safranin, crystal violet, malachite green, and others, and is referred to as “simple or direct stains.”

The basic stains contain a positive auxochrome, which charges the stain’s chromogen particles and causes them to bind with the specimen. The stain’s chromophore group adds colour to the microscopic image. Because the basic stain has a positive charge, it is also known as a positive or cationic stain. The goal of direct staining is to add contrast to the specimen by staining the bacterial cells directly with a colourless background.

The simple stains make the organism visible and allow us to examine its shape, size, and arrangement, which are required to distinguish a specific group of organisms. In general, the procedure consists of three sequential steps: smear preparation, heat fixing, and bacteria staining.


Simple staining of bacteria is defined as a common yet popular method for elucidating the bacterial size, shape, and arrangement in order to differentiate the various bacteria groups. It uniformly stains the bacterial cell, increasing an organism’s visibility. The terms simple staining of bacteria and direct, positive, or monochrome staining are sometimes used interchangeably. Let us now look at why simple staining of bacteria is referred to by such different names.

Direct staining: Because it is a direct method that directly stains the bacterial cell with a colourless background.

Positive staining: Because it uses positively charged basic dyes that bind with the negatively charged bacterial cell.

Monochrome staining: Monochrome staining adds contrast to the specimen by using a single stain only.

Simple Stains

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 colour most bacteria. As the simple stain carry a positive charge, it firmly adheres to a negative bacterial cell and makes the organism coloured by leaving a background colourless. 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.


Simple staining of bacteria can be used to quickly and easily determine the shape, size, and arrangement of bacteria cells. Because most bacterial cells and cytoplasm have a negatively charged surface, these positively charged stains adhere easily to the cell surface.

The principle of simple staining of bacteria is based on the principle of creating a distinct contrast between the organism and its surroundings through the use of basic stain. A basic dye is made up of a positive chromophore that attracts negatively charged cell components and molecules such as nucleic acids and proteins.

The bacterial smear is stained with a single reagent in simple staining of bacteria, resulting in a clear contrast between the organism and its background. Because bacterial nucleic acids and certain cell wall components have a negative charge that attracts and binds to the cationic chromogen, basic staining with a positively charged chromogen is preferred. Simple staining of bacteria is used to clarify the morphology and arrangement of bacterial cells. Methylene blue, crystal violet, and carbon fuchsin are the most commonly used basic colours.

Simple Staining Technique



A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells significant contrast so they are much more visible. Different stains have different affinities for different organisms, or different parts of organisms. They are used to differentiate different types of organisms or to view specific parts of organisms.


Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.


Fixation by itself consists of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and makes them permeable so it will accept stains.


Smear preparation

The first step in most bacterial staining procedures is the preparation of a smear. In a smear preparation, cells from a culture are spread in a thin film over a small area of a microscope slide, dried, and then fixed to the slide by heating or other chemical fixatives. A good smear preparation is the key to a good stain.

Simple Staining Technique

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: Heat fixing helps in the fixation of a specimen to the glass slide. Heat fixing helps the stain to penetrate the smear.

Simple Staining Technique

Staining of Bacteria

Simple Staining Technique

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: 

(i) Add stain to the heat fixed smear. 

(ii) Allow the stain to stand for at least 1 minute so that it can penetrate between the cells. 

(iii) Wash off the glass slide carefully. 

(iv) Blot dry the slide with absorbent paper (do not wipe the slide). 

(v) 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 immersion over the glass slide’s stained area to observe it under 100X objective.

Some Common Stains

Loeffler’s Methylene Blue

Method of Staining: Flood the smear with methylene blue, allow for 2 minutes, pour off the stain and allow the air to dry by keeping in a slanting position and by this the organism will retain the methylene blue stain.

Use: Methylene blue staining is used to make out clearly the morphology of the organisms eg. H.influenzae in CSF, Gonococci in urethral pus.

Polychrome Methylene Blue

Preparation: Allow Loeffler’s Methylene blue to ‘ripen’ slowly. Methylene blue stain is kept in half filled bottles, aerate the content by shaking at intervals, slow oxidation of methylene blue forms a violet compound and Stain gets polychrome property. The ripening nearly takes 12 months and this is hastened by addition of 1% potassium carbonate.

Use: Polychrome Methylene Blue is used to demonstrate Mc Fadyean reaction of B.anthracis and in this the blue bacilli is surrounded by purple capsular material.

Dilute Carbol Fuchsin

Preparation: Prepare carbol fuchsin and dilute it to 1/15 using distilled water Method of staining Flood the smear and let stand for 30 seconds, wash with tap water and blot gently to dry.

Use: To stain throat swab from patients of suspected Vincent’s angina, (Borrelia are better stained), it is used as a counter stain in Gram stain and to demonstrate the morphology of Vibrio cholerae (comma shaped).


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.


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