Negative Staining Method and its Mechanism

Introduction

• 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.

Procedure

1. A grease-free slide is taken and a smear is made on the slide by using Nichrome wire loop.
2. The slide is allowed to air dry.
3. Here there is no Heat fixation step.
4. 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.
5. These film is allowed to air dry and observed under oil immersion.

Note

•  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

Mechanism

1. A bacterial cell surface has negative charges.
2. So in this negative staining method, the acid stain which we use has a negative charge on its chromophore group.
3. 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.
4. So here negative charges repel and stain don’t bind to the cell surface and get deposited around the cell.
5. Hence background gets coloured and the cell remains colourless.

Application

1. This method is used to study morphological characters of cell size, shape, and arrangement of bacterial cells.
2. This method is easy and useful in observing the bacteria that are difficult to stain. For example- Spirilli.
3. By using this method we can observe spirochetes.