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Module II
CLASSIFICATION AND IDENTIFICATION OF BACTERIA, FUNGI AND
VIRUSES

Microorganisms are classified and identified to distinguish one from another and
to group similar organisms by criteria of interest to microbiologists or other
scientists. The science that involves classification, identification, and
nomenclature of microorganisms is referred to as Taxonomy. For many years,
studies were conducted for the purpose of bringing order to a diverse group of
microorganisms until these have been organized into subspecies, species, genera,
families, and higher orders of classification.
Bacteria were identified routinely for many years by morphological,
biochemical tests, and other phenotypic criteria as well as DNA relatedness. In
phenetic approaches to classification, strains are reportedly grouped on the basis
of a large number of phenotypic characteristics. DNA relatedness is used to group
strains on the basis of overall genetic similarity while identification is
supplemented by specialized tests such as serotyping and antibiotic inhibition
patterns. In the last 2 decades, newer molecular techniques have permitted the
identification of many species of microorganisms by their genetic sequences.

Objectives:
On successful completion of the module, students will be able to:

1. Recognize the importance of classification and identification in the study of

microorganisms.

2. Identify the different criteria or bases in the classification of microorganisms for

study.

3. Explain the part of nomenclature in classification and identification of

microorganisms

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There are 3 important terms that are commonly encountered in the study of
bacterial classification and identification. These include classification,
identification and nomenclature.
Classification involves the orderly arrangement of microorganisms into
groups. This involves arranging together or distinguishing microorganisms that are
different from each other as a means of bringing order to a variety of organisms.

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There is no unified basis of classification, different groups of scientists and experts

have the capacity to classify even the same organisms differently. Some
microbiologists can classify organisms based on serotype, antimicrobial resistance
pattern, toxins and invasiveness factors in one pathogen while others may be
interested on genes which are concerned with mutations and plasmids. For many
years, the following taxonomic groups have been recognized and used in many
fields of study:

Taxonomic groups of bacteria (Taxa)

1. Species (basic taxon)

2. Genus (Plural: genera) is a group of microorganisms that possess characteristics

analogous to other species within the genus

3. Family is a group of microorganisms that possess characteristics analogous to

other genera within the family

4. Order is a group of microorganisms possess characteristics analogous to other

families within the order

5. Class includes groups of microorganisms that possess characteristics analogous

to other orders within the class

6. Division composes of a group of microorganisms that possess characteristics

analogous to other classes

7. Kingdom constitutes a group of microorganisms that possess characteristics

analogous to other members of a division

Identification involves the practical use of classification criteria to

distinguish certain organisms from others, to verify the authentic nature of a strain
and to isolate and identify the organism that causes an infection.
Nomenclature involves the giving of an official name to a species of
microorganism by which the characteristics of that species are defined and
communicated among members of the scientific community. The designated names
are often derived from names of persons, shapes or forms of microbes.

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Rules in designating official names for bacteria

1. Names are always written in binomials (2 words).
2. Binomials are italicized or underlined.
3. First letter of the generic name is capitalized while that of the specific name is
not capitalized.
Example: Staphylococcus aureus or Staphylococcus aureus

Three domains of classification

There exists a three-domain in the classification system of microorganisms
and this was reportedly introduced by Carl Woese and his colleagues in 1990. The
classification system divides all organisms into three large groups or domains.
These domains include Archaea, Bacteria and Eukarya. Domains Bacteria
and Archaea are reportedly made up of prokaryotic cells. Domain Eukarya is
reportedly made up of eukaryotic cells. The understanding is that archaea,
bacteria, and eukaryotes each reportedly arose from a common ancestor.

Fig. 1. The three-domains in the classification system of microorganisms based on

the analysis of the ribosomal RNA gene

The bacterial domain includes microorganisms that are associated with

human or animal diseases. Most bacterial species do not cause disease but many
of them reportedly play beneficial roles by producing antibiotics and food. The soil
is inhabited by free-living bacteria that perform many essential functions in the

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biosphere (nitrogen fixation). The human body is covered with bacteria that make
up the normal flora.

