Negative Staining- Principle, Procedure and Result Interpretation

Negative staining is a technique that allows us to visualize unstained microorganisms by creating a contrast between them and the surrounding medium. Unlike other staining methods, negative staining does not require heat fixation or chemical treatment of the specimens, which may alter their morphology or damage their structures. Therefore, negative staining can preserve the natural shape and size of the microorganisms and reveal details that are otherwise difficult to observe.

Some of the objectives of negative staining are:

• To study the morphological characteristics of bacteria, such as shape, size, arrangement, and presence of capsules or flagella.
• To examine bacteria that are resistant to conventional staining methods, such as some spirilla.
• To prepare biological samples for electron microscopy, which requires high contrast and resolution.
• To measure the size of bacteria cells accurately, since they are not distorted by heat or chemicals.

In this blog article, we will explain the principle of negative staining, the reagents and equipment needed for this technique, and the procedure of negative staining. We will also discuss some of the advantages and limitations of negative staining and its applications in microbiology.

Negative staining is a technique that uses an acidic stain, such as India ink or nigrosin, to create a contrast between the background and the unstained cells. The acidic stain has a negatively charged chromogen, which is the colored part of the stain. The chromogen does not react with the cells because they also have a negative charge on their surface. Therefore, the stain is repelled by the cells and accumulates around them, forming a dark halo.

The advantage of negative staining is that it does not require heat fixation or other treatments that may alter the shape and size of the cells. It also allows the visualization of bacteria that are difficult to stain by other methods, such as some spirilla. Negative staining can also be used to prepare biological samples for electron microscopy, by using a solid matrix such as potassium phosphotungstate to enhance the contrast between the specimen and the background.

Negative staining can reveal the morphology, arrangement, and external structures of bacteria, such as capsules, flagella, and pili. However, it cannot differentiate between different types of bacteria or show their internal structures. Negative staining is also not suitable for observing living cells, as the acidic stain may damage them. Moreover, negative staining requires careful handling of the slides, as the cells are not fixed and may be infectious.

Reagent and Equipment’s for Negative Staining

To perform a negative staining procedure, you will need the following reagents and equipment:

• Nigrosin: This is an acidic stain that has a dark color and a negative charge. It will not penetrate the bacterial cells, but will form a contrasting background around them. You can also use India ink as an alternative to nigrosin.
• Microincinerator or Bunsen burner: This is used to sterilize the inoculating loop before and after transferring the bacterial culture. A microincinerator is preferred over a Bunsen burner as it reduces the risk of aerosol formation and contamination.
• Inoculating loop: This is a metal wire with a loop at one end that is used to pick up and transfer a small amount of bacterial culture from the broth or agar medium. It should be flamed until red hot before and after each use to avoid cross-contamination.
• Staining tray: This is a plastic or metal tray that holds the slides during the staining process. It prevents the stain from spilling over the bench and protects the slides from dust and dirt.
• Glass slides: These are thin, flat pieces of glass that are used to hold the bacterial smear for microscopic examination. They should be clean and free of scratches or fingerprints. You can use lens paper to wipe them gently before use.
• Lens paper: This is a soft, lint-free paper that is used to clean the glass slides and the lenses of the microscope. It prevents scratching or damaging the glass surfaces. You should not use tissue paper or cloth as they may leave fibers or dust on the slides or lenses.
• Microscope: This is an optical instrument that magnifies the image of the bacterial cells using a series of lenses. You will need a compound microscope with an oil immersion objective (100x) to observe the negative stained cells. You should also adjust the light source, the condenser, and the diaphragm to obtain a clear and bright image.

Procedure of Negative Staining

To perform a negative staining procedure, you will need the following steps:

1. Place a small drop of nigrosin near one end of a clean, grease-free, and dry glass slide. Nigrosin is an acidic stain that will create a dark background for the unstained cells.
2. Using aseptic technique, take a loopful of inoculum from the bacterial culture and mix it with the drop of nigrosin. Do not spread the drop on the slide.
3. Use another clean slide to spread the drop of stain containing the organisms across the slide. Hold the clean slide at a 45° angle and touch the edge of the drop. Then push the slide away from the drop, dragging it along the slide and forming a thin smear.
4. Allow the smear to air-dry completely. Do not heat-fix the slide, as this will distort the cells and alter their natural shape and size.
5. Examine the slide under oil immersion using a light microscope. You should be able to see clear cells against a dark background.

Some additional points you may want to include in your blog article are:

• The advantages and disadvantages of negative staining compared to other staining techniques.
• The applications of negative staining in microbiology, such as visualizing bacterial capsules, flagella, spirochetes, and viruses.
• The precautions and safety measures to be taken when handling live microorganisms and acidic stains.

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