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The Gram stain procedure was developed in the 1880s by Christian Gram (Walker, 1998) and was then updated to provide a better differentiation of bacteria by Hucker in 1921(Murray, 2003). It is used widely in both the medical and food industries to help in the identification of bacteria by staining samples with dye. This colours the bacteria, enabling the size, shape and morphology to be clearly seen when it is examined under a microscope (Murray, 2003). It also helps to differentiate between the two main types of bacteria (Gram +ve e.g. Bacillus, and Gram -ve e.g. Pseudomonas) by the colour of dye that they take up and retain.
This essay will look at the Gram stain method and why and how it works. It will also compare and contrast the structure of the bacterial cell walls of Gram +ve and Gram -ve bacteria, explaining how the differing structures enable the method to produce the results it does; Gram +ve bacteria showing purple under a microscope whereas Gram -ve appear a pink colour.
Gram stain method
Step one: Apply a thin smear of the bacteria to a sterilised glass slide (if too much is used then the result will be hard to read).
Step two: Heat fix the bacteria to the glass slide by passing it over a Bunsen burner a
couple of times, then leave it to cool.
Step three: Flood the slide with 2% Crystal Violet dye and leave it for one minute. Then wash it thoroughly with sterile water.
Step four: Apply Gram's iodine solution and leave for one minute.
Step five: Wash the slide with acetone for a couple of seconds, then wash it again
with sterile water.
Step six: Flood the slide with the counter-stain Safranin 2.5%. Leave for 10-15
seconds, then wash it off with sterile water.
Step seven: Blot the slide gently (so as not to disturb or remove the bacteria) with a paper towel.
Step eight: Allow the slide to dry if not thoroughly dried by the blotting. Then add a drop of Immersion oil on to the stained bacteria.
(Jones et al, 2007)
The slide is now ready to be examined under the microscope.
The Gram +ve and Gram -ve cell walls have a few similarities and many differences. Both have a peptidoglycan layer consisting of n-acetylgylcosamine and n-acetylmuramic acids (Greenwood et al, 2007). In the Gram +ve bacterium this layer makes up 50-60% of the weight of the cell wall and is very rigid. This is because of a high concentration of cross-linking of the polysaccharide layers that make up the peptidoglycan layer. In the Gram +ve bacterium it is the outer part of the wall, whereas in the Gram -ve cell wall it is the inner part and only accounts for 5-10% of the weight of the cell wall (Boyd et al, 1991). In the Gram +ve bacteria this layer makes up one of only two layers (the other being the cytoplasmic membrane). The Gram -ve bacterium, however, has a three layered wall. These layers consist of an inner peptidoglycan layer and an outer membrane consisting of mainly lipolysaccharides with a space between them called the Periplasmic space which contains hydrolytic enzymes (Boyd et al, 1991; Walker, 1998).
The cell wall of the Gram -ve bacterium acts as a protective layer enabling it to resist the action of lysosomes and many antibiotics. Porin proteins located in the outer layer allow selective permeability and the attachment of certain molecules, therefore stopping molecules that could damage the cell from getting through (Greenwood et al, 2007). The next layer down, the periplasmic space, acts as a secondary barrier as it contains hydrolytic enzymes which can break down unwanted molecules that make it through the outer membrane. In comparison the Gram +ve bacterium's cell wall has little defence against such attacks as it doesn't contain that same outer membrane. It also allows the diffusion of metabolites in and out of the cell (Boyd et al, 1991), some of which could potentially damage it. Due to these reasons the Gram stain method can give the knowledge of whether an antibiotic is going to be of use in the control of a particular infection.
The other differences between the two types of bacteria are that the Gram +ve bacteria possess teichoic and lipoteichoic acids attached to the cell wall, whereas the Gram -ve do not (Boyd et al, 1991). However the Gram -ve have flagella and pilli which the gram +ve are lacking (Walker, 1998).
The Gram stain works by dying the cell's organelle and cell wall, enabling them to be clearly seen under a microscope. In the clinical treatment of many infections just knowing if the bacteria is Gram +ve or -ve is enough to effectively treat the problem. If the bacteria show a purple/blue colour it is a Gram +ve bacteria and therefore it is more likely that antibiotics such as Penicillin will effectively treat the infection.
The third step of the procedure allows Crystal Violet and iodine to enter the cell and form a complex (CV-I), dying the cell a purple/blue colour (Fankhauser, 2006). This is the colour that identifies the Gram +ve bacteria. It retains the colour as in the fifth step; the alcohol that is used to decolourise the cell dehydrates the cell wall due to the low lipid content. As the cell wall is dehydrated, the permeability is decreased so that the CV-I complex is trapped inside the cell making it appear purple, even after the attempted dying with the counter-stain Safranin in step six (Walker, 1998).
Gram -ve bacteria appear pink under the microscope; this is because unlike in the Gram +ve bacteria, the CV-I complex is able to leave the cell wall. In contrast to the Gram +ve cell wall, which is dehydrated and has its permeability reduced, the Gram -ve cell wall has its outer membrane washed away by the alcohol. This, in combination with the higher lipid content in the Gram -ve cell wall, means that it is not dehydrated but the permeability of the cell wall is actually increased. This increased permeability allows the CV-I complex to be washed out of the cell and the counter-stain Safranin to be the dominant colour of the cell wall and organelle when examining under the microscope (Walker, 1998).
There are a few things to take into account when identifying bacteria in this way. Gram +ve bacteria may appear Gram -ve if they are taken from an aging culture or if they have been exposed to antibiotics. This is due to a breaking down of the peptidoglycan layer which, as in the Gram -ve bacteria, increases the permeability of the cell wall allowing the CV-I complex to be washed away and the pink from the Safranin to be seen under the microscope. These are referred to as Gram-variable.
There are also bacteria that resist the Gram stain, either due to a waxy outer layer e.g. Mycobacteria, or the lack of a cell wall capable of retaining a dye e.g. Mycoplasma (Walker, 1998).
The Gram stain is an invaluable procedure in the medical industry. It can provide a one test way of identifying if an infection can be treated by certain antibiotics, enabling fast treatment of the infection. If more identification is needed it still provides a good starting point for deciding upon other tests. In the food industries the Gram stain is used regularly, often being the only test needed to identify a contamination in ingredients, finished products or even in the factory environment itself. It is essential however that samples are collected aseptically and used within the shortest time scale possible. Otherwise it is possible that the identification of the bacteria could prove false, either due to contamination of the sample, or the age of the sample giving a Gram -ve result due to the peptidoglycan layer breaking down. This could lead to the problem not being rectified or even exacerbated.