Negative Staining Procedure in Bacteria

In this negative staining procedure in bacteria post we have briefly explained about negative staining procedure principle, objectives, requirements, negative staining procedure, uses and limitations.


Negative staining procedure is used to observe intact microbial structures without affecting their cellular shape. Negative staining procedure is named from the fact that the bacterium cells are not stained, but the glass background containing cells. The chemical properties of the dye and the specimen being viewed are used to select dyes for staining.

In most circumstances, a positive stain, a dye that is absorbed by the cells or organisms being studied, is desirable, as it adds colour to things of interest and helps them stand out against the backdrop. However, there are times when using a negative stain, which is absorbed by the background but not by the cells or organisms in the material, is useful.

Negative staining procedure results in the organisms’ outline or shadow against a colourful background. Acidic colours such as acid fuchsin, eosin, nigrosin, and rose bengal are commonly utilised. A drop of acidic stain is deposited on the slide and cells growing on solid media are mixed into it in the negative staining procedure. A second slide is used to apply the stain thinly across the slide, which is then allowed to dry.


Acidic dyes, which dissolve to create H+ and a negative chromogen, are used in negative staining. Negatively charged chromogens resist negatively charged cell walls, leaving the cell uncoloured and surrounded by a hued background.



1. Microscopic glass slide

2. Inoculating loop

3. Spirit Lamp

4. Staining Rack

5. Wash bottle

6. Microscope


1. Nigrosin stain: Add 10 g Nicrosin (C.C.) to 100 ml distilled water and dissolve by placing in a boiling water bath for 30 minutes. Replace water lost by evaporation and add 0.5 ml of formalin. Filter twice through double filter paper.

Negative Staining Procedure in Bacteria

Slide Method

1. Place a drop of India ink or Nigrosin on one end of a clean, dry, scratch- and grease-free Microscopic glass slide at the edge.

2. Using a sterilised Inoculating loop, take a little part of the bacterial colony. Mix the culture with the dye on the Glass slide thoroughly.

3. Take another Microscopic Glass slide and place it near the specimen-dye combination at a 30°- 45° angle.

4. Move the slide closer to the drop of the specimen-dye mixture until it makes contact with it at the correct angle.

5. Then, smoothly and quickly advance the spreader slide over the specimen slide, pulling the dye mixture behind it into a thin film.

6. Allow the smear to air dry before observing a thin region under oil immersion and the unstained cells surrounding by the grey stain under the microscope using a high power objective (45X) and an oil immersion objective (100X).

Negative Staining

Loop Method

Negative Staining

1. Place a drop of nigrosine or India ink in the centre of a clean and grease-free glass slide.

2. Transfer a little part of the specimen to the slide containing a Drop of Dye using a sterile inoculating loop.

3. Using the sterilised straight wire, thoroughly mix the Specimen with the Dye and spread equally over an area of about 1 – 2 cm.

4. Allow the smear to air dry before examining it under a microscope with a high power objective (45X) and an oil immersion objective (100X).


If you are utilising a mixture of bacteria against a dark backdrop, the Bacterial cells observed as clear transparent bodies or objects in Negative staining Preparations may be of different size and shape.


1. Negative staining procedure is often used to investigate the morphological characteristics of bacterial cells, such as their size, shape, and arrangement, without distorting their true characteristics.

2. Negative staining procedure is simple and effective for examining bacterial cells that are difficult to stain using conventional methods. Spirillum, Spirochetes, and so forth.

3. The bacterium capsules can also be observed using the Negative staining procedure.


1. Negative staining procedure does not give much information rather than the morphological characteristics of bacteria. Through simple staining, we cannot classify a particular type of organism.


Heat fixing, which shrinks and distorts cells, is not required. The drying process destroys the majority of cells, but not all of them. As a result, make sure to dispose of the spreading slide in the proper container as soon as possible after usage. As an added precaution, we shall not use this procedure with harmful organisms.

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