What is the purpose of using oil in Gram staining?

In light microscopy, oil immersion is a technique used to increase the resolving power of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

How will a Gram stain of a gram negative bacteria appear under oil immersion?

This requires oil immersion to obtain a clear imaging of material on the slide. Gram positive cells will appear dark purple and Gram negative will appear red or pink. If done properly, Gram positive baceria remain purple at this stage, while Gram negative bacteria become colorless.

What is the purpose of each stain in Gram staining?

What is Gram Staining? Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet.

What is used as decolorizing agent in Gram’s staining?

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.

What is the purpose of the oil in the oil immersion objective?

Immersion oil increases the resolving power of the microscope by replacing the air gap between the immersion objective lens and cover glass with a high refractive index medium and reducing light refraction.

Why is oil used in oil immersion objective?

It is best to use an oil-immersed objective at high magnification as the oil compensates for short focal lengths associated with larger magnifications. The oil has a similar refractive value to the glass slides and slipcovers.

What is the importance of Gram staining in microbiology?

The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between gram positive organisms and gram negative organisms. Hence, it is a differential stain. Gram negative and gram positive organisms are distinguished from each other by differences in their cell walls.

Why do certain bacteria stain differently when stained with Gram stain?

Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis.

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 dead cells in a sample.

Why is Gram iodine used in Gram staining?

The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.

What is the purpose of crystal violet in Gram staining?

The gram stain utilizes crystal violet as the primary stain. This basic dye is positively charged and, therefore, adheres to the cell membranes of both gram negative and positive cells. After applying crystal violet and waiting 60 seconds the excess stain is rinsed off with water.

What is the purpose of using immersion oil with the 100x objective?

Oil immersion is the technique of using a drop of oil to wet the top of the specimen or slide cover and the front of the objective lens. This effectively immerses or bathes the light path between the lens and object viewed, allowing finer details to be seen.

What does a Gram stain tell you?

Use of the gram stain in microbiology The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be “gram-positive,” whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be “gram-negative.”

How do you remove the secondary stain from Gram staining?

Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope.

How do you stain Gram positive bacteria with safranin?

Add the secondary stain, safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope.

How are the categories of Gram staining used to classify bacteria?

The categories are diagnosed based on the how the bacteria reacts to the Gram stain. A Gram stain is colored purple. When the stain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red. If the bacteria stays purple, they are Gram-positive. If the bacteria turns pink or red, they are Gram-negative.