when staining the microbes it has access stain the slide, place the slide under bibulous paper to blot up the excess without ruining your slide. The use of the positively charged stain allows it to bind the negatively charge cell wall, thus staining the cell.

The preparation of a smear is required for many laboratory procedures, including the Gram-stain. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium.

Just as in preparing a smear, you only need a small amount of organism. If you have too many organisms, you won’t be able to see the morphology of individual cells. It is also important not use too much nigrosin. If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun.

Chemical Compounds Commonly Used as Antiseptics and Disinfectants

Bacterial smear. – Obtaining bacteria, spreading it on a slide, and staining for microscopic examination. – Bacterial smear is the first step of the staining process. Accomplishments. Causes bacteria to adhere (attach) to slide allowing staining and observations.

An ideal bacterial smear should be thin, semi-transparent, whitish layer or film, circular with diameter 1 cm. (approximately) free from dirt, dust or any contamination.

Steam helps to loosen up the waxy layer and promotes entry of the primary stain inside the cell. The smear is then rinsed with a very strong decolorizer, which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms.

PRINCIPLE OF ZIEHL-NEELSEN STAIN The phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbol fuchsin is further enhanced by steam heating the preparation to melt the wax and allow the stain to move into the cell.

Most bacterial capsules are composed of polysaccharide however some genera produce polypeptide capsules. Capsular material is very moist (slimy) and any heating will cause it to shrink – it is for this reason that we will not heat fix the slide before staining.

A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background.

The capsules exclude the ink and appear as clear halos surrounding the bacterial cells. Capsules are usually polymers of simple sugars (polysaccharides), although the capsule of Bacillus anthracis is made of polyglutamic acid.

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. We use India ink and Gram crystal violet.

If the color portion of the dye resides in the positive ion, as in the above case, it is called a basic dye (examples: methylene blue, crystal violet, safranin). If the color portion is in the negatively charged ion, it is called an acidic dye (examples: nigrosin, congo red).

black

In this method, two dyes, crystal violet, and India ink are used. The capsule is seen as a clear halo around the microorganism against the black background.

Acidic/Basic Dyes The most widely used histological stains differentiate between the acidic and basic components of cells and tissues. Basic dyes have a net positive charge and bind to components of cells and tissues that are negatively charged.

decolorizer for acid-fast stain. water. decolorizer for capsule stain. Only $2.99/month. safranin.

During the acid fast stain, heat is used as a mordant to allow the primary stain to penetrate the waxy mycolic acid layer. The heat will prevent the cells from being destained using acid-alcohol. Because these cells hold fast to the primary stain with acid alcohol treatment, they are termed acid fast positive.

Because the cell wall is so resistant to most compounds, acid-fast organisms require a special staining technique. The primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. This is further assisted by the addition of heat.

Acid fast bacteria have a high content of mycolic acids in their cell walls. Acid fast bacteria will be red, while nonacid fast bacteria will stain blue/green with the counterstain with the Kinyoun stain.

Acid-fast bacillus (AFB) is a type of bacteria that causes tuberculosis and certain other infections. Tuberculosis, commonly known as TB, is a serious bacterial infection that mainly affects the lungs. It can also affect other parts of the body, including the brain, spine, and kidneys.

[1] Bacteria displaying acid fastness include:

• Genus Mycobacterium – M. leprae, M. Tuberculosis, M. smegmatis, M. Avium complex, M. kansasii.
• Genus Nocardia – N. brasiliensis, N. cyriacigeorgica, N. farcinica, and N. nova.