The Definitions, Methods, And Procedures Involved In Capsule Staining

Capsule staining is a method used to determine whether a bacterium produces a capsule. Capsules are usually made of polysaccharides and are found around many bacteria.

Some bacteria also produce a slime layer instead of a capsule. Slime layers are thinner than capsules and are not easily detected using standard stains.

The Definitions, Methods, And Procedures Involved In Capsule Staining

Capsule staining involves the use of two types of dyes. One type of dye reacts with acid groups, while another type reacts with basic groups. Acidic groups are present in both capsules and slime layers.

However, capsules contain significantly higher levels of acid groups than slime layers. Hence, capsule staining will result in a bright red color when the capsule is stained. If the bacteria do not produce a capsule, then there will be no red color.

Capsule stains can be removed using either a manual or automatic method. Manual removal involves soaking the stained area in a capsule of stain remover until the stain disappears.

Automatic removal uses a machine that automatically applies the capsule stain remover. This article explains both these methods and more so that you can understand everything there is to know about how the best methods actually work.

What Is A Capsule Stain Exactly?

Capsule staining is a method of differentiating Gram-positive and negative bacteria. It involves using two stains, one acidophilus and another basic.

Acidophilus stains negatively charged molecules such as DNA and RNA, while basic stains stain positively charged molecules like proteins.

By combining the results of both stains, you can determine whether there is a polysaccharide capsule around the bacterium or not. In the capsule stain procedure, they do not heat fix and wash the smear with water.

Heat and water may dislodge the capsules from the bacteria.

They, therefore, do not heat fix, and then rinse the smear with water. This is very important to remember when working with capsule stains, as some of this terminology can be easily interpreted in the incorrect way if not understood correctly.

What’s The True Definition?

Capsule staining is a special type of staining method that highlights the capsule of a bacterium. Some bacteria like Bacillus anthrax, Streptococcus pneumonia, etc., have capsules that can cause pathogenicity.

Thus, it becomes essential to identify the presence of a capsule. 

Capsule staining can be done using India ink, Anthony’s, Manaval’s, and Hissa’s method. The most popular methods are the India ink method and Anthony’s method, as you will see below when we dive into each method and how they best work.

The Principle Of A Capsule Stain

Primary Stain 1: Crystal Violet (1%) A violet stain is applied at this step. At this point, cells and the capsular material should turn dark purple.

To remove the purple stain, we need to add a decolorizing agent. The decolorizing agent is copper sulfate.

Primary Stain 2: Giemsa (10%) A pink stain is applied at this stage. The Giemsa stain binds to both the capsular material and the cell walls. As the slide dries, the Giemsa stain turns red.

We use copper sulfate to wash away the purple stain. If you were using a different dye, you might use another decolorizing agent instead. For example, if you were using an orange stain, you could use sodium carbonate.

Bacterial capsular polysaccharides are not ionic, therefore neither acid nor base will stick to their surface. Capsules are usually stained with two different dyes: one dye stains the background and another one stain the capsule.

Different staining techniques are available. The results (stains of the capsule and background) depend on the type and concentration of the dye used. The most commonly used techniques are described here:

The India Ink Method

In this exact method, there are two dyes, which are crystal violet, and the India inks. The capsules are seen and interpreted as an extremely clear hale around the microorganisms against the stark black style background.

The first step in this method actually is to place a single drop of the India Ink onto the slide. Then, you must add a drop of Crystal Violet to the slide. Next, you must cover the entire area with a coverslip.

After covering the area, allow the slide to dry for at least 2 minutes. Once the slide is dry, remove the coverslip and gently tap the slide on a clean surface to dislodge any air bubbles on the slide.

Now, put another coverslip on top of the slide. Be careful when putting the second coverslip because the India Ink and Crystal Violet will run down the sides of the slide.

Make sure there are no bubbles underneath the coverslip before placing the slide under the microscope. If there are bubbles, you can remove them using a razor blade.

