![]() ![]() Gram stain is the most important and widely used microbiological differential stain, published by Hans Christian Gram in 1884, Gram-positive and Gram-negative bacteria ◉ What is a Gram stain ? Perform the Stain procedure as stated below.Fr Gram Staining : Principle, Procedure and Results.Heat-fix the slide by holding the slide at one end with your fingers and quickly moving it back and forth a few times over the flame.Let the slide air-dry totally before proceeding on with the procedure.Place a piece of tape on the side of the smear so you know which way is UP.You might think about marking the smear area with a surrounding wax pencil mark so that you can find the smear under the microscope easily. ![]() Spread the suspension on the slide so that it covers an area at least the size of a nickel, preferably a quarter.If taken from a solid agar medium (plate or slant), suspend the inoculum in a drop of water on the slide and mix it well.If taken from a broth, use 1-2 loopfuls of the broth solution.Make the bacterial smear from broth, slant, or plate.There are also prepared, gram stained slides of bacteria of different shapes and sizes of bacteria to look at. You can use 2 slides, 1 for each bacterium, or you can divide one slide in half and smear each bacterium on the divided slide. This will give you gram + and gram – controls to check your procedure against. Using old cultures ( preferably, the cultures should be 18-48 hours old).Over decolorizing the smear, too long a time.Some bacterial species tend towards gram variable, and will show both colors although most often gram +.The most common reasons for false gram reactions? 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. 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.Īlthough 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 acetone-alcohol actually causes the peptidoglycan molecules (arranged in a latticework) to shrink, thereby holding the crystal violetiodine even tighter. During the crystal violet-iodine step, the bound molecules within the peptidoglycan of the gram + cell wall and within the membrane are held tightly. Take a look at the accompanying diagram of the stain procedure and its effects on the bacterial color. Both gram + and – bind to the crystal violet: the key step to their differentiation is the decolorization. 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. Because of the 2 dyes used in the procedure–crystal violet and safrinin-as well as the decolorizer acetone-alcohol, bacteria will fall into 2 groups based on their gram reactivity. As a result of the use of 2 dyes, making this procedure a differential stain, bacteria will either become purple/blue or pink during the procedure.īefore staining, the specimen must be mounted and fixed on the slides, as previously done in the simple staining technique. The Gram stain is a differential stain, as opposed to the simple stain which uses 1 dye. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. It has to be one of the most repeated procedures done in any lab. The gram stain, originally developed in 1884 by Christian Gram, is probably the most important procedure in all of microbiology. ![]()
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