Endospore Staining

introduction image

Endospore staining is a type of special staining method that makes the use of differential staining procedure to highlight the presence or absence of endospores. For the staining of endospores, a first article was published by Dorner in the year 1922. Later, the Dorner method of staining endospores has been modified by the two scientists, Sheffar and Fulton in the year 1933. Sheffar and Fulton had introduced a faster process for staining endospores by the use of differential stain, which selectively focuses on the bacterial endospore.

The main objective of endospore staining is to differentiate the bacterial endospore from the vegetative cell, which in turn allow us to distinguish between the endospore formers and non-endospore formers.

Content: Endospore Staining

  1. Definition of Endospore Staining
  2. Definition and types of Endospore
  3. Methods of Endospore Staining
    1. Sheffar and Fulton’s Method
    2. Dorner’s Method
  4. Significance

Definition of Endospore Staining

Endospore staining can define as one of the unique staining methods which are used to differentiate between the spore formers and non-spore formers by selectively staining the endospore from the vegetative cell.

Clostridium perfringes, Bacillus cereus, Sporosarcina sp etc. are the examples of endospore formers. Escherichia coli, Salmonella sp etc. are the examples of non-endospore formers. Endospore can be stained by the two methods, namely Dorner’s method and Sheffar and Fulton’s method.

Definition and types of Endospore

These are the structures that form within the cell. It is the unique structure of the bacteria, which forms during unfavourable condition approximately within 6-8 hours.

The endospores are thick, dormant, metabolically inactive and the refractile bodies which can be produced by the few genera of the bacteria like Bacillus and Clostridium species.

Endospores can remain in a resting phase or dormant state before the germination into the new individual. The position of endospore is different in different species, but commonly of three types:

  • Central: The location of an endospore is towards the centre of the vegetative cell.
  • Terminal: The location of an endospore is towards an edge of the vegetative cell.
  • Sub-terminal: The location of an endospore is between the central and terminal position of the vegetative cell.

position of endospore

The structure of endospore also varies with an individual to individual, but are of three types:

  • Elliptical: An endospore appears long or elongated.
  • Spherical: The endospore appears small and pear-shaped.
  • Ovoid: An endospore appears small and egg-shaped.

structure of endospore

Endospores are resistant to extreme physical conditions like radiation, heat, desiccation etc. and chemical conditions like staining, disinfection etc. These can resist at boiling temperature for a few hours and heat treatment at 80 degrees Celsius for 10 minutes.

Endospores consist of a colossal amount of DPA (Dipiclonic acid) which constitute about 10-15% of spore’s dry weight. DPA is found in the core of endospore in combination with a large amount of calcium and forms a Ca-DPA complex. The Ca-DPA complex provides heat resistance to an endospore. During germination, endospore loose resistance to heat and staining.

Methods of Endospore Staining

There are two methods of endospore staining, namely Sheffar and Fulton method and Dorner’s method.

methods of endospore staining
The process of endospore staining follows a general staining procedure and makes the use of three kinds of stain. A primary stain helps in the staining of the endospore.

general process of endospore staining

Sheffar and Fulton’s Method

It is the most common method of endospore staining, which makes the use of three reagents, namely primary stain, decolourizer and counterstain.

  • Malachite green (As a primary stain): To prepare 0.5% of an aqueous solution, take 0.5g of malachite green in 100ml of distilled water.
  • Distilled water works as a decolourizer.
  • Safranin (As counterstain): To prepare 2.5% of an aqueous solution, take 2.5g of safranin into 100ml of 95% ethanol.

Procedure

Sheffar and Fulton’s method includes the following steps:

Sterilization of glass slide: Take a clean and sterilized glass slide.

Smear preparation: Prepare smear by taking a bacterial culture onto the glass slide.

Heat fixing: It is the fixing of the smear by moving the glass slide to and fro movement over the flame of Bunsen burner.

Saturation with primary stain: Place a blotting paper to the size of a glass slide and saturate it with malachite green dye.

Steaming: Steam the saturated blotting paper for up to 5 minutes. During this step, more dye can be added as required.

Decolourizing: Decolourize the glass slide by adding distilled water thoroughly.

Counterstaining: Flood counterstain safranin over the glass slide and allow to stand for 1min. and then wash off the slide.

Air drying: After counterstaining, allow the glass slide to air dry.

Microscopic observation: Add a drop of oil immersion on the stained area of a glass slide and observe it under the microscope of a 100X objective.

Principle

In Sheffar and Fulton method, the endospore is subjected to heat because unlike vegetative cell, it cannot be stained directly. The heat treatment allows the penetration of the primary stain, i.e. Malachite green into the cortex of an endospore. Once they take up the dye, they retain the colour of the dye or resistant to de-staining.

Result

Endospore retains the colour of primary stain of malachite green and appears green in colour, whereas vegetative cells lose up the primary stain and take up the colour of counterstain, i.e. safranin and appear pink in colour.

Result of sheffar and fulton method

Dorner’s Method

It is an old method of endospore staining, which makes the use of three reagents, namely primary stain, decolourizer and counterstain.

  • Carbol fuschin (As a primary stain): Take 0.3g of basic fuschin, 10ml of ethanol (95%) and 95ml of distilled water.
  • Acid alcohol (As decolourizer): Take 97ml of 95% ethanol in 3ml of concentrated hydrochloric acid.
  • Nigrosine (As counterstain): Take 10ml of nigrosine in100ml of distilled water.

Procedure

Dorner’s method includes the following steps:

Sterilization of glass slide: Take a clean and sterilized glass slide.

Smear preparation: Prepare smear by taking a bacterial culture onto the glass slide.

Heat fixing: It is the fixing of the smear by moving the glass slide to and fro movement over the flame of Bunsen burner.

Saturation with primary stain: Place a blotting paper to the size of a glass slide and saturate it with malachite green dye.

Steaming: Steam the saturated blotting paper for up to 5-10 minutes.

Decolourizing: Decolourize the glass slide by adding acid alcohol thoroughly.

Counterstaining: Place a drop of nigrosine on one end of the glass slide and take another slide and drag it to make a thin film of stain. Allow the dye to stand for 1 min. and then wash off the slide.

Air drying: After counterstaining, allow the glass slide to air dry.

Microscopic observation: Add a drop of oil immersion on the stained area of a glass slide and observe it under the microscope of a 100X objective.

Result

Endospore retains the colour of primary stain, i.e. Carbol fuschin and appears red coloured, whereas vegetative cells appear colourless with a dark background.
result of Dorner's method

Significance

Endospore staining helps in the microscopic study of the endospore by which we can differentiate and classify the bacteria. It also plays a significant role in the medical science and food industry as the spores can be present in canned food, which leads to food spoilage and ultimately cause food diseases in humans and animals.

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