What is a Decolorizer in microbiology?

A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria

Gram positive bacteria

Eubacterium is a genus of Gram-positive bacteria in the family Eubacteriaceae. These bacteria are characterised by a rigid cell wall. They may either be motile or nonmotile. If motile, they have a flagellum. A typical flagellum consists of a basal body, filament, and hook.

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Eubacterium - Wikipedia

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What is the purpose of a Decolorizer?

The decolorizer dissolves the lipids, increasing cell-wall permeability and allowing the crystal violet-iodine complex to flow out of the cell. The color of the counterstain must contrast with that of the primary stain. A counterstain such as safranin stains gram-negative cells red.

What is a Decolorizer in gram staining?

The decolorizer, ethyl alcohol, is the most crtitical step. Ethyl alcohol is a nonpolar solvent, and thus penetrates the cell walls of Gram negative cells more readily and removes the crystal violet-iodine complex.

What is decolorizing agent in microbiology?

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed.

What's a Decolorizer?

Definition of 'decolorizer'

1. a substance that removes colour. 2. a substance that removes the greeny-brown colour in glass that is caused by impurities.

Gram stain: Preparation of decolorizer

Why is decolorization important in gram staining?

The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria. The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification.

What is the purpose of the iodine and Decolorizer in the Gram stain procedure?

Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed. This is referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells.

Why is acid alcohol used as a decolorizing agent?

Acid alcohol is used as a decolorizing agent because of its interaction with the bacterial cell wall.

What is alcohol used for in Gram staining?

Decolorizer-Alcohol

Either acetone or ethyl alcohol can be used as the decolorizing agent. The alcohol dissolves lipids found in the outer cell membrane of Gram-negative bacteria, allowing the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer.

What is the best procedure for decolorization?

What is the best procedure for decolorization? Add decolorizing agent until run-off is clear. This method allows the decolorizing agent to dissolve the outer membrane of Gram-negative cells and rinse out the crystal violet from the thin layers of peptidoglycan. This causes the run-off to be purple.

What is the Decolorizer in the Gram stain quizlet?

The decolorizer is Ethanol. It is added to chemically change the shape of the dye molecule and trap it in the cell wall. Iodine is used in the Gram stain.

What is the purpose of Decolorizing in any differential stain?

Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell.

What is the decolorizing agent in acid fast stain?

Principle of Acid-Fast Stain

Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution.

What happens to the gram positive cell wall during decolorization?

What happens to the Gram-positive cell wall during decolorization? The decolorizing agent dehydrates the peptidoglycan..... Removing water from or dehydrating the peptidoglycan allows the decolorizing agent to shrink the spaces through which the crystal violet-iodine complexes might be able to pass.

What does alcohol do to Gram-negative bacteria?

Alcohols kill germs by destroying the cell membranes and denaturing proteins of bacterial cells. Because of this, gram-negative bacteria (e.g. E. coli and salmonella) are more susceptible to sanitizers, since they have a thin peptidoglycan cell wall surrounded by an outer membrane, which can be dissolved by alcohols.

What happens to the gram negative cell wall during decolorization?

What's Going On? The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye.

What happens if you don't Decolorize a gram stain?

Terms in this set (83) What happens when you decolorize for too long or not long enough? If you leave the decolorizer on too long: gram positive bacteria will come out pink. If you don't leave it on long enough: gram negative bacteria will come out purple.

How do you make a gram Decolorizer?

Gram Decolorizer Solution: Mix equal volumes of 95 % ethanol and acetone. Gram Safranin Solution: Dissolve 2.5 g of safranin O in 100 ml of 95 % ethanol to make a stock solution. Working solution is obtained by diluting one part of the stock solution with five parts of water.

Why 70% alcohol is used in Gram staining?

70% denatured alcohol penetrate the cell wall more completely which permeates the entire cell, coagulates all proteins, and therefore the microorganism dies. Extra water content slows evaporation, therefore increasing surface contact time and enhancing effectiveness.

Why acid alcohol is used for decolorization rather than ethyl alcohol?

Why is acid-alcohol rather than ethyl alcohol used as a decolorizing agent? The primary stain is more soluble in cellular waxes than acid-alcohol, so the stain is able to penetrate mycobacteria and not be washed away with the use of acid-alcohol.

Why do we use acid alcohol?

Acid alcohol is a differentiation reagent. It is used in various staining methods, most frequently in regressive hematoxylin eosin (HE) staining and provides excellent differentiation between nuclear and non-nuclear structures. Differentiation rinses dyes from cytoplasm while the nucleus remains stained.

What substance is responsible for the acid fastness of an organism?

Mycolic acid is the mechanism responsible for the acid-fast property. The walls of the bacteria produce this waxy substance called mycolic acid. This acid links with substances within the cell walls of the bacteria, making them resistant to decolorization with acid alcohol treatment.

What happens if you don't add iodine to a Gram stain?

If the iodine is not applied during the Gram stain, then gram positive cells will likely stain pink.

Why do we use 95 alcohol in Gram staining?

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone.

Why crystal violet is used in Gram staining?

Crystal violet (being violet) is taken first and safranin as a counterstain basically to bring the colour difference between G+ and G - . its not for thickness of peptidoglycan, the content of lipid is more important.