Biophysics Lab

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PSBPP103

Differential Gram’s staining
EXPERIMENT NO. 1 AIM: THEORY:

Page No: C1 Date:

To Gram stain the given bacterial suspension and to differentiate between gram positive and gram negative organism. Visualization of microorganisms in the living state is very difficult, not just because they are minute, but because they are transparent and almost colorless when suspended in an aqueous medium. To study their properties and divide microorganisms into specific groups for diagnostic purposes, biological stains and staining procedures, in conjunction with light microscopy, have become major tools in microbiology. Chemically, a stain may be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome. Stains are of 2 types: 1. Acidic stains e.g., picric acid 2. Basic stains e.g., methylene blue. Types of staining techniques: 1. Simple staining. (Use of a single stain)This type of staining is used for visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads). 2. Differential staining. (Use of 2 contrasting stains)It is divided into two groups: (a) Separation into groups, Gram stain and acid-fast stain. (b) Visualization of structures, Flagella stain, capsule stain, spore stain, nuclear stain. The Gram Stain The Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple with the Gram-staining procedure are termed Gram-positive; those that stain pink are said to be Gram-negative. The terms positive and negative have nothing to do with electrical charge, but simply designate 2 distinct morphological groups of bacteria. Grampositive 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. The material in the bacterial cell wall that confers rigidity is peptidoglycan.

PSBPP103

Page No: C2

In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20–80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Chemically, 60% to 90% of the Gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of Grampositive are teichoic acids. Teichoic acids that extend through and beyond the rest of the cell wall are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium. The Gram-negative cell wall, on the other hand, contains only 2–3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10%–20% of the Gram-negative cell wall is peptidoglycan. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A: embedded in the membrane, and a polysaccharide portion extending outward from the bacterial surface. The outer membrane also contains a number of proteins that differ with the strain and species of the bacterium.

PRINCIPLE
The Gram-staining procedure involves 4 basic steps: 1. The bacteria are first stained with the basic dye crystal violet. Both Gram-positive and Gramnegative bacteria become directly stained and appear purple after this step. 2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gramnegative bacteria remain purple after this step....
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