Hi Very much appreciated Would be great if u also add some points about the modifications and variations and also gram variable bacteria and some general info about why gram staining is needed and where it is not indicated!! Because in medicine what accompanies practicals are viva and these questions can be very important Cheers Gud luck. Greatly impressed by this article. Painstakingly put together in a simple language. All enterobcteriaceae are gram negative bacteria but not all gram negative are enterobcteriaceae to distinguish the enterobcteriaceae you have to do oxidase test because enterobcteriaceae are oxidase negative bacteria.
But if i may ask, which step in Gram staining tech can be omited without affecting the final result? Mr Len artifacts results from poor methods of rinsing of slides. Beside, what is the color of safranin? More of a controlled decolorization. If Gram positive organisms have such complex cell wall that could defy decolorisation,why then is a mordant used?
Moreover, the test procedure is performed to differentiate between the two groups of bacteria. Save my name and email in this browser for the next time I comment. Principle of Gram Staining When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol.
Show Caption Hide Anthrax gram stain. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet.
Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. The Gram stain is a differential stain, as opposed to the simple stain which uses 1 dye.
Before staining, the specimen must be mounted and fixed on the slides, as previously done in the simple staining technique. Because of the 2 dyes used in the procedure--crystal violet and safrininas well as the decolorizer acetone-alcohol, bacteria will fall into 2 groups based on their gram reactivity. Gram positive bacteria retain the crystal violet even through the decolorizor step: gram negative bacteria do not retain the crystal violet, are decolorized, and then pick up the safrinin dye.
Take a look at the accompanying diagram of the stain procedure and its effects on the bacterial color. The acetone-alcohol actually causes the peptidoglycan molecules arranged in a latticework to shrink, thereby holding the crystal violetiodine even tighter. In the gram — cell, the outer lipopolysaccharide layer of the wall is dissolved by the decolorizer agents, and because the peptidoglycan layer is so thin in that group of bacteria, the crystal violet is leached out of the wall.
Although there is a standard routine and set reagents used in this stain, each person has to find a particular method that works best for them.
The many variables that can affect this stain are age of the culture, amount of decolorizer used, the time of decolorization, the type of organism acid-fast bacteria and spores do not stain well , thickness of the smear, and the general care of the stainer. Objectives Become proficient at performing the gram stain consistently and accurately. Differentiate among various shapes, sizes, arrangements, and gram reactions of bacteria. Note Make sure that your smear is not too thick: otherwise, either the dyes cannot penetrate or the cells will not be decolorized adequately.
Be sure that you properly time the decolorization step.
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