Question

3. You have used morphological observations and biochemical tests to identify two unknown bacterial isolates in your practical sessions, briefly describe three other techniques which can be used to identify unknown bacteria (you can use any reliable online resources).

Answer 1

Other than morphological observations and biochemical tests, there are various molecular techniques which can be implied for bacterial identification. Three of them are described below:

1) PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism)

This technique consists of PCR for the amplification of a gene or parts of it, along with the subsequent digestion of the PCR products with the help of restriction enzymes. After this, the electrophoretic mobility of the restriction products is analysed which reveals the polymorphisms of the gene or gene fragments and thereby the genetic changes among the test isolates. This technology is very rapid,simple and possess high reproducibility.

2) Amplified Fragment Length Polymorphism (AFLP)

AFLP is basically a genomic fingerprinting technique. It is based on the selective amplification of a subset of DNA fragments which are generated after digestion by restriction enzyme. There are two variations of AFLP:

i) with 2 restriction enzymes and 2 primers for the amplification,

ii) with only 1 primer and 1 restriction enzyme.

Purified bacterial DNA is subjected to digestion with two different restriction enzymes. The restriction fragments, thus generated, are bound to adaptors which contain each restriction site and a sequence, homologous to a binding site of the PCR primer. The PCR primers contain DNA sequences homologous to the adaptor and contain 1 or more selective bases at 3’ end. The poor resolution of this method is the main drawback associated with this method.

3) Multilocus Sequence Typing (MLST)

MLST is a genetic method based on sequencing fragments of 7 housekeeping genes (450-500 bp size). This technique is basically used for epidemiological analysis of pathogenic bacteria i.e. for comparing the strains of different geographical regions and large time scales.