4th November 2019

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Why do Gram positive and Gram negative bacteria stain differently?

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Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.

In respect to this, what is the purpose of a differential stain?

Differential Staining is a staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.

Is the Endospore stain a differential stain?

The endospore stain is a differential stain used to visualize bacterial endospores. Endospores are formed by a few genera of bacteria, such as Bacillus . By forming spores, bacteria can survive in hostile conditions. Spores are resistant to heat, dessication, chemicals, and radiation.

Why is Gram staining important for classifying bacteria?

Importance of a Gram Stain: The Gram stain is a very important preliminary step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope.

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Why Gram negative bacteria are resistant to antibiotics?

Although all bacteria have an inner cell membrane, gram-negative bacteria have a unique outer membrane. This outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram-negative bacteria are generally more resistant to antibiotics than are gram-positive bacteria.

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What color does safranin stain?

A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red.

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Why does gram negative bacteria stain pink?

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.

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Why Gram stain is not used for acid fast bacteria?

They cannot be stained by the Gram stain because of their high lipid content. Acid fast staining is used to stain mycobacteria. Bacteria are treated with a red dye (fuchsin) and steamed. (This drives the stain into the cell and because of the lipid, they become difficult to destain.)

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Are bacteria Gram positive or negative?

In contrast to Gram-positive bacteria, all archetypical Gram-negative bacteria are bounded by a cytoplasmic membrane and an outer cell membrane; they contain only a thin layer of peptidoglycan (2–3 nm) between these membranes.

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Why is Gram staining important for classifying bacteria?

Importance of a Gram Stain: The Gram stain is a very important preliminary step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope.

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Why is the Gram stain so important?

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 positive cells take up the crystal violet, which is then fixed in the cell with the iodine mordant.

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What is the purpose of a gram stain?

Purpose: is to determine the Gram stain of your bacterial sample. Introduction: Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. The cell wall can stain either positive or negative, depending on its chemistry.

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What is the mordant used in Gram staining?

The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily.

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What does it mean to be Gram positive or negative?

This stain will either stain the cells purple (for positive) or pink (for negative). Gram-positive bacteria have a very thick cell wall made of a protein called peptidoglycan. These bacteria retain the crystal violet dye (one of the 2 main chemicals used for gram staining).

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What is the purpose of a differential stain?

Differential Staining is a staining process which uses more than one chemical stain. Gram staining uses two dyes: Crystal violet and Fuchsin or Safranin (the counterstain) to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria.

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What is the Counterstain in the Gram stain?

Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining technique and the eosin counterstain to haematoxylin in the H&E stain.In Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, allowing the

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Can the iodine be added before the primary stain in a Gram stain?

Can iodine be added before the primary stain in a Gram stain? No because the iodine allows the crystal violet stain to bind to the peptidoglycan in cell walls.

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Why are most staining procedures not useful for living cells?

Cell staining is a technique that can be used to better visualize cells and cell components under a microscope. By using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell.

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What is the purpose of the iodine in the Gram stain?

What is the purpose of iodine in the Gram's stain procedure? It forms an insoluble crystal violet iodine complex which allows the stain to be retained better. E. What is a mordant? It is a chemical that can deepen the reaction of another dye.

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Which organisms stain reddish pink?

Gram-positive bacteria appear dark blue or violet due to the crystal violet stain following the Gram stain procedure; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain (usually safranin).

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What is a gram negative organism?

Example species. The proteobacteria are a major phylum of gram-negative bacteria, including Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc.

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How is Gram staining done?

Place slide with heat fixed smear on staining tray.
Gently flood smear with crystal violet and let stand for 1 minute.
Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
Gently flood the smear with Gram's iodine and let stand for 1 minute.

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What is the most crucial step in the performance of the Gram staining procedures?

The most critical phase of the Gram stain procedure is the decolorization step, which is based on the ease with which the crystal violet-iodine complex is released from the cell. Over-decolorizing will result in the loss of the primary stain, causing Gram positive bacteria to appear Gram negative.

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When would you use a negative stain?

The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed. Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion and becomes negatively charged.

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