Traditional and Molecular Methods for Classifying Bacteria

 Higher organisms are easily studied according to their observable characters. Micro- organisms, especially, bacteria are difficult to separate according to size and shape. The reason being, diverse micro-organisms share common morphology.

  Likewise, study of endospore, flagella and capsules are also less helpful. So, study of biochemical, physiological and genetic characters can yield useful Information for classification. The characters that are used to study, classily and identify bacteria can be broadly divided into two categories -

  1. Classical (traditional) method,
  2. Genomic (molecular) method.

1. Traditional methods :

  Traditional or classical methods involve study different characters of organisms. They are being used for routine identification for many years. They may provide phylogenetic details too.

a). Morphological characters : 

Microbiologists have often used morphological characters in microbial taxonomy. Some of the morphological features that are used in classification and identification are -

  1. Shape and size of cells
  2. Arrangement of cells
  3. Ultra structural features
  4. Staining characteristics
  5. Occurrence of cilia or flagella
  6. Mode of motility
  7. Endospore formation,shape and location
  8. Cellular Inclusion, etc.
  These characters have been studied by various techniques of light microscopy and electron microscopy.

b). Physiological and metabolic Characteristics :

Physiological and metabolic characteristics are very useful in classification. It is because they are directly related to the nature and activity of microbial enzymes as well as transport proteins. These include :

  • Mode of nutrition - an organism can be autotrophic, heterotrophic and fastidious. It can be a phototroph or chemotroph with respect to energy source. 
  • Growth characteristics in liquid media and solid media are characteristic of each species . Colony morphology and pigmentation are useful characters.
  • Physiological characters of each species are unique. Thus, they show differences with respect to growth temperature, pH, oxygen requirement. osmotic tolerance, antibiotic sensitivity and tolerance etc. 
  • Biochemical characteristics include activity of microbial enzymes and transport proteins. Enzymes confer ability to catalyze a specific biochemical reaction. Various biochemical tests used in microbiology laboratory are helpful in identification of bacteria.

  Some of the biochemical tests that can be used are -

  • Sugar fermentation
  • Hydrolysis of polymeric substances
  • Gelatin liquefaction
  • Production of catalase and peroxidase
  • Indole production
  • Hydrogen sulfide ( H₂S ) production
  • Mixed acid fermentation
  • Diacetyl formation
  • Nitrate reduction
  • Urease, phenylalanine deaminase, etc. production
  •  Utilization or specific nutrients.

c). Ecological characteristics : 

   Ecology is the study of organisms with reference to their specific environment. Some micro-organisms that resemble in many respects but they inhabit different environments. It means that these organisms may be closely related.
  The taxonomically important properties include.
  1. Pattern of life cycle
  2. Nature of symbiotic relationship
  3. Ability to cause disease in a given host
  4. Ability to participate in biogeochemical transformations
  5. Habitat preferences such as requirements for temperature, pH, oxygen and osmotic concentration.

d). Antigenic characteristics :

  Bacterial cell is clustered with several antigens. Each set of antigen is characteristic of the bacterial cell. Therefore , antigenic characters of bacteria can be useful for classification and identification of bacteria.
  When different strains of bacteria are identified by serological reaction the method is called serotyping.

e). Phage typing :

   Phage typing is concerned with Identification of strains or species of bacteria on the basis of their susceptibility to bacteriophage or phage. Phage typing gives various phagovars

f). Genetic analysis :

   Genetic analysis aims at determining genetic seminaries or differences in the organism, and there by use it for identification or classification of organisms.
The genetic tools used for the purpose include .....

2. Molecular methods :

Micro-organisms have more or less no record of fossils. However their phylogenetic and evolutionary relationship can be established by determining their genetic similarities. Organism showing a close genetic similarity, suggest their evolutionary closeness.

Following methods are available for comparison of microbial genomes. 

1] Nucleic acid Base Composition or GC content of the DNA 

2] Nucleic acid hybridization :

  • This involves hybridization between single stranded DNA obtained from two different organisms.
  •  The extent of hybridization between the DNA obtained from different organisms, show degree of their genetic similarity.

3] Nucleic acid sequencing :

  • The determination of G + C content as well as DNA homology studies have yielded useful informations. 
  • However, studies of sequence of nucleotides in small subunit rRNA ( 16S and 18S rRNA from prokaryotes and eukaryotes respectively ) ( SSU rRNAs ) have proved more useful in inferring microbial phylogeny and taxonomic assignments. 

4] Genomic Fingerprinting :

  • Genomic fingerprinting is a technique useful for classification and determination of phylogenetic relationship.
  • It does not depend on determination of nucleotide sequence. 
  • The assays include the study of restriction fragments generated by specific restriction endonucleases and conserved repetitive sequences.

5] Amino acid Sequencing :

  • The amino acid sequences of proteins directly reflect mRNA sequences and in turn that of the genes. 
  • Different varieties of the proteins can be compared. 
  • The organisms having similar amino acid sequence and functions may be closely related.