Negative staining procedure, principle, and results

Negative staining procedure is a technique used in microbiology to examine the morphological characteristics of bacterial cells. This technique uses a negatively charged stain to color the background, leaving the cells light-colored and unstained.

The stain is used at a pH where interaction between the stain and biological materials is negligible. The background stains and leaves the biological elements of the specimen untouched and visible.

This staining method allows for determining the morphology and structure of subcellular components and isolated macromolecules. Scientists use it to study bacterial cells that are difficult to stain or too delicate to be heat-fixed and to prepare biological samples for electron microscopy. 

The test provides valuable information about bacterial cells’ shape, size, and arrangement.

Positive staining vs. negative staining

The main difference between negative and positive staining is that in positive staining, a positively charged stain colors the negatively charged cells, making them stand out against a light background. In contrast, negative staining uses a negatively charged stain to color the background, leaving the cells light-colored and unstained.

For example, positive staining of viruses is like negative staining. The difference is that in positive staining, the virus image is darker and formed on a light background, while in negative, a light viral particle image forms on a dark background.

Negative staining principle

The stain applied scatters electrons strongly. The stain gives up a hydrogen ion (proton), making the chromophore of the dye negatively charged. The negatively charged chromogen of the acidic stain will not penetrate cells because of the negative charge on the surface of bacteria. This results in a contrast between the stained background and the unstained specimen.

Stains used for Negative staining

The test uses an acidic dye such as India Ink. Other suitable stains include ammonium molybdate, uranyl acetate, phosphotungstic acid, uranyl formate, osmium tetroxide, osmium ferricyanide, and auroglucothionate. 

Ammonium molybdate is a 1-2% solution in distilled water with the pH adjusted with ammonium or sodium hydroxide to pH 7.02. It is handy for staining osmotically sensitive organelles.

While Ammonium molybdate gives excellent results for many specimens, it produces a lower electron density than other stains.

Also Read: India ink test procedure, principle, and results for capsule staining.

Negative staining procedure

1. Place a drop of your acidic stain at the edge of a clean slide.
2. Aseptically inoculate the test bacteria on the drop of the stain without dispersing it.
3. Using a second slide, make a thin smear of the preparation.
4. To create a thin smear on a microscope slide, start by placing one end of a clean slide with the stain on top of the slide. Tilt the clean slide to form a small angle with the stained slide. Then, slide the clean slide towards the drop of stain until it touches the drop and causes it to spread along the edge of the clean slide. Keeping the angle between the slides small, push the clean slide towards the other end of the stained slide, dragging the drop of stain behind it. This will create a broad, even, and thin smear on the stained slide.
5. Allow the slide to air dry. DO NOT heat.
6. Examine the slide under oil immersion with a microscope.

Observation under a Transmission Electron Microscope

One technique for viewing under a TEM is negative staining coupled with heavy metallic elements coating. The metallic layer scatters electrons that appear on the photographic film, while uncoated sections are used to study bacterial and viral cell morphologies and structures.

Negative staining works by washing tissue specimens with a concentrated heavy metal salt solution and letting them dry. The specimen will be covered with a fragile layer of the metal salt, excluded in areas where an adsorbed macromolecule is present.

The metallic coating scatters electrons that appear on the photographic film, while uncoated sections help study bacterial and viral cell morphologies and structures.

Here is a general procedure for using a TEM:

1. Hold the grid coated with film in a pair of self-clamping forceps with the film side facing up.
2. Mix the sample with an equal amount of negative stains, such as 2% uranyl acetate or 2% sodium or potassium phosphotungstate (pH 7.4).
3. Add 5µl of this mixture to the grid. Smaller particles will stick to the grid surface faster than larger particles. Alternatively, you can add the sample mixed with fixative to the grid first, then add the negative stain afterward.
4. Let the grid sit for 30-90 seconds, then use filter paper to remove any excess liquid.
5. Let the grid air dry, then examine it using a transmission electron microscope (TEM).

Negative staining results

Negative staining results in an unstained or clear specimen with a dark-colored background. Repulsion occurs between the negatively charged stain and the specimen, after which you can observe cells of different shapes and sizes as unstained outlines against a stained background.

Bacterial cells have clear transparent bodies or objects, which may be of variable size and shape if you use a mixture of bacteria against a dark background.