Sometimes it is further helpful to determine the overall bacterial cell morphology without any use of harsh staining or heat fixing methods that alters the shape of the bacterial cells.

The negative staining is favored in cases when the bacterium does not stain well (i.e. some of the spirochetes) or when it is acceptable to prove observations made on the shape and size or Morphology of the bacteria observed either in a wet mount or hanging drop preparations etc.

In negative staining, the bacterial cells are not stained, they appear as a clear zone against a dark background. The background of the specimen is stained dark due to the use of negatively charged dye.

The negatively charged bacterial cell wall repulse the negatively charged stain, that’s why the stain does not penetrate the cell and cell appears as a clear zone. 

During negative staining, we can use different types of acidic dyes such as Nigrosin, India ink, Eosin or Congo red.

In negative staining, the heat fixed does not apply to smear, because heat fixation can change the morphological characters of bacteria cell by melting the bacterial capsule and slime layer. 

Negative Staining Objective

Negative Staining method is used to study the morphological characters of cells such as shape, size, and arrangement of bacteria cells which are difficult to stain such as Spirilla.

Negative Staining Principle

In negative staining method, an acidic dye is used known as India Ink or Nigrosin. When the bacterial cells are exposed to this stain, due to the presence of acidic nature it readily gives up a hydrogen ion (proton) and the chromophore. As a result, the dye becomes negatively charged, now the bacterial cell surface deflects the stain. 

Hence the glass slide retains the color of dye but the bacterial cells will not. As a result, when we observe the cell under microscope the cell appear clear against a drak background.

Negative Staining Requirement

• Nigrosin (Nigrosin 100 gm/L, Formalin 5 ml/L in water)
• Bunsen burner
• Inoculating loop
• Staining tray
• Glass slides
• Lens paper
• Microscope.

Negative Staining Procedure

1. Place one or two drops of Nigrosin at one end of the slide.
2. Use a sterile inoculating needle to transfer a loop full bacteria culture from the parent slant tube or broth culture in the drop of nigrosin and mix it properly by using this loop.
3. Take a clean slide and place it against the drop of suspended organisms and nigrosin mixture at a 45° angle and allow the drop to spread along the edge of the applied slide.
4. Now make a thin smear by pushing the 2nd slide away from the drop of suspended organisms.
5. Air dry the slide without applying any heat to the slide.
6. Finally, the slide is ready to observe under the microscope. Observe the slide by using oil immersion technique.

Observation 

Under microscope, the bacterial cells appear as a clear transparent body of variable size and shape (If you are using a mixture of bacterial culture) against a dark background.

Result Interpretation of Negative Staining 

Importance of Negative Staining

1. Negative staining method is used to stain cells that are too delicate to be heat-fixed. 
2. Used to prepare biological samples for electron microscopy.
3. Used to observe viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power.
4. Used for the examination and identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical micelles (M) and inverted hexagonal HII cylindrical (H) phases by Negative staining transmission electron microscopy.
5. Can be used to observe the bacterial capsule.
6. There are several bacterial cells (Spirillum, Spirochetes etc.), that are difficult to stain by other staining techniques, but this negative staining method can be used to stained them easily.