The Archean domain includes methanogens (methane-maker), extreme

halophiles (salt lovers) and extreme thermophiles (heat/cold lovers). The
methanogens reportedly live in swamps, marshes, gut of cattle, termites, and other
living substrates. One organism (Methanococcus jannaschii) isolated from the
deeper part of the sea use only CO2, H and N to produce energy to live and as a
result gives off methane gas. Methanogens are decomposers and can be explored
for use in sewage treatment. The extreme halophiles require an environment as
salty or even 10 times saltier (more salty) than ocean water as some reportedly
tolerate up to 30% salt concentrations. These bacteria reportedly include the
inhabitants of the Dead Sea, the Great Salt Lake and salt evaporation ponds. The
extreme thermophiles prefer temperatures above 60ºC up to 110ºC or near or below
freezing. Some thermophiles die at room temperature. Thermophiles live in hot
sulfur springs, deep sea hydrothermal vents (black smokers), geothermal power
plants and in ocean waters.

The Eukarya domain covers the protista, fungi, plants and animals. The
protists include the single celled eukaryotes like euglena, amoeba, paramecium
and protozoa. Protists are purportedly found on land, in water or living inside other
organisms. Some protists are photosynthetic like the phytoplankton and produce
more oxygen than plants. Other protists are parasites like the protozoan
Trypanosoma brucei (causes African Sleeping Sickness and transmitted to man by
the bite of the tsetse fly), Entamoeba histolytica (a parasite of the stomach which
kills cells and drains blood from their host) and Plasmodium vivax (causes malaria
also carried by female mosquitos. Fungi include mushrooms, bread molds, water
molds and yeasts. Fungi work best in breaking down dead organic material and
they recycle nutrients through ecosystems. Other fungi provide drugs such as
penicillin and other antibiotics, foods like mushrooms, truffles and morels, and in
the production of the champagne, beer, wine and alcohol. A number of fungi and
yeasts, are important "model organisms" for studying problems in genetics and
molecular biology. Plants include flowering plants, gymnosperms (conifers), ferns,
mosses and others. Most plants use chlorophyll to capture light energy, which fuels
the manufacture of food (sugar, starch and other carbohydrates).
Animals include land dwelling domestic animals, sponges, jellyfish, corals, fish,
arthropods (include insects with jointed appendages) and tetrapods (animals with
4 feet like the amphibians, reptiles, birds and mammals).

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Fig. 2. The tetrapods

(https://www.biology.iupui.edu/biocourses/N100/2k23domain.html)

Classification of bacteria by morphology, biochemistry and other methods

Bacteria are classified on the basis of many characteristics. The classification

of bacteria serves a variety of different functions where bacteria may be grouped.

1. Morphological-based classification. Cell shape, color, nature of cellular

aggregates, motility-related organelles and formation of spores are important
criteria in bacterial classification. Morphological features such as shape and
color of colonies are purported to be not always constant and can be influenced
by environmental conditions.

2. Staining reactions. Gram staining and the application of other stains for
bacteria are important methods in evaluating bacterial morphology. Of all the
different classification systems, the Gram stain has reportedly withstood the
test of time.

The method has been reportedly discovered by H.C. Gram in 1884 and it still
remains as an important and useful technique to this day. It allows a large
proportion of clinically important bacteria to be classified as either Gram-
positive or Gram-negative based on their morphology and different staining
properties. Slides are sequentially stained with crystal violet, iodine, then
decolorized with alcohol and counter-stained with safranin. Grampositive
bacteria stain blue-purple and Gram-negative bacteria stain red.

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The difference between the two groups is believed to be due to a larger

peptidoglycan content of the cell wall of Gram-positive bacteria. The application
of the iodine and crystal violet precipitate in the thickened cell wall and are not
eluted by alcohol in contrast with the Gram-negative bacteria where the crystal
violet is readily eluted from the bacteria. As a result, bacteria can be
distinguished based on their morphology and staining properties. Some bacteria
such as mycobacteria (the cause of tuberculosis) are not reliably stained by the
Gram due to the high lipid content of the peptidoglycan. An alternative staining
technique (acid fast stain) is often recommended. Evaluation on bacterial
morphology and staining reactions provide relevant data for the phenotypic-
based classification systems of many clinically important bacteria.

3. Biochemical features. Important in the classification of bacteria is the

conduct of biochemical tests including the determination of the kinds of
nutrients a bacterium can use, the products of its metabolism, the response
to specific chemicals, and the presence of particular characteristic enzymes.

4. Growth requirements. Microorganisms can be grouped on the basis of their

need for oxygen to grow. Facultatively anaerobic bacteria can grow in high
oxygen or low oxygen content and are among the more adaptable bacteria. In
contrast, strictly anaerobic bacteria grow only in conditions where there is
minimal or no oxygen present in the environment. Bacteria such as
Bacteroides and Fusobacterium are examples of anaerobes. Strict aerobes only
grow in the presence of significant quantities of oxygen. Pseudomonas
aeruginosa is an example of a strict aerobe. Microaerophilic bacteria grow
under conditions of reduced oxygen and sometimes require increased levels of
carbon dioxide. Neisseria species (cause of gonorrhea) is an example of a
micro-aerophilic bacterium.

5. Antigenic composition, habitat and disease production. Bacterial antigens

include surface proteins, lipopolysaccharides and peptidoglycan structures
that help bacteria invade host cells by gaining access between epithelial cells.
Studies on surface structures are important in recognizing the ability of
bacteria to infect hosts while this also helps in the recognition and
classification of potential pathogens. Evaluation for the antigenic properties of
bacteria is necessary as it aids in the identification unique tags where
antibodies and bacteriophages can be mobilized as possible strategies against
disease prevention.
6. Serologic systems: Selected antisera are reportedly used to classify different
bacterial species. This may be based on either carbohydrate or protein
antigens from the bacterial cell wall or the capsular polysaccharide. (Group A
streptococcal M proteins or O and H polysaccharide antigens of Salmonella).
Some scientists were convinced that serological techniques could be more
generally used in the systematic study of all groups of micro-organisms. The

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high specificity of serological reactions is determined by the chemical nature

of the antigen and it is possible to detect differences between complex
molecules particularly proteins. Serology is reported as a delicate tool for
comparing and contrasting antigenic components of the microbial cell
providing information of use both in identification and classification.
Serological analysis of superficial antigens associated with flagella and
capsules has detected slight differences between strains, and within bacterial
genera on the basis of somatic antigens. Scientists in earlier years understood
the sharing of some antigens between genera like Escherichia and Klebsiella
where the members of these groups are closely related.

7. Electron microscopy. Microorganisms can also be classified based on the

ultrastructure of the bacteria revealed under the electron microscope. This
utilizes samples which are processed as thin sections and subjected to
negative staining before electron microscopic evaluation.

8. Genotypic and molecular analysis: The classification of the 3 domains for all
living organisms– bacteria, archaea and eucarya was reportedly based on the
comparison of 16s ribosomal RNA sequences. These sequences are highly
conserved and undergo change at a slow, gradual and consistent rate which
makes comparisons among the different living organisms easier.

Ribosomal RNA (rRNA) sequence analysis has emerged as a major method

for classification. It has been used to establish a phylogenetic tree. Other
systems embrace studies on DNA homology and composition and DNA
sequences which evaluate surface polymers (Ex: capsules, teichoic acids and O-
antigens) and genes as references for comparison of bacterial relatedness. It is
necessary to determine whether strains from the same species are the same or
different. Evidences can be obtained by examining the biochemical studies or
the antibiotic susceptibility profile of a bacterial pathogen, but a more reliable
method is by molecular analysis.

Pulsed field gel electrophoresis (PFGE) has been used as molecular technique
in Microbiology for many years. Chromosomal DNA is digested with a restriction
enzyme that makes relatively infrequent cuts in the DNA and as a result creates
large DNA fragments. The DNA fragments from the different strains are then
run on a gel and compared. Other technics allow for the comparison of highly
conserved genes among different species. As a result of these comparisons a
phylogenetic tree can be developed where the degree of relatedness of different
organisms are displayed.
9. Genetic basis of classification. This classifies microorganisms-based gene-
controlled metabolic patterns, production of cell polymers and organ
structures. It embraces studies on DNA homology and composition, DNA

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sequences which evaluate surface polymers (Ex: capsules, teichoic acids and
O-antigens) and used as references for comparison of bacterial relatedness.

Evaluation of G+C base composition, DNA analysis that uses genetic probes,
nucleic acid sequencing and rRNA analysis are some common methods
employed to classify groups of bacteria. DNA relatedness can be determined by
comparing the frequency of nucleic acid bases (G + C content), perform nucleic
acid hybridization to determine the degree to which DNA sequences are the
same, perform nucleic acid sequencing, or perform other advanced molecular
techniques.

10. Phylogeny based on nucleic acid homology. Phylogeny estimates and

analyzes genetic relationships between different organisms. The approach
compares nucleic acid or protein sequences from different organisms using
computer programs and estimate the relationships based on the degree of
homology between the sequences. Nucleic acids and proteins are linear
molecules made of smaller units called nucleotides and amino acids,
respectively. The nucleotide or amino acid differences within a gene reflect the
evolutionary distance between two organisms. In other words, closely related
organisms will exhibit fewer sequence differences than distantly related
organisms. In particular, the sequence of the small-subunit ribosomal RNA
(rRNA) is widely used in molecular phylogeny.

Fig. 3. An example of an informative site analyzed by phylogenetic approach

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11. Numerical taxonomy. This is a classification system in which deals with the
grouping by numerical methods of taxonomic units based on their character
states. It aims to create a taxonomy using numeric algorithms like cluster
analysis rather than using subjective evaluation of their properties. The concept
was first developed by Robert Sokal and Peter Sneath in 1963.
The field has been reportedly divided into phenetics in which classifications
are formed based on the patterns of overall similarities; and cladistics
classifications are based on the branching patterns of the estimated
evolutionary history of the taxa. The method involves the choice and implicit or
explicit weighting of characteristics which are influenced by available data and
research interests of the investigator. This requires explicit steps to create
dendrograms and cladograms using numerical methods rather than subjective
synthesis of data.

Fig. 4. An example of a data analyzed by numerical taxonomy

12. Bacteriophage typing. This differentiates serologically identical species of

bacteria in terms of their susceptibility or resistance to the lytic effects of
species-specific bacterial viruses known as phages.

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Fig. 5. Lytic effect of a bacteriophage shown on a specific bacterium

13. Pathogenicity in animals. This differentiates certain organisms in terms of

their lethal effects on laboratory animals (Example: test that differentiates
between pathogenic from non-pathogenic strains of Mycobacterium
tuberculosis that uses guinea pigs.

Fig. 6. Pathogenicity testing in laboratory animals

14. Antibiotic sensitivity. The test differentiates bacteria in terms of their

sensitivity or resistance to the effect of antibiotics. The differences of bacterial
responses that may be related to various concentrations applied may also be
evaluated with the antibiotic sensitivity test.

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Fig. 7. Antibiotic sensitivity testing

Identification of bacteria
Accurate and definitive identification of microorganism is essential for
correct disease diagnosis, treatment of infection and in tracing back disease
outbreaks associated with microbial infections. Bacterial identification is
reportedly used in a wide variety of applications such microbial forensics, criminal
investigations, bioterrorism threats and environmental studies.
Traditional methods of bacterial identification rely on phenotypic
identification of the causative organism such as the following:
1. Gram staining
2. Culture/cultivation
3. Biochemical methods

Fig. 8. An example of data gathered and presented in the traditional

method of bacterial identification

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Microscopic evaluation of bacterial morphology

The microscopic morphology of bacteria can be evaluated in terms of the
following parameters:
1. Size of bacteria
The length of bacteria usually varies from 0.5 μm to 500 μm and the width
also varies form 0.2 μm to 5 μm.

2. Shapes of bacteria
2.1. Cocci or spherical bacteria
2.1.1. Diplococci are round or spherical bacteria seen in pairs

Fig. 8. Cocci in pairs (Diplococcus and Neisseria species)

2.1.2 Streptococci are spherical bacteria in chains

Fig. 9. Cocci in chains (Streptococcus species)

2.1.3. Staphylococci are spherical bacteria in clusters resembling

bunch of grapes

Fig. 10. Cocci in clusters (Staphylococcus species)

2.1.4. Sarcinae are round or cocci in tetrads or packets and are usually seen in
groups of 4 to 8 cells.

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Fig. 11. Coccoid bacteria in groups of 4 (Sarcina species)

2.2. Bacilli or rod-shape bacteria come in different forms which include the
following:

2.2.1. Cocco-bacilli are seen as short rods

Fig. 12. Cocco-bacillary bacteria

2.2.2. Some bacilli are seen with rounded ends

Fig. 13. Bacilli with rounded ends (Salmonella typhosa)

2.2.3. Fusiform bacilli have tapering ends

Fig. 14. Bacilli with tapering ends (Fusobacterium necrophorum)

2.2.4. Some bacilli are seen with square ends

Fig. 15. Square-ended bacilli (B. anthracis)

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2.2.5. Some filamentous bacilli (These usually do not separate and

tend to form “long threads”

Fig. 16. Bacilli that form long threads

2.2.6. Some bacilli are club-shape

Fig. 17. Club-shape bacilli (C. diphtheria)

2.3. Helical/Vibroid-shape bacteria

2.3.1. Rigid, helical bacterial forms coil or turn several times

Fig. 18. Rigid helical bacteria (Spirillum minus)

2.3.2. Vibroid/comma-shape bacteria have less than one complete twist

Fig. 19. Vibroid/comma-shape bacterium (Vibrio cholera)

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2.3.3. Highly flexible helical forms

Borrelia Treponema Leptospira

Fig. 20. Highly flexible helical Spirochetes

2.3.4. Miscellaneous shapes of bacteria

2.3.4.1. Pear-shape bacteria

Fig. 21. Pear-shape bacteria (Pasteuria species)

2.3.4.2. Lobe-shape bacteria

Fig. 21. Lobe-shape bacteria (Sulfolobus species)

2.3.4.3. Bacteria that appear as discs and arranged like stacks of coins

Fig. 21. Bacteria seen as discs and arranged like stacks of coins
(Caryophanon species)

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Staining reactions
Staining reactions of bacteria also serve as a parameter in the identification of
bacteria. Staining reflects ion exchange communication between the stain and the
active sites at the surface or within the bacterial cell.

a. Simple staining
This method involves the application of a single stain solution to a fixed
bacterial smear.

b. Differential staining

This involves application of a dye or a stain that makes the differences between
cells and its ultra-structures visible.

Gram staining

This is a staining procedure that classifies microbes into Gram-positive

and Gram-negative groups. It uses crystal violet (primary stain), iodine
(mordant), acetone alcohol (decolorizer) and safranin (counterstain).

Principle

The cell walls of Gram (-) bacteria thin and contain a high amount of lipids.
Alcohol treatment of smears extracts the lipid contents of these bacteria and
results in increase porosity and permeability of the cell wall. The blue color
imparted by the primary stain is washed out and decolorized thus the color of
the counterstain safranin is retained. Conversely, the cell wall of Gram-positive
bacteria which are made up of large amounts of teichoic acid are neither easily
washed out nor decolorized by alcohol, thus the color of the primary stain
remains and imparts color to Gram-positive bacteria, even with e application of
the decolorizer.

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Table 1. Differences between a Gram-positive from a Gram-negative bacteria

Criteria Gram (+) Gram (-)

Susceptibility to sulfonamides Marked Much less

and penicillin
Inhibition by basic dyes (crystal Marked Much less
violet)
Susceptibility to lysozymes many species pretreatment of cell
wall
Ratio of RNA and DNA in the cell 8:1 almost equal

Susceptibility to sodium azide much less marked

Susceptibility to streptomycin much less marked

Other staining methods for bacteria

1. Acid fast stain differentiates acid fast from non-acid-fast bacteria
2. Endospore stain demonstrates shapes and location of spores in the cytoplasm
structures
3. Capsule stain differentiates bacteria in terms of the thickness of the cell
structure
4. Flagellar stain demonstrates presence, number and location of flagellae in
bacteria
5. Cytoplasmic inclusion stain demonstrates intracellular deposits of starch and
glycogen
6. Lactophenol cotton blue stains fungal spores and other structures
7. Giemsa stain is recommended for rickettsial and protozoan specimens

The above methods of bacterial identification are associated with two major
limitations. One of these is that the application of Gram staining,
culture/cultivation and biochemical methods is only possible for organisms
cultivated in vitro. Second, unique biochemical characteristics are only exhibited
by some strains which may not fit into patterns that have been used as a
characteristic of other bacterial genus and species.

A number of terms are used to describe different appearances of bacteria

(Gram-positive or Gram-negative, coccus or bacillus growing both aerobically and
anaerobically). This description may appear as a pointless verbiage or jargon. It
means that if a microorganism is found to be Gram-negative, it cannot be one of
the Gram-positive bacteria, therefore any suspected Gram-positive organism is
ruled out. It is the same with a microscopically evaluated bacillus shaped

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microorganism, which would require elimination of suspected cocci. If the

microorganism is described growing anaerobically, the aerobes are discarded while
if it is described growing aerobically then the anaerobes are ruled out. discarded.
This would finally give an identification of a Gram-negative bacillus growing as a
facultative anaerobe as the most likely cause of an infection.

Phenotypic method of identification

1. Bacterial colonial morphology
1.1. Bacteria with wavy edges and long filaments that form loops

Fig. 22. A bacterium that forms loops (B. anthracis)

1.2. Bacteria with folding and snapping colonies

Fig. 23. Bacteria with folding and snapping colonies

(P. pestis and C. diphtheria)

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1.3. Bacteria with slipping, smooth and spreading growth

Fig. 24. Bacteria with slipping, smooth and spreading growth

(P. vulgaris and E. coli)

1.4. Mucoid (M) colonies are exhibited by bacteria that possess slimes and
capsules. The colonies have moist glistening appearance.

Fig. 25. Bacteria that form mucoid colonies

(K. pneumonia, D. pneumonia, H. influenza and C. neoformans)

1.5. Smooth(S) colonies demonstrate the appearance of homogeneity and

uniform texture.

Fig. 26. Bacteria forming smooth colonies

(Gram-negative coli-typhoid-paratyphoid-dysentery group of bacteria and
Salmonella spp.)

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1.6. Rough(R) colonies appear granulated and are exhibited by bacteria that lack
a part of the carbohydrate polymer structure.

Fig. 27. Bacteria forming rough colony

(Mycobacterium spp. on a solid medium [Left picture, right side] and in a liquid
medium [Right picture, right side] compared to a smooth colony [Left side of each
picture])

1.7. L-colonies are seen in organisms which are devoid of a rigid cell wall

Fig. 28. Bacteria forming L-colonies various sizes and shapes

Recent developments in bacterial identification

Genetic approaches used in the identification of bacteria are aimed at

defining a degree of relatedness between organisms since these organisms
reportedly diverged from a common ancestor. DNA-based approaches used in the
identification of bacteria include DNA-DNA hybridization, DNA fingerprinting and
DNA sequencing.

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DNA-DNA hybridization was developed in the 1980s and is used to determine

the similarity of DNA sequences from different organisms. The degree of similarity
is reflected in the degree to which a strand of DNA from the organism of interest
passively hybridizes with (attaches to) a single strand of DNA from a known
organism. The less stable the hybridization is, the more quickly the DNA strands
dissociate when heated. This indicates that low DNA melting temperatures suggest
low degrees of sequence similarity. DNA-DNA hybridization is most valuable for
determining genetic relatedness at the genus and species levels.

DNA fingerprinting methods for bacterial identification is centered on the use

of the polymerase chain reaction (PCR). Repetitive element-PCR targets specific
DNA segments that are repeated at random in the bacterial genome. The
identification of repetitive elements is powerful and is capable of resolving bacteria
at intraspecies levels.

DNA sequencing methods like whole genome sequencing and multi-locus

sequence analysis (MLSA) have been proven to be useful in the identification of
bacteria. MLSA which involves DNA sequencing of subsets of so-called
housekeeping (or conserved) genes has been shown to provide resolution down to
intraspecies levels.

Sequencing of the 16S rRNA assesses evolutionary relatedness among

bacteria. The gene 16S rRNA encodes the RNA component of the smaller subunit
of the bacterial ribosome (16S refers to the rate of sedimentation in Svedberg units
of the RNA molecule in a centrifugal field). The 16S rRNA gene is present in all
bacteria and a related form occurs in all cells. For example, the 16S rRNA gene of
E. coli is 1,542 nucleotides long and some of its regions are double stranded while
other regions are single-stranded. Single-stranded regions reportedly form loops,
because there is a lack of complementary bases on the opposing strand. Since 16S
rRNA makes very specific contacts with many different ribosomal proteins and with
other parts of itself, the pace at which spontaneous random mutation can change
the sequence of the bases in the rRNA is slow. Any change in sequence at one site
has to be compensated for by another change elsewhere within the rRNA or in a
ribosomal protein, lest the ribosome fail to assemble properly or to function in
protein synthesis and the cell die.

Analysis of the 16S rRNA sequences from many organisms has revealed that
some portions of the molecule undergo rapid genetic changes, thereby
distinguishing between different species within the same genus. The comparison
of 16S rRNA sequences between organisms is quantitative and is based on a
defined set of assumptions. The assumption that the rate at which base changes
occur and are established within a species is constant is unlikely to be true. A

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limitation of the 16S rRNA sequence analysis includes its poor resolution below the
genus level. Populations of organisms in a given genus that reside within the same
habitats can have unique 16S rRNA genotypes. However, whether those unique
genotypes are indicative of distinct species typically cannot be determined from
rRNA information alone.

The analysis of rRNA sequences from bacteria that are closely related to one
another has revealed several surprising relationships between those organisms.
For example, Mycoplasma which appear to be different from other bacteria (in that
they are very small, lack a cell wall, have a very small genome, and have sterols in
their cell membranes) are related to some Gram-positive Clostridia on the basis of
their nucleic acid sequences. These circumstances underscore the hazard of relying
on phenotypic traits (observable characteristics such as the absence of a cell wall)
for the assignment of evolutionary or genetic relationships.

In the past decade, molecular techniques which may require non-culture-

based methods have emerged. These have been proven beneficial in overcoming
some limitations of traditional phenotypic procedures for the identification of
bacteria. Several PCR platforms and DNA microarrays are currently reported as the
most commonly employed molecular techniques. In using DNA-based assays, one
can easily detect bacterial strains directly from clinical samples or from small
amounts of cultured bacterial cells, improving diagnosis and recognition of bacteria
decreases the time required for bacterial identification. PCR has been particularly
useful and it relies on primer sequences designed to facilitate bacterial
identification at any level of specificity: strain, species or genus.

Microarrays combines the potential of simultaneous bacterial identification

and speciation. This method is versatile and makes it possible to detect and
discriminate different bacterial samples on a single slide. DNA microarray-based
approach is used for the quick detection and identification of bacteria using
species-specific oligonucleotide probes designed for specific regions of various
targeted genes.

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Fig. 29. Data generated from molecular techniques like PCR and DNA
sequencing as recent methods of bacterial identification

Classification scheme for viruses

The Baltimore classification is a system that classifies viruses based on the

manner by which messenger RNA (mRNA) is synthesized. By studying viruses
based on the manner of mRNA production, it is possible to study viruses that
behave similarly as a distinct group. Seven Baltimore groups are reportedly
described based on their deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
contents, the number of DNA strands (single- or double-stranded) and whether the
sense of a single-stranded RNA genome is positive or negative.

Baltimore classification closely corresponds to the means a virus replicates

its genome making itself useful for grouping viruses with similar patterns for
transcription and replication together. Other subjects or criteria that pertain to
viruses are associated with multiple and specific forms in the translation of mRNA
and the host range of different types of viruses where specific Baltimore groups
may be created. Baltimore classification provides insights into the transcription
and replication parts of the viral life cycle. Structural characteristics such as the
shape of the viral capsid which stores the viral genome and the evolutionary history
of viruses are not necessarily related to Baltimore groups.

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The Baltimore system classifies viruses according to the aforementioned

characteristics. There are seven Baltimore groups that are currently recognized,
namely:

• Group I: double-stranded DNA viruses

• Group II: single-stranded DNA viruses
• Group III: double-stranded RNA viruses
• Group IV: positive sense single-stranded RNA viruses
• Group V: negative sense single-stranded RNA viruses
• Group VI: single-stranded RNA viruses with a DNA intermediate in their life
cycle
• Group VII: double-stranded DNA viruses with an RNA intermediate in their life
cycle

Fig. 30. Representation of Baltimore classification

Other recommended guides in the identification of bacteria

The main source for determining the identity of prokaryotic organisms

particularly bacterial species that uses characterizing parameters is the Bergey’s
manual of systematic bacteriology. The Bergey’s manual was published subsequent
to the Bergey's Manual of Determinative Bacteriology which is still reportedly
published as a guide for identifying unknown bacteria. The author David Hendricks
Bergey used to classify bacteria based on their structural and functional attributes
by arranging them into specific familial orders. Classifying bacteria based on their
structural and functional attributes by arranging them into specific familial orders
has become more empirical in recent years.

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Evaluation
1. Identify the simplest method of classification that a Microbiology student can
conduct based on his/her present training.

2. Explain and discuss the importance of learning the basic principles of bacterial
classification and identification.

References
Woese CR, O Kandler and ML Wheelis. June 1990. Evolution towards a natural
system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya.
Proc. Nati. Acad. Sci. USA Vol. 87, pp. 4576-4579.

https://www.biology.iupui.edu/biocourses/N100/2k23domain.html

https://www.ck12.org/biology/bacteria-
classification/lesson/prokaryoteclassification-advanced-bio-adv/

https://courses.lumenlearning.com/boundless-
microbiology/chapter/methods-of-classifying-and-identifying-microorganisms/

https://www.google.com/search?q=numerical+taxonomy&tbm=isch&chips=q:n
umerical+taxonomy,g_

https://study.com/academy/lesson/the-evolutionary-relationships-oforganisms.

https://en.wikipedia.org/wiki/Baltimore_classification

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