Dorner's staining - Introduction, Spore, Reagent, Principle, Procedure, Result

Introduction to Dorner's staining

Dorner's staining is used for endospore staining and it differentiates bacterial spores from other vegetative cells. It also functions to differentiate spore-forming bacteria from non-spore-forming bacteria.

Spore

Spores are highly resistant, metabolically inactive cell types that microorganism produces. Some spore-producing genera include anaerobes such as Clostridium, Desulfotomaculum, and aerobes such as Bacillus.

Usually, spores are formed when environmental conditions turn unfavorable to sustain a vegetative state such as scarcity in nutrients including carbon sources. These cells undergo sporogenesis, giving rise to an intracellular structure called an endospore, and are surrounded by impervious layers known as spore coats. As the environment continues to become unsuitable, endospores are released from the degenerating vegetative cells. Once released, these structures are called spores.

The chemical composition (calcium, dipicolinic acid) of spore layers makes it resistant to extreme desiccation, heat, freezing, radiation, and chemical components. When favorable environmental conditions return, these spores revert back to the metabolically active vegetative stage.

On the basis of the location of endospores, they are classified into three types:

• central - spore located at the center of vegetative cell

• subterminal - spore located between the center and edge of the microbial cell

• terminal - spore located at the edge of the bacterial cell

Reagents for Dorner's staining

The reagents used for Dorner's staining include:

• Primary stain: Carbol Fuchsin

• Counterstain: Nigrosine

Principle of Dorner's staining

The principles of Dorner's staining are based on the use of primary stain and counter stain.

Primary stain

Carbol Fuchsin is the primary stain used in Dorner's staining. With the help of heat application, the carbol fuchsin is able to penetrate the microbial cytoplasm as well as the endospores. Both cells and spores appear red at this stage.

Counterstain

Nigrosine is used as a counterstain as it provides dark background for contrast. However, the sporangium is decolorized by nigrosine while the spores retain the color of the primary stain and appear red.

Procedure of Dorner's staining

The procedure for Dorner's staining is as follows:

1. Take a heavy suspension of test bacteria and mix it with 5 drops of sterile water in a test tube

2. Add 5 drops of primary stain (carbol fuchsin) into the suspension

3. Place the test tube containing the suspension in a bear of boiling water for 10 minutes

4. Place 1-2 drops of counterstain (nigrosine) on a clean grease free glass slide and mix it with the bacterial suspension

5. Emulsify the mixture and make a thin smear with the help of another slide.

   * Hold a second slide at 30° and place it on the mixture till the mixture spreads alongside the edge of the second slide touching it. Slide the second slide firmly, quickly, and in one single swipe over the first slide.

6. Let the smear air dry

7. Examine the slide under the microscope- first at low magnification and then at 100x under oil immersion.

Result of Dorner's staining

After Dorner's staining, in the case of sporing bacteria, the spores appear red-colored, sporangium is colorless while the background is dark due to nigrosine. In non-sporing bacteria the sporangium is colorless, the background is dark due to nigrosine and there is an absence of red coloration.