Negative Staining Method and its Mechanism
- Negative staining procedure is also called as Relief staining.
- Some bacteria have non-ionic charge or slime layer on their surface.
- Because of which the stain doesn’t penetrate inside the cell and the bacterial cells are not easily stained.
- In case of such bacterial cells negative method is used for observing the morphological characters of the cell.
- In negative staining method actually, the background is stained and the bacterial cell remains colourless.
- This is the only staining method in which bacterial cells are not stained but they are made visible against a dark background.
- Generally, the acidic stain such as 10 % Nigrosin, Eosin, India ink and Congored is used.
- A grease-free slide is taken and a smear is made on the slide by using Nichrome wire loop.
- The slide is allowed to air dry.
- Here there is no Heat fixation step.
- After air drying a drop of stain is added on an end of a slide and a thin film of stain is made by using another slide.
- These film is allowed to air dry and observed under oil immersion.
- Here we stain background and not cells so after drying there is no water wash step slide is directly observed under oil immersion.
- In negative staining, heat fixation step is avoided because heat fixation can alter morphological characters.
Flowchart of Negative staining method
- A bacterial cell surface has negative charges.
- So in this negative staining method, the acid stain which we use has a negative charge on its chromophore group.
- The negative charges of acidic stain get repelled by the negative charges present on the cell surface.As we know opposite charges get attracted and negative charges repel each other.
- So here negative charges repel and stain don’t bind to the cell surface and get deposited around the cell.
- Hence background gets coloured and the cell remains colourless.
- This method is used to study morphological characters of cell size, shape, and arrangement of bacterial cells.
- This method is easy and useful in observing the bacteria that are difficult to stain. For example- Spirilli.
- By using this method we can observe spirochetes.