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Information about Staining1

Published on September 5, 2010

Author: aditya0291


Staining : Staining Increase contrast of microorganisms Types of staining Simple staining Differential staining Structural or special staining Stain : Stain - A stain is a substance that adheres to a cell, giving the cell color. - The presence of color gives the cells significant contrast so are much more visible. - Different stains have different affinities for different organisms, or different parts of organisms - They are used to differentiate different types of organisms or to view specific parts of organisms Dyes : Dyes Organic salts with positive and negative charges One ion is colored -chromophore Basic dye: positive ion is colored MeBlue+ Cl- Acidic dye: negative ion is chromphore Basic Dye : Basic Dye Works best in neutral or alkaline pH Bacterial cell wall has slight negative charge at pH 7 Basic dye (positive) attracted to cell wall ( negative) Crystal violet, methylene blue, safranin Acidic Dye : Acidic Dye Chromophore repelled by negative cell wall Background is stained, bacteria are colorless Negative stain-look at size, shape Acidic dye will stain bacteria if grown at lower pH Eosin, India ink Simple Staining : Simple Staining Simple easy to use One dye, one step Direct stain using basic dye Negative stain using acidic dye Features of dyes: give coloring of microorganisms; bind specifically to various cell structures. Simple Staining : Simple Staining Principle: The surface of a bacterial cell has an overall acidic characteristic because of large amount of carboxyl groups located on the cell surface due to acidic amino acids. Therefore, when ionization of carboxyl groups takes place it imparts negative charge to the cell surface as per the following equation. COOH → COO- + H+ H+ is removed and the surface of the bacteria becomes  negatively charged and a positively charged dye like (methylene blue) attaches to the negatively surface and gives it a coloured appearance. Methylene blue chloride →  Methylene Blue++ Cl‑ Differential Staining : Differential Staining To distinguish different kinds of bacteria into separate groups based on staining properties. More than one dye Principle: Differentiation is due to the different chemical and physical properties of cell and as a result, they react differently with the staining reagents. Differential staining procedure utilizes more than one stain. In some techniques the stains are applied separately, while in other as combination. Fixation : Fixation -The purpose of fixation is to make the stained cells resemble living cells as closely as possible. - Structures of microorganisms are preserved. - Inactivates Enzymes that disrupt cell morphology and toughens cell structures. - Fixation usually results in the death of the attached microorganisms. Chemical fixation: Preserves fine substructures and morphology Fixative chemicals penetrate cells and react with proteins and lipids; make them inactive, insoluble and immobile. Heat fixation: gentle heat of air-dried film of bacteria; preserves morphology not structure. GRAM STAIN : GRAM STAIN A method of differentiating bacteria by staining and brightfield microscopy into two broad, phylogenetically relevant groups: gram-negative bacteria and gram-positive bacteria. Gram Stain : Gram Stain Principle: - Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of ellwall),whichstains purple - while gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane which contains lipids. Gram Stain : Gram Stain Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color the cell. Slide 14: When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell will lose its outer membrane and the lipopolysaccharide layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from by alchohol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red color. Gram-positive cell walls Gram-negative cell walls : Thick peptidoglycan 90% peptidoglycan Teichoic acids 1 layer Not many polysaccharides In acid-fast cells, contains mycolic acid Gram-positive cell walls Gram-negative cell walls Thin peptidoglycan 5-10% peptidoglycan No teichoic acids 3 layers Outer membrane has lipids, polysaccharides No acid- fast cells (mycolic acid) The characteristic compound found in all true bacterial cell walls is peptidoglycan. The amount of PPG is among one of the differences between the GP and GN cell walls. Slide 17: Figure 4.13b, c Slide 18: The process includes the use of: a primary stain (crystal violet) a mordant (helper) iodine solution, a decolorizer (95% ethanol), a counterstain (safranin). Slide 19: The Gram stain Thin smear/heat fix Gram stain: a. Flood slide with crystal violet and let stain for 1 minute.   b. Drain off crystal violet and rinse off with distilled water; flood slide with Gram's iodine for 1 minute.   c. Rinse off Gram's iodine with distilled water.   d. Hold the slide on an angle (preferably with a clothes pin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no longer has a purple tint; be sure to drop the alcohol onto the upper portion of the slide so that the smears are subjected to uniform decolorization. Be careful not to "decolorize" dye from the clothes pin!!   e. Rinse with distilled water and flood the slide with safranin and let stain for 2-3 minutes.   f. Rinse with distilled water and blot dry with bibulous paper. Gram positive Gram negative Slide 20: Demos: Gram stained slides of Neisseria, Streptococcus, Pseudomonas, Actinomyces species. Neisseria Pseudomonas Streptococcus Differential Stains: Acid-Fast Stain : Differential Stains: Acid-Fast Stain Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them. Figure 3.12 Acid-fast Stain : Acid-fast Stain Principle:Acid fast staining is another widely used differential stain­ing procedure in bacteriology. This stain was developed by Paul Ehrlich in 1882. Some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms. Acid alcohol is very intensive decolourizer. This staining technique divides bacteria into two groups (i) acid-fast and (ii) non acid-fast. This procedure is extensively used in the diagnosis of tuberculosis and leprosy.Acid-fastness property in certain Mycobacteria and some species of Nocardia is correlated with their high lipid content. Due to high lipid content of cell wall, in some cases 60% (w/w), acid-fast cells have relatively low permeability to dye and hence it is difficult to stain them. For the staining of these bacteria, penetration of primary dye is facilitated with the use of 5% aqueous phenol which acts as a chemical intensifier. In addition, heat is also applied which acts as a physical intensifier. Once these cells are stained, it is difficult to decolourize - Acid-fast Stain : Acid-fast Stain The waxy cell envelopes of these bacteria prevents the penetration of most stains - They do not bind easily to simple stains; must be stained by harsher methods - Heat with a mixture of fuchsin and phenol: Acid-fast cells: not decolorized with acid-alcohol wash => Red Non Acid-fast cells: decolorized with acid-alcohol; with Methylene blue counterstain => blue - Method used to identify M. tuberculosis and M. leprae Special Stains : Special Stains Negative staining is useful for capsules. Heat is required to drive a stain into endospores. Flagella staining requires a mordant to make the flagella wide enough to see. Figure 3.13a–c

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