Introduction to Lab Ex. Differential Stains Gram Staining
Introduction to Lab Ex. Differential Stains Gram Staining Introduction to Lab Ex. Differential Stains Gram Staining Basic classification of bacteria is based on the cell wall structure. There are 2 main groups: Gram positive and Gram negative. Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. The staining technique, developed in the late 1700s by Christian Gram classifies the rigid cell walled bacteria into one of two groups based on whether they are able to resist the decolorizing action of an alcoholic solution. Those that resist decolorization by 95% ethanol are arbitrarily termed Gram positive and those that do not are Gram negative
(the terms positive and negative have nothing to do with charges of the cell but based on differences in the cell wall structure of these two groups of bacteria). 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. Gram-positive cell walls walls
Thick peptidoglycan 90% peptidoglycan Teichoic acids 1 layer Not many polysaccharides In acid-fast cells, contains mycolic acid Gram-negative cell
Thin peptidoglycan 5-10% peptidoglycan No teichoic acids 3 layers Outer membrane has lipids, polysaccharides No acid- fast cells (mycolic acid) Figure 4.13b, c The process includes the use of: a primary stain (crystal violet) a mordant (helper) iodine solution,
a decolorizer (95% ethanol), a counterstain (safranin). 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!!
Gram positive Gram negativ The crucial step in the staining process is the decolorizing step. The most accepted theory about the rationale for the Gram staining process is the one proposed by Salton. This theory relies on the fact that the PPG is found in layers and the stain molecules are trapped within the many layers of the GP CW when they form the complex with the mordant Iodine molecules. Since the GN CWs lack much PPG the amount of stain captured in those CWs is much lesser.
When the cells are treated with the decolorizer the ethanol this causes denaturation of the proteins in the outer membrane of the GN CWs resulting in gaping holes in these CWs that lead to the removal of the crystal violet-iodine complexes easily, leaving these cells unstained. The counterstain -safranin- thus is used to make these cells visible. There are 4 conditions to be followed for a valid Gram staining procedure: Young cultures - must be young within 18-24hrs old (older cultures lose their Gram staining properties due to changes in the CWs as the cells get older) Thin smear thicker or uneven smears will result in uneven staining and decolorization
Fresh reagents - of proper strength Control cultures - for a known GP bacterium and GN culture (S.aureus & E.coli) Demos: Gram stained slides of Neisseria, Streptococcus, Pseudomonas, Actinomyces species. Pseudomonas Neisseria Streptococcus
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