How does Gram staining identify bacteria?

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. … This gives rise to Gram-variable and Gram-indeterminate groups.

Why does gram positive stain purple?

gram stain test

Gram-positive bacteria remain purple because they have a single thick cell wall that is not easily penetrated by the solvent; gram-negative bacteria, however, are decolorized because they have cell walls with much thinner layers that allow removal of the dye by the solvent.

What is the main purpose of Gram staining?

A Gram stain is most often used to find out if you have a bacterial infection. If you do, the test will show if your infection is Gram-positive or Gram-negative. A Gram stain may also be used to diagnose fungal infections.

What does an Endospore stain tell you?

Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them highly resistant to chemicals.

What is difference between gram positive and negative?

Gram positive bacteria have a thick peptidoglycan layer and no outer lipid membrane whilst Gram negative bacteria have a thin peptidoglycan layer and have an outer lipid membrane.

What is the principle behind the Gram stain reaction?

The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.

What happens if mordant step is skipped?

Mordant step skipped: Gram-positive bacteria will appear Gram-negative. … Slide not decolorized: Gram-negative bacteria will appear Gram-positive. 5.

What is the importance of stain in microbiology?

The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and deceased cells in a sample.

What is the most critical step in the process of Gram staining?

The thickness of the smear used in the Gram stain will affect the result of the stain. The step that is most crucial in effecting the outcome of the stain is the decolorizing step.

What is the most important reagent in the Gram stain method?

crystal violet
The primary stain of the Gram’s method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. The microorganisms that retain the crystal violet-iodine complex appear purple brown under microscopic examination.

What is the function of each one of the Gram stain reagents?

The first reagent is called the primary stain. Its function is to impart its color to all cells. The second stain is a mordant used to in- tensify the color of the primary stain. In order to es- tablish a color contrast, the third reagent used is the decolorizing agent.

Why do we add alcohol in Gram staining?

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.

Why do we add iodine in Gram staining?

Add iodine (Gram’s iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test. … The counterstain stains both gram-negative and gram-positive cells.

What mordant is used in Gram staining?

Iodine
The mordant is Gram’s Iodine. This binds to the crystal violet making a large complex that adheres to the cell membrane.

Why 95% ethanol is used in Gram stain?

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 happens if you skip the Decolorizer in a Gram stain?

During the decolorization step, the gram positive bacteria become dehydrated, trapping the crystal violet-iodine complexes within the multi-layered peptoglycan wall. If you skip this step, gram negative bacteria retain the purple dye. If you decolorize properly, the saffrin dye will dye the gram negative bacteria pink.

Why is acetone used in Gram staining?

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.

Why is the decolorization important in Gram staining?

The Gram stain is the most important staining procedure in microbiology. … This layer makes up 60-90% of the gram positive cell wall. 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 function of 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.

Why do we use positive and negative controls in the Gram stain?

The positive and negative controls are bacterial smears we use to test if the Gram stain was performed properly. If the positive and negative controls are not as expected, you can not trust that the stain was performed properly. As a Positive Control we use a known Gram-positive bacteria.