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Full Version: Beyond Gram Stain - Structural differences in bacterial cell wall
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Gram Stain is a differential staining technique used in routine microbiological laboratory procedures which was developed by Hans Gram. It's a preliminary method in bacterial identification which divides the bacteria into gram positive and gram negative depending on the retained colour of the smear. This technique makes use of two basic stains; Crystal violet and Safranin, a mordant (Iodine), and a decolourizing agent which is alcohol. However there are bacterial species which do not give a clear difference in the colour or which doesn’t stain at all and known as gram variable or acid fast bacteria. It’s also possible to differentiate gram positive and gram negative bacteria using Potassium Hydroxide (3%) in which gram negative bacteria form a mucoid string.

Basic dyes such as Crystal Violet, Saffranin consist of coloured positive ions called as chromophores. These chromophores get attracted to the bacterial cells which are negatively charged at pH 7. In this procedure, Crystal Violet stains the smear in purple colour and in the subsequent step Iodine is applied which intensifies the purple colour of the stain. Iodine increases the affinity of the stain to the cells and coats the cell structure. Crystal Violet forms a complex with Iodine known as CV-I complex, which is larger in size than Crystal Violet molecules. In the decolourizing step, gram positive bacteria can retain the colour of the primary stain. When Safranin, the counterstain; is applied gram negative bacteria which are decolourized stains in pink. However it’s the structure of the cell wall of bacteria which results in this differential staining.

In case of gram positive bacteria, the peptidoglycan layer is thicker when compared to gram negative bacteria. Peptidoglycan is made up of two monosaccharaides; N- acetylglucosamine (NAG) and N- acetylmuramic acid (NAM) linked in an alternating fashion to form a carbohydrate backbone. Alternating disaccharide chains are linked by polypeptide chains of which the composition is different in gram positive and gram negative bacteria. Amino acids in the tetrapeptide side chains attached to NAM and NAG units occur in D and L forms which is unique about proteins.

Thicker peptidoglycan layer in gram positive bacteria helps to retain the CV-I complex in staining. In contrast, gram negative bacteria have higher lipopolysaccharide content in their cell wall which disrupts in alcohol treatment. The lipopolysaccharide layer is composed of Lipid A, Core polysaccharide and O polysaccharide. The disruption of lipopolysaccharide layer creates pores in the cell wall and the cell wall permeability increases resulting in escape of CV-I molecules. So the decolourized cells, takes the colour of Safranin. Age of the bacterial culture is a key factor in determining the gram character. Old cultures may produce unacceptable results.

Apart from the difference in the peptidoglycan layer, there are many differences in gram positive and gram negative bacteria. Gram positive cell wall contains Techoic acids which are important for antigenic specificity. This is correlated with identification procedures. Gram negative bacteria lack Techoic acids. O polysaccharide in gram negative cell wall lipopolysaccharide layer is important to distinguish bacterial species. Possessing a periplasmic space is unique to gram negative bacteria. Because of these structural differences, gram character is important in identification of bacteria in laboratory procedures.

However, this technique is not widely accepted as a standard due to existence of gram variable and acid fast bacterial species. These include; Mycobacterium which are acid fast bacteria, some Actinomycetes strains. Gram variable results may also produce because of mistakes made during the staining procedure such as excessive heating of the smear, excessive decolourizing, old cultures, uneven smears etc.
Gram negative bacteria are bacteria are generally more resistant to antibiotics due to the presence of lipopolysaccharide layer. These include clinically important bacteria such as E. coli, Salmonella, and Shigella. Gram positive bacteria are more sensitive to antibiotics like Penicillins and Cephalosporins. Gram positive bacteria include Bacillus sp., Streptococcus sp., Staphylococcus sp. which are known as human pathogens.

Gram staining along with other biochemical tests such as catalase, oxidase, fermentation of different sugars, IMVIC tests is used for further bacterial identification. Gram character is also important in prescribing medicine for human bacterial diseases. Apart from gram staining, other staining techniques used in the laboratory include acid fast staining, endospore staining, flagella staining etc.