ISOLATION, IDENTIFICATION & DETECTION

OF MICROBES

Meenakshi Kashyap
M.Phil Botany
Course 2B: Research
Methodology in 2A
 CONTENTS
• Introduction
• Culture Dependent analyses of Microbes
• Culture Media
• Isolation in Pure Cultures
• Limitations of Culture Dependent analyses of Microbes
• Culture Independent analyses of Microbes
• Phenotypic Methods of Identification
• Immunological Methods of Identification
• Genotypic Methods of Identification
• Conclusion
• Future Developments
 INTRODUCTION
Microbiology is a specialized area of biology that deals with
microorganisms that include bacteria, fungi, viruses, protozoa and
some parasitic worms.

Molecular biology is the study of the macromolecules of life (particularly

DNA, RNA and proteins) and their interactions at the molecular (molecule)
level.

Physiology Immunology
Energy/Environment Disease Causing

MOLECULAR MICROBIOLOGY
(Interaction of microbes with their
‘environment’ at the molecular level)

Interaction with human

Genetics &
Industry & Agriculture
Biotechnology
 MICROBIAL ANALYSES

CULTURE DEPENDENT CULTURE INDEPENDENT

MEDIA PREPARATION
EXTRACT EXTRACT
STRAIN ISOLATION NUCLEIC ACID FATTY ACID

STRAIN PURIFICATION PCR & DGGE

FAME

IDENTIFICATION T-RFLP
GC
PHYLOCHIPS

PHENOTYPIC IMMUNOLOGICAL GENOTYPIC

 CULTURE DEPENDENT ANALYSES OF
MICROBES

CULTURE MEDIUM : A preparation used to grow, store & transport microbes.

Criteria of selecting a culture media:

 Knowledge of microbe

 Habitat of microbe

 Nutrients, growth factors (vitamins) required for growth

 Sources of energy (C, N, P, S & various minerals) & electrons.

 CLASSIFICATION OF CULTURE MEDIA

Physical Nature Chemical Composition Functional Types

Solid Defined (synthetic) Supportive

Liquid Complex Enriched

Selective

Differential
 CULTURE MEDIA CLASSIFICATION
Based on Physical Nature

SOLID MEDIUM LIQUID MEDIUM

Semisolid medium prepared by addition of No solidifying agent

a solidifying agent such as agar.

The minimal medium is colorless LB (Luria and Bertani) Broth is for

(left), while the nutrient agar is tan liquid culture.
coloured (right).
 CULTURE MEDIA CLASSIFICATION
Based on Chemical Composition

DEFINED / SYNTHETIC MEDIUM COMPLEX MEDIUM

 A medium with all known components  Media containing some ingredients of unknown
chemical composition (peptones, yeast extract
etc…)

 Sources of carbon, nitrogen, sulphate,  Source of carbon, nitrogen & energy

phosphate & other minerals.

 Sustains the growth of photoautotrophs &  Growth of fastidious microbes

chemoorganotrophs.

 Used to know what experimental organism is  Used to find out the nutritional requirement of
metabolizing. microbe.

 Eg: BG-11 medium for cyanobacteria  Eg: Nutrient broth for bacteria
 CULTURE MEDIA CLASSIFICATION
Based on Functional Types

SUPPORTIVE ENRICHED SELECTIVE DIFFERENTIAL

MEDIUM MEDIUM MEDIUM MEDIUM

 Specially fortified  Medium that inhibits  Medium that displays

 General purpose medium. the growth of certain visible differences
medium. microbes as it contain (colony size, gas bubble
 Special nutrients are some agents (bile salts & formation and precipitate
dyes) that suppress formation etc) among
added to general
bacteria. different groups of
purpose medium.
microbes.

 Favours the growth of  Helps in tentative

Sustains the growth  Favours growth of
a particular microbe. identification of microbes
of many microbes. fastidious microbes based on biological
characteristics.

 Eg: Tryptic Soy  MacConkey agar is

 Eg: Blood agar,
Broth, Tryptic Soy used for E.coli detection.
MacConkey agar
Agar etc.
SUPPORTIVE ENRICHED
MEDIUM MEDIUM

Enterobacter sp. growing on Large, golden colonies of