When viewing the slide through the microscope, look for purple cells surrounded by an opaque white circle. These cells are encapsulated in yeasts.

Capsules are found in many species of bacteria, including those that cause disease. Capsules help protect bacteria from environmental factors like heat, cold, radiation, chemicals, and antibiotics. 

They also help bacteria survive within the host. Some capsules are made out of proteins, while others are made out of polysaccharides.

In Gram-negative bacteria, capsules are usually composed of something called lipopolysaccharide and colonic acid. In Gram-positive bacteria, capsules are typically composed of teichoic acids.

These acids are important when working with capsule stains because this is how they are removed correctly and in the safest way possible, if that makes sense.

Reagents And Materials Required

Reagents And Materials Required

The results are as follows: Stained bacteria appear purple because of the dye. Place a drop of bacteria culture on the slide and cover with a coverslip.

If you cannot see any bacteria, increase the number of bacteria until you can see them. Be careful when handling the slide, as the bacteria will not be dead yet.

Bacteria should be placed on a clean surface, like a piece of paper. Once the slide is covered, place it under the microscope and focus on the area containing the bacteria.

Make sure to adjust the brightness and contrast settings to get the best view. When you see the bacteria, you will notice that they are surrounded by a clear or pale blue halo. This is the capsule. Dispose of staining waste in an appropriate container.

It’s extremely important to dispose of the staining waste appropriately to keep you and the surrounding environment as safe as possible, cross-contamination is never a good thing, so it’s always best to avoid it at all costs by just sticking to the cleaning procedures.

The Anthony Stain Method

Place a single drop of reagent on the main part of the slide. Make sure the top coverslip is clean and dry before placing it on the slide. If you do not have an antifog cover slip, place a piece of paper towel under the slide.

Be careful not to let the slide touch anything else. Use tweezers to pick up the slide. Tweezers are the best tool to use so that you can avoid handling it wrong.

This method uses two reagents, namely Congo red and Maneval’s stain. After staining: 

A bacterial cell appears to positive staining method that stains both the capsule and the bacterial cells with a darker background.

As a result, a capsule appears as a dark violet color between a bright violet-colored bacterial cell and a colorless background.

The Maneval Method

This method is also called Gram staining. It is a simple and quick test that can help you identify different types of bacteria. By using this method, we can see if there is a capsule around the bacteria.

If there is a capsule, then it means that the bacterium is gram-positive, otherwise, it is gram-negative.

The Hiss Method

This method is commonly used to detect and identify different types of microorganisms. It works by using an acidified dye called crystal violet.

Crystal violet binds to negatively charged molecules found in all gram-positive bacteria, including Staphylococcus aureus, and Clostridium.

The reaction also occurs with other gram-negative species, such as Escherichia coli and Salmonella typhi. However, it does not bind to any gram-negative organisms.

Gram-positive stain purple due to the binding of the crystal violet to the polysaccharide matrix. Gram-negative rods appear unstained because the capsule protects them from the dye.

Things You Need To Remember

Use a clean bench cover to keep dirt out of the microscope. Use a lens cleaning solution to remove any oil from the microscope lenses. If you stain the slide, dispose of the staining solution properly.

After using the slide, wash your hands thoroughly before touching other surfaces. Also, be careful when handling the slide because the cells haven’t been killed yet.

These are simple rules to stick by so for the sake of your own safety, as we’ve talked about several times in this article, stick to them if possible. 

Final Thoughts

Staining methods are very useful for identifying and classifying bacteria. They are easy to use and can provide information about the shape of the bacteria.

There are many ways to stain bacteria, but each one has its own advantages and disadvantages.

Using the right method depends on what you want to achieve. For example, if you want to know which type of bacteria is present, then you need to use the Gram stain method.

On the other hand, if you want to determine whether the bacteria are alive or dead, then you should use the Live/Dead kit.

Jennifer Dawkins

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