After having studied the various aspects of characterization of
microbes
discussions on certain important features related to their classification, one may now have an explicit
broader vision on the
systematized classification. An extensive and intensive survey of literature
would reveal that the microorganisms may be classified in a systematized manner under the following
eight
categories, namely:
(
i) Natural classification,
(
ii) Phyletic classification,
(
iii) Linnean binomial scheme,
(
iv) Phenotypic classification,
(
v) Microscopic examination,
(
vi) Cataloguing rRNA,
(
vii) Computer-aided classification, and
(
viii) Bacterial classification (Bergey’s Manual of Systematic Bacteriology).
The aforesaid
eight categories in the systematized classification of microorganisms would nowbe dealt with individually in the sections that follows.
Natural Classification
The
natural classification may be considered as one of the most desirable classification systems
which is broadly based upon the anatomical characteristic features of the specific microorganisms. In
actual practice, the
natural classification predominantly helps to organize and arrange the wide spectrum
of organisms into various categories (or groups) whose members do share several characteristics,
and reflects to the greatest extent the intricate and complex biological nature of organisms. In reality, a
plethora of taxonomists have concertedly opined that a larger segment of the so called
natural classification
is importantly and essentially the one having the maximum informations incorporated into it or
the emanated predicted values obtained thereof.
Phyletic* Classification
Phyletic classification
usually refers to the evolutionary development of a species. Based upon
the most spectacular and master piece publication of Darwin’s—
On the Origin of Species (1859),
microbiologists across the globe started making an attempt much to sincere and vigorous, so as to
develop phyletic (or phylogenetic) classification systems. Interestingly, the present system serves
exclusively as a supporting evidence on the evolutionary relationships in comparison to the general
resemblance. It has offered an appreciable hindrance for bacteria and other microorganisms basically on
account of the paucity of reliable and authentic
fossil records. Nevertheless, the availability of most
recent up to date copious volumes of genuine information(s) with reference to comparison of
genetic
material
and gene products, for instance: DNA, RNA, proteins etc., mostly circumvent and overcomea large segment of these problems invariably encountered.
Linnean Binomial Scheme
The microorganisms are invariably classified according to the
Linnean Binomial Scheme of
various
genus and species. The International Code of Nomenclature of Bacteria (ICNB) particularly
specifies the scientific nomenclature (names) of
all categories (taxa) solely based upon the following
guidelines, namely:
(1) The ‘
words’ used to refer to any taxonomic group are either to be drawn from Latin or are
Latinized
, if taken from other languages.
(2) Each distinct species is assigned a name comprising of
two words viz., Salmonella typhi;
Bacillus subtilis
; and the like. Here, the first word is the name of the genus and is always
written with a
capital letter, whereas the second word is a particular epithet (i.e., a descriptive
word) which is
not capitalized at all.
(3) A taxonomic sequence of taxonomic groups is usually employed to categorize the intimately
related microorganisms at different stages of similarity
Explanations:
The terminologies, species or genus are invariably employed as in the case of
other types of classification. A
species may be defined as a single type of bacterium, whereas a genus
essentially includes a cluster of species all of which predominantly possess substantial resemblance to
one another to be considered intimately related; and, therefore, may be distinguished very conveniently
from the respective bonafide members of the other genera. Importantly, the boundaries of certain
genera
are defined explicitly and sharply; whereas, the boundaries of
species are relatively difficult andcumbersome to define precisely.
(1) Extraction of DNA from the cells by causing rupture very carefully and meticulously.
(2) The resulting DNA is subject to purification to get rid of the non-chromosomal DNA.
(3) Subsequently, the base composition may be estimated by adopting either of the following
two
methodologies, namely:
(
a) Subjecting the purified DNA to a gradually elevating temperature and determining the
ultimate enhancement in
hypochromicity*, and
(
b) Centrifuging the resulting DNA in cesium chloride in density gradients.
Principle of Melting Point Method [
i.e., Method 3(a)] : In an event when the double-stranded
DNA is subject to enhancing temperature, the two DNA strands undergo separation at a characteristic
temperature. The critical melting temperature solely depends on the actual (G + C) content of the DNA.
It has been duly observed that higher the (G + C) content, higher shall be the melting point.
(4)
Melting Point (Tm) : The particular mean temperature at which the thermal denaturation
of DNA
takes place is usually termed as the Melting Point (Tm). However, Tm may be
determined by recording carefully the ‘
observed change’ in the optical density of DNAsolution at 260 nm in the course of heating period,
(5)
Density Gradient Centrifugation: The % (G + C) composition may also be calculated by
estimating the relative rate of sedimentation in a cesium chloride solution. In actual practice,
the DNA preparations on being subjected to
ultracentrifugation in the presence of a heavy
salt solution
, shall emerge as a sediment at a specific region in the centrifuge tube where its
density is equivalent to the density of the medium. Importantly, this method is particularly
suitable for such DNA samples that are heterogeneous in nature, and hence could be separated
simultaneously. It has been observed that the ensuing
buoyant density is an extremely
characteristic feature of each individual type of DNA; and hence is solely dependent on the
% (G + C) values
By the help of
buoyant density, it is quite easy and convenient to arrive at the % (G + C) content
precisely by employing the following empirical formula:
P = 1.660 + 0.00098 [% (G + C)] g . cm
–3
(6)
Chromatographic Method: Another alternative method of estimating % (G + C) is accomplished
by the
controlled hydrolysis of DNA in the presence of acids, separating the
nucleotides
by ultracentrifugation, and ultimately assaying the nucleotides by chromatography.
Though this method is apparently lengthy and tedious, yet is quite simple and gives
reasonably accurate results.
