How are bacteria identified?

Since bacteria are smaller than anything that can be seen by the human eye, identifying the genus and species of bacteria can be difficult.

For identifying bacteria outside the body, two common chemical techniques are as follows.

Gram Staining.

This technique is named after a pioneering Danish microbiologist. It works as follows.

1. The bacteria are stained with a purple dye (Crystal Violet). Most bacteria are then stained red.
2. The bacteria are stained again with Potassium Iodide.
3. The bacteria are then washed with alcohol. Those bacteria that retain the dye after washing are known as Gram Positive bacteria. Those that lose the dye are known as Gram Negative bacteria.
4. The bacteria are further stained with a pink dye (Safranin). Gram Negative bacteria will go pink after this dyeing, whereas Gram Positive bacteria will remain purple, from the original purple dye.

Ziehl-Neelsen Staining.

This technique is also referred to as acid-fast staining. This technique is necessary because some bacteria, notably all Mycobacteria, have waxy coats on their cell walls that prevent them taking in the dye from the Gram Staining procedure. As a part of the acid-fast staining process, detergents are applied which remove this waxy coat.

1. The bacteria are stained with hot Carbol-Fuchsin, a red dye which contains detergents. All bacteria are then stained red.
2. The bacteria are washed with acid alcohol. Those bacteria that retain the red dye are known as acid-fast bacteria.
3. The bacteria are then stained with Methylene Blue, a blue dye. Those bacteria that retain the red dye from the original stain are known as acid-fast bacteria, all others go blue.

All Mycobacteria test positive using the Ziehl-Neelsen acid-fast stain test, i.e. all species of Mycobacteria stain red using this procedure. Thus, this procedure cannot be used to differentiate between species of Mycobacteria.

Genetic testing.

Staining techniques, as described above, are useful for identifying the genus of a bacterium, but not for identifying the species of a bacterium, within a genus. Differentiation of a mycobacterium from a streptococcus can be done with stain testing, but to differentiate a Mycobacterium tuberculosis from a Mycobacterium leprae requires much more specific testing.

Recent advances in genetic technology enable highly specific testing for bacteria. What is required is that a segment of the DNA structure of the bacterium be identified. These segments are referred to as "insertion sequences", and consist of a sequence of proteins that makes up the DNA of the bacterium. Although the DNA structure of Mycobacterium tuberculosis will have a lot in common with the DNA structure of Mycobacterium paratuberculosis, there will be some parts of the DNA structure of each organism that is unique to that organism.

To identify the bacterium by testing, its DNA is "multiplied", using the Polymerase Chain Reaction. This technique creates large numbers of copies of the DNA to be tested. This delivers enough DNA to be tested for the presence of the "insertion sequence". This presence can be tested by techniques such as "Nucleic Acid Hybridization" and "Autoradiography". I will not attempt to describe these techniques here.

The IS900 insertion sequence.

Recently discovered "insertion sequences" that are important are the IS900 and IS902 sequences. The IS900 sequence is unique to Mycobacterium paratuberculosis. The IS902 sequence is unique to Mycobacterium avium subspecies silvaticum. Even though Mycobacterium paratuberculosis and Mycobacterium avium are very closely related, IS900 will test positive with Mycobacterium paratuberculosis but will test negative with Mycobacterium avium. Similarly, IS902 will test positive with Mycobacterium avium subspecies silvaticum, but will test negative with Mycobacterium paratuberculosis. Insertion sequences have been identified for many mycobacteria, including IS6110 for Mycobacterium tuberculosis and IS901 for Mycobacterium avium type A/I.

Source: http://crohn.ie/archive/PRIMER/bactid.htm
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