Determining Bacteria – Introducing the G Stain

Organisms found in pharmaceutical and healthcare environments require identification in order to determine the species. This is important so that the origin associated with contamination can be assessed and the foundation of contamination determined. This is typically performed by using a standing technique the Gram stain, which is based is really a type of “phenotypic identification method” plus it undertaken so that the microbiologist can understand the general profile for microorganisms.

The initial step of most identification schemes is to describe the colony and cellular morphology of the microorganism. Colony morphology is normally described by directly observing growth on agar, where the colony can look as a particular shape (such since raised, crenated, spherical and so on) and the colony will have a particular color. Some microbiologists will attempt to recognize the microorganism based on such visual identification. This is not normally encouraged because considerable experience is required to do this as well as the variety of microflora cannot be characterised with any degree of accuracy. Furthermore, the functions of a microorganism are often dependent upon the type of culture medium used. Nevertheless, a description of the morphology can assist with further stages of identification.

Mobile staining provides important information relating to the composition of the microbial cell walls, as well as the shape of the organism. Of those, the most frequently used method is the G stain.
The Gram stain technique employed includes the four-step method: Crystal violet (primary stain); iodine (mordant); alcohol (decolorizer); and safranin (counter stain).
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Done correctly, Gram-positive organisms retain the crystal violet spot and appear blue; Gram negative organisms lose the crystal violet spot and contain only the counter-stain safranin and thus appear red. Common stumbling blocks in this method are that high temperature fixation may cause Gram-positive cells to stain Gram-negative and older cultures may give Gram-variable reaction; using a lot of 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 spot (the Gram-positive bacteria) have a higher peptidoglycan and lower lipid articles than those that do not retain the spot (the Gram-negative bacteria). The effect from 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 particular stain. There are now several automated Gram stain devices available on the market that can reduce the labour requirement required when executing several multiple Gram stains plus, possibly, improve accuracy.

In addition to the distinction based on cell wall, microscopic study of the stains allows the mobile shape to be determined. Bacteria typically fall into the categories of coccus (spherical), rod, vibrio (curved), spirilla (spiral) and plemomorphic (variable).

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