Friday, 28 March 2014

What Is The Gram stain?

What Is The Gram stain?

Tim Sandle
By Tim Sandle
The primary staining technique used to differentiate bacteria is the Gram stain. The Gram stain method employed includes the four-step technique: Crystal violet (primary stain); iodine (mordant); alcohol (decolorizer); safranin (counter stain) or the three-step method in which the decolorization and counter-staining step are combined. Done correctly, Gram-positive organisms retain the crystal violet stain and appear blue; Gram negative organisms lose the crystal violet stain and contain only the counter-stain safranin and thus appear red. Common pitfalls in this method are that heat fixation may cause Gram-positive cells to stain Gram-negative and older cultures may give Gram-variable reaction; using too much decolorizer could result in a false Gram-negative result and not using enough decolorizer may yield a false Gram-positive result.
The Gram reaction is based on the differences in the cell wall composition for the two cellular ‘groups'. The bacteria that retained the stain (the Gram-positive bacteria) have a higher peptidoglycan and lower lipid content than those that do not retain the stain (the Gram-negative bacteria). The effect of the solvent is to dissolve the lipid layer in the cell wall of the Gram-negative bacteria, thereby causing the crystal violet to leach out; whereas for Gram-positive bacteria the solvent dehydrates the thicker cell walls, blocking any diffusion of the violet-iodine complex, which closes the pores of the cell and retains the stain. There are now several automated Gram stain devices available on the market that can reduce the labour requirement required when performing several multiple Gram stains and, possibly, improve accuracy.
In addition to the difference based on cell wall, microscopic examination of the stains allows the cellular shape to be determined. Bacteria commonly fall into the categories of coccus (spherical), rod, vibrio (curved), spirilla (spiral) and plemomorphic (variable).
Tim Sandle, Ph.D, M.A., BSc (Hons), CBiol, MSBiol., MIScT – Dr. Sandle is the Head of Microbiology at the UK Bio Products Laboratory. Dr. Sandle is a chartered biologist and holds a first class honors degree in Applied Biology; a Masters degree in education; and obtained his doctorate from Keele University. His role involves overseeing a range of microbiological tests, batch review, microbiological investigation and policy development. In addition, he is an honorary consultant with the School of Pharmacy and Pharmaceutical Sciences, University of Manchester and is a tutor for the university's pharmaceutical microbiology M.Sc course. Dr. Sandle serves on several national and international committees relating to pharmaceutical microbiology and cleanroom contamination control (including the ISO cleanroom standards). He is currently chairman of the PharMIG LAL action group and serves on the NBS cleaning and disinfection committee. He has written over eighty book chapters, peer reviewed papers and technical articles relating to microbiology. He is currently the editor of the Pharmaceutical Microbiology Interest Group Journal and runs an on-line microbiology forum (www.pharmig.blogspot.com). Dr. Sandle is an experienced auditor and frequently acts as a consultant to the pharmaceutical and healthcare sectors.

Posted by Tim Sandle