Principles And Techniques Of Negative Staining

Negative staining is a method used for the primary purpose of studying bacteria cells that are otherwise difficult to stain. By staining the background, rather than the sample itself, the specimen can be viewed without damaging the cell morphology.

Principles And Techniques Of Negative Staining

Against the dark background, the shape of the cell will appear bright, and the size and structure can be observed. Due to the delicacy of the method, negative staining can also be used to view samples that can’t be heat fixed.

Negative staining can further be used to prepare samples for transmission electron microscopy. Select negative stains have high electron scattering properties, while also adsorbing biological matter.

This technique can be used to study and observe low-electron scattering materials such as viruses, bacterial flagella, and proteins.

The technique can also be used to identify aqueous lipid aggregates, for example, lamellar liposomes, and inverted spherical micelles. 

How Does Negative Staining Work?

Negative staining requires the use of an acid stain that has a negatively charged chromogen. As the surface of bacteria has its own negative charge, the cell won’t be penetrated by the dye.

Instead, it is repelled. As a result, the glass of the slide becomes stained, but the bacteria remains clear.

When observed, the bacteria cell will appear as a bright area contrasting strongly against the dark of the stain. Common acidic dyes used include Nigrosin and India Ink. 

This is in contrast with positive staining, in which the negative charge of the bacteria cell attracts the positive charge of the stain, absorbing the dye.

Positive staining is the more common technique in microbiology, and can also be used for the staining and observation of pathogens. 

Common Reagents Used In Negative Staining

Acidic stains must be used in negative staining. Common choices include India Ink and Nigrosin. India Ink consists of carbon particles that are suspended in water.

Nigrosin is a synthetic dye, created from a mixture of nitrobenzene, anilini, and hydrochloric acid, which are heated in the presence of copper or iron. 

When preparing a sample for transmission electron microscopy, common staining agents include uranyl acetate and uranyl formate. These have a fine grain size that allows for a higher resolution of image. 

Techniques For Negative Staining

  1. Clean and heat a glass slide. Place a small drop of your chosen reagent (for example, nigrosin), at one end of the slide. The drop must be small: less than a single drop from a dropper, but more than a standard loop full.
  2. Using an inoculating loop, apply a small amount of culture to the stain. Be careful not to spread the drop of stain when adding the culture.
  3. Spread the culture and stain using a clean slide. To do this, gently rest one end of the clean slide at the center of the stained slide. Angle the clean slide to roughly 45 degrees, and carefully pull the slide forward until it reaches the drop. The stain and culture should spread across the edge of the slide. Keeping the slides at 45 degrees, move the spreader slide back to the center. It should drag the stain along with it, creating an even smear. 
  4. Leave the smear to dry. Do not heat.
  5. To view, apply immersion oil to a small area, and observe under a microscope. The cells should contrast clearly against the background stain. 

Negative Staining And Transmission Electron Microscopy (TEM)

  1. Prepare your sample for use by mixing one part sample with one part chosen negative stain. Uranyl acetate and uranyl formate are popular choices for TEM, as the fine grain size improves the clarity of the image.
  2. Place a TEM mesh grid in a pair of self clamping forceps, with the support film layer facing upwards.
  3. Apply 5μl of your prepared sample to the grid, and allow it to absorb and incubate for roughly 60 seconds.
  4. Blot the stain with a small amount of filter paper to remove any excess liquid. 
  5. Leave the stain to air dry. Do not heat. It can now be viewed in the transmission electron microscope.
  6. For an alternate method, apply a drop of the sample, without stain, to the grid. Allow the sample to absorb, and then blot away any excess. Apply your staining reagent, and leave for 10 to 40 seconds. Blot the excess liquid, and leave the grid to air dry. Once dry, the grid can be placed in the TEM. 

Expected Results Of Negative Staining

Performed correctly, negative staining should create an image with a clear contrast. As the cells have repelled the dye, they appear as bright spots against a dark background.

In some cases, negative staining will allow you to observe the bacteria spores as well. An outline or shape of the cell should be clearly visible in this contrast. This technique is mild, and shouldn’t cause damage to delicate samples. 

Jennifer Dawkins

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