Microscopic Examination
In general, microorganisms have been duly classified by
microscopic examination based upon
their shape, size, and various staining characteristics. It has been abundantly proved that the
stained
preparations
have obviously provided much better and clear information ; however, the unstained
preparations
may also be employed for these investigations to a certain extent as well.
The
size and shape of microbes invariably may provide sufficient valuable informations thatmay be gainfully utilized for the presumptive diagnostic identification
Cataloguing rRNA*
Since mid seventies,
progressive comparative analysis of the 16 S rRNA sequences had gained
a tremendous momentum which enabled its proper and legitimate usage to explore the
prokaryotic
phylogeny.
The ribosomal RNA (i.e., rRNA) molecules are found to be of immense choice due to the
following
three cardinal reasons:(a) They exhibit a constant function,
(
b) They are universally present in all organisms, and
(
c) They seem to have changed in sequence extremely slowly.
Salient Features.
The various salient features in cataloguing rRNA are as enumerated under:
(1)
5S rRNA Molecule: Because of its relatively smaller size it has been taken as an accurate
indicator
of the phylogenetic relationship.
(2)
16S rRNA Molecule: It is sufficiently large ; and, therefore, quite easy to handle with a
reasonably high degree of precision.
(3)
23S rRNA Molecule: Because of its relatively much larger size it is rather more difficult to
characterize, and hence used in the comparative analysis.
(4) In the last two decades, the 16 S
rRNA has been critically examined, explored, and extracted
from a large cross-section of microorganisms and duly digested with
ribonuclease T1. The
resulting nucleotide are meticulously resolved by
2D-electrophoresis* technique, and
sequenced appropriately.
(5) The advent of latest sophisticated instrument
e.g., DNA-Probe** which may sequence
nucleic acids have further aided in the phenomenon of sequencing of 16 S
rRNA from
microorganisms.
(6) The skilful comparison of
rRNA catalogues predominantly designates genealogical relationship
existing amongst the wide range of microbes.
(7) The aforesaid
genealogical relationship may be suitably quantified in terms of an association
coefficient,
designated as SAB, which proves to be a typical characteristic feature for apair of microorganisms. The association coefficient SAB may be expressed as follows :
S
AB = AB
A B
2N
N +N
where, N
AB = Number of residues existing in sequences common to two rRNA catalogues.
N
A and NB = Total number of residues duly represented by oligomers of at least 6 nucleotides
in catalogues A and B respectively.
(8) As to date, the
rRNA sequences of more than 200 species of microbes and eukaryotes have
been duly characterized and documented adequately.
(9) It has been observed that most of the microorganisms strategically give rise to a coherent but
also a very large segment including the
eubacteria. Importantly, the methanogens, halophiles,
and thermoacidophiles do not necessarily fall within the domain of
eubacteria***.
(10) The aforesaid kind of
rRNA sequencing has in fact duly permitted the methodical and logicalcharacterization of archaeobacteria.
Computer Aided Classification
In the latest spectacular and astronomical growth in the field of
computer technology, it hasinducted a tremendous impetus and great help in the proper grouping of microorganisms, and eventually
classifying them with an utmost accuracy and precision. One may come across a host of problems in
comparing a relatively huge number of characteristic features as may be seen in the very instance of
numerical taxonomy
or the Adansonian approach under the perview of the general classification of
microbes. In order to circumvent such difficulties and problems, the proper usage of
computer-aided
programmes
and devices have been rightly pressed into service for determining the differentiating
capacity of the tests
and also for determining the overall similarity with the known organisms. As to
date, the commendable extremely high
speed and memory of computer conveniently allows it to
accomodate very swiftly a host of possible species in the identification/classification phenomenon by
judiciously comparing the characteristic properties of an
‘unknown microorganism’ with those stored
duly in the
computer. In fact, the advent of the utility of computer, definitely and grossly minimizes the
probability of error in the identification/classification by virtue of either infrequent occurrence of a
microorganism or the critical presence of a rather more frequent microbe with not-so similar or superficial
resemblance to other organisms. A good number of highly sophisticated, modern, and advanced
computer softwares
(systems) for microbiology have now been duly developed and put into practice
across the world profusely. The
‘microbiological laboratories’ strategically attached to most modern
hospitals and
research and development (R & D) laboratories have gainfully commenced the utilization
of the elaborated
computer facilities in the handling/processing of ‘test samples’ to obtain most
reliable, dependable, and reproducible results meant to be used in correct diagnosis and research activities
with certainly more confidence and fervour.
Bacterial Classification [Bergey’s Manual of Systematic Bacteriology]
Microorganisms
represent an exceptionally large conglomerate of minute living body with enormous
diversity having a procaryotic cellular organization. Several sincere intensive and extensive studies
were duly made with particular reference to their broad spectrum physical, structural, and functional
characteristic qualities, but none of them could ever produce and evolve an overall satisfactory generally
acceptable classification.
Chester (1899 and 1901) initiated and took active interest in the classification of bacteria, and
subsequently published for the first time—
‘The Manual of Determinative Bacteriology’. The said
manual was painstakingly and meticulously revised, substantiated, and modified by David Hendrick’s
Bergey (1923) and entitled as—
‘Bergey’s Manual of Systematic Bacteriology’, later on commonly
termed as
‘Bergey’s Manual’. In fact, Bergey’s Manual is being recognized as the ‘official compendium
of all identified and classified bacteria, and serves as an indispensable and valuable guide to the
microbiologists
across the globe.
The latest edition of
‘Bergey’s Manual’—(1994) provides a more rational and emperical approach
for the classification of bacteria. Besides, it gives rise to an effective system of keys for establishing
the precise
genetic position of an unknown organism. Table 3.4 gives a comprehensive account ofthe classification of bacteria (Division II)* upto the generic level.