Comparison of Traditional and Modern Approaches to the Identification of Bacteria.

1. the rRNA gene sequence approach has limitations of its own.  

Identification tests should always be performed on single colonies or pure cultures

A colony or a pure culture is one that consists of only one type of microorganism and is derived from a single cell. Aseptic techniques must be used to isolate and maintain pure cultures of microorganism.

Bacteria are identified by simple characteristics and biochemical properties. A preliminary identification of many of the bacteria of medical importance can be made on the basis of the following few simple characteristics of the cells

• Gram reaction.

• Cell morphology and arrangement.
• Ability to grow under acrobic or anaerobic conditions.
• Growth requirements (simple or fastidious)

Further identification is made on the basis of biochemical properties such as:

• Ability to produce enzymes that can be detected by simple tests (eg. Coagulase, catalase, oxidase, lecithinase,)
• Ability to metabolise sugars oxidatively or fermentatively (eg. The Huge and Liefson oxidation / fermentation test)
• Ability to use a range of substracts for growth (e.g. glucose, lactose, sucrose). These tests can be done individually (e.g. in broth media containing the test sugar) or in commercial kits, which allow a range of different tests to be set up simultaneously on each organism

API

The API system (BioMerieux) is widely used and while it is not as sophisticated as the vitek, particularly with sample inoculation, it has been semi-qutomceted as the ATB system with an accompanying change from 20 to 32 biochemical test, options per strip. Lapage (1973) designed a computer program which showed that the average of 29-32 tests were required for reliable identification of aberrant strains of Euterobacteriaceae and non-fermenting Gram negative rods end that additional test did not improve the accurency.

Two system consists of a densitometer, inoculator reader and data-handler and allows a choice of rapid or overnight incubation, depending on the test type. It has both identification and antimicrobial susceptibility testing options. A particularly useful output feature is the typically index which provides that user with culture identification and indicates whether the isolate is typical or compared with the base profile for that species.

API systems

API systems are available for: Enterobacteria (20E, Rapid, 20E, 10S); Non-enteric Gram-negative rods (20NE); Staphylococci (20/STAPH); Streptococci (20/STREP); Anearobes (20A); Heterotrophic aerobes (20B); Yeasts (20C/AUX); Fermentation, oxidation, assimilation (50CH); Enzyme tests (ZYM).

Suspensions of the organisms are added to cupules in plastic galleries. There are incubated overnight. Some tests then require reagents to be added. Results are read from codes and a Profile Register. There is a computer identification service.

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--From Chocollins Datricia, M lyne, T. M. Grange, Colins and Lyne’s Microbiological Methods 7th edition, P122

Classification

Classification is the process of recognizing and describing groups of living organism.

Classification is important. It is an activity essential to all scientific work. It would be impossible to make any generalizations about microorganisms and their role in nature if we could only refer to each strain by a different and arbitrary name. We must arrange microorganisms into groupsthat share common properties, so that we can talk about sets of strains that have properties in common.

Identification

Identification is a branch of systematics. It is, however, different from classification, which tests of the way living organisms are grouped together into taxonomic groups or taxa (singular taxon). Identification deals with the process of allocating a new specimen (an ‘unknown’) to the correct and previously described taxon.

Importance:  Identification is a very important practical activity, which will concern most microbiologists from time to time. Large areas of microbiological work are heavily dependent on good identification. Some areas, such as hospital microbiology, are almost entirely concerned with identification and are collectively referred to as diagnostic microbiology. It may be emphasized that numerous kinds of microbiological work……..

Culture of Microorganisms

Many microorganisms, especially those with simple morphology, must first be isolated in pure culture before they can be identified. The importance of the purity of these cultures cannot be overemphasized: most cultural tests become grossly misleading with mixed cultures. The commonest cause of admixture is isolating a microorgnaism from a selective medium that inhibits, but does not kill, unwanted species.

The main classes of data used in microbial identification are morphological, chemical, and serological.

---From Professor J. R. Norris and Professor M. H. Richmond, 1978, Essay in Microbiology, 9/1-10/1

Ribosomal genes

Contain regions that are conserved among all self replicating  organisms, together with highly variable regions

Are large enough to contain adepqate amount of information

Phylogenetic relationships among prokaryotes

Carl Woese and his colleagues demonstrated that the nucleotide sequences occurring in ribosomal RNA could be used to suggest phylogenetic relationships among prokaryotes. In other words, Woese reasoned that changes in rRNA sequences that still permitted protein synthesis would occur so slowly that such changes could be used as a clock to compare one organism with another. Thus, if six different genera of bacteria all contained the same specific changes in a segment of their rRNA, one could suppose that all had a common ancestral origin. To put it another way, the more widespread the existence of a specific rRNA sequence, the longer ago it occurred.

Why are rRNA sequences so highly conserved? Keep in mind that such RNA must maintain a fairly restricted secondary structure in order to function in protein synthesis and that this structure is dependent on base pairing within the RNA molecule. Any mutational change incompatible with such secondary structure will be lethal to the organism. In addition, rRNA must be able to bind to protein within the ribosome, and any changes that interfere with such binding would also be lethal.

Woese’s sequnce analyses led to the conclusion that all living organisms could be fitted into one of three distinct kingdoms. One, comprising all of the eucaryotic cell types was termed the EUCARYTOTES and the other two kingdoms, contaning prokaryotic cells, were designated the Eubacteria (true bacteria) and the Archaebacteria (ancient bacteria). Moreover, it was subsequently shown that rRNA existing in mitochondria and chloroplasts (obtained from eucarytoic cells) is closely related to that found in the Eubacteria. In addition, at least one protein sequence occurring in eucaryotic cells is closely related to a similar protein found in the Archaebacteria. Thus , eucaryotic cells appear to contain genes derived from both eubacteria and archaebactria as well as distinct gene sets that have evolved in eucaryotic cells.

The general structure of ribosomes from prokaryotes and eukaryotes is summarized. Figure

Although ribosomes from prokaryotes such as bacteria are smaller (70S) than those from eukarytoes (80S), all share the same basic structure. They are composed of two major subunits of different size and with different sedimentation coefficients. Ribosomes can be completely dissociated into their component parts in solutions of urea or detergents. Each subunit contains at least one single-stranded RNA molecule and numerous proteins. Naturally enough, the RNA’s structural components of the ribosomes, are referred to as ribosomal RNA’s or rRNA’s. In both prokaryotes and eukaryotes the larger ribosomal subunit contains two rRNA molecules,……… The small ribosomal subunit contains one single-stranded rRNA molecule—16S in prokaryotes and about 1500 nucleotides long.

FROM 1

REFERENCE

1. James W. Fristrom, Michael T. Clegg. 1989. Principles of genetics. Second edition. 347-349
2. Professor J. R. Norris and Professor M. H. Richmond. 1978. Essay in Microbiology, 1/9-1/10
3. C. H. Chollins, Patricia M. Lyne, J. M. Grange. 1984. Collins and Lyne’s Microbiological Methods, 7th edition. 122
4. Abigail A. Salyers, Dixie D. Whitt. 2001. Microbiology, diversity, disease, and the environment. 440-444
5. Wesley A. Volk. 1992. Basic Microbiology. 7th Edition. 184-185
6. Mims Playfair Roitt, Wakelin Williams. 1998. Medical Microbiology. 2nd Edition. 174