Simple staining is one of the conventional methods of staining techniques. As from the name, it is quite clear that it is very simple and direct staining method which makes the use of a single stain only. The microorganism is invisible to the naked eye, therefore to make it visible, the staining is performed, which gives divergence to a microscopic image. Simple staining makes the use of basic dyes like methylene blue, safranin, crystal violet, malachite green etc. which refers as “Simple or Direct stains”.
The basic stains are having a positive auxochrome which charges the chromogen particle of the stain to bind with the specimen. The chromophore group of the stain imparts colour to the microscopic image that has to study.
As the basic stain carries a positive charge, it also refers as Positive or Cationic stain. The purpose of simple staining is to add contrast to the specimen by directly stain the bacterial cell with a colourless background.
Therefore simple stain not only able us to observe the organism but also helps us to examine the organism’s shape, size and arrangement, which is necessary to distinguish a particular group of organism. The process generally involves three sequential steps like smear preparation, heat fixing and staining of the bacteria.
Content: Simple Staining
- Definition of Simple Staining
- Simple Stains
- Procedure of 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.
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|
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:
- Smear preparation
- Heat fixing
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:
- Take a clean, grease-free glass slide.
- Add a drop of distilled water at the centre of the glass slide.
- Then add inoculum from the bacterial culture with the help of sterilized inoculating loop on the glass slide.
- After that, mix the inoculum with a drop of distilled water to make a thin film by uniformly rotating the inoculating loop.
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:
- Add stain to the heat fixed smear.
- Allow the stain to stand for at least 1minute so that it can penetrate between the cells.
- Wash off the glass slide carefully.
- Blot dry the slide with absorbent paper but do not wipe the slide.
- 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.
- 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.
- It does not give much information rather than the morphological characteristics of bacteria.
- Through simple staining, we cannot classify a particular type of organism.
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.