The first 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 will appear as a particular shape (such as raised, crenated, spherical and so on) and the colony will have a particular pigment. Some microbiologists will attempt to identify the microorganism based on such visual identification. This is not normally encouraged as considerable experience is required to do this and the variety of microflora cannot be characterised with any degree of accuracy. Furthermore, the characteristics 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.
Cellular staining provides important information relating to the composition of the microbial cell wall, as well as the shape of the organism. Of these, the most frequently used method is the Gram stain.
The Gram stain method employed includes the four-step technique: Crystal violet (primary stain); iodine (mordant); alcohol (decolorizer); and safranin (counter stain). 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).
Posted by Tim Sandle