Compilation of Microbiology Staining Q&a

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Introduction
Identification and classification of bacteria is important to make easier manipulation on the bacteria for various purposes such as for medical, research, developmental, and biotechnicalfieldsThe cell wall is the basis for classification of bacteria according to the Gram stain. Accordingto the chemical make up of bacterial cell wall, a staining procedure, Gram stain, helps usclassify bacteria into two subgroups, The cell wall can stain either positive or negative,depending on its chemistry. If the bacteria stains positive it will retain a purple/blue color. If the bacteria stains negative, the bacteria will not retain the purple/blue color, but rather have a pinkish/red color.Gram-positive bacteria have a thick layer of peptidoglycan external to the cytoplasmicmembrane. In contrast, Gram-negative bacteria have a thin layer of peptidoglycan located between the cytoplasmic membrane and a second membrane called the outer membrane. Thisregion is known as the periplasmic space. Figure 1 shows schematic representation of twotypes of bacterial cell wall structures. Other important constituents of the cell wall include thefollowing: Peptidoglycan:

This is a polymer of alternating N -acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). Long strands of this alternating polymer may be linked by L-alanine, D-glutamic acid, L-lysine, D-alanine tetrapeptides to NAM . Gram-positive cellshave a much more highly cross-linked peptidoglycan structure than Gram-negative cells.Peptidoglycan is also the "target" of antimicrobial activity. For example, penicillins interferewith the enzymes involved in biosynthesis of peptidoglycan while lysozyme physicallycleaves the NAM-NAG bond. Lipoteichoic acids:

Lipoteichoic acids (LTA) are found only in Gram-positive bacteria.These polysaccharides extend though the entire peptidoglycan layer and appear on the cellsurface. As a consequence, these structures can serve as antigenic determinants. Lipopolysaccharides:

Lipopolysaccharides (LPS) are found only in Gram-negative bacteria.These structures are composed of lipid A, which binds the LPS in the outer membrane and isitself the endotoxic portion of the molecule. The polysaccharide moiety appears on the cellsurface, serving as an antigenic determinant Periplasmic space:

The region between the peptidoglycan and LPS layers is termed the periplasmic space (coloured grey in the figure); it is a fluid or gel-like zone containing manyenzymes and nutrient-carrier proteins. Figure 1

Cell wall structures of Gram positive and Gram negative bacteriaThe Gram staining method, named after the Danish bacteriologist who originally devised it in1882 (published 1884), Hans Christian Gram, is one of the most important staining techniquesin microbiology. It is almost always the first test performed for the identification of bacteriaGram stain includes several staining and negative staining steps. The primary stain of theGram's method is crystal violet. Gram staining is based on the ability of bacteria cell wall toretaining the crystal violet dye during solvent treatment. The cell walls for Gram-positivemicroorganisms 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 removedeasily. This step is commonly 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. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. Gram-positive cells retain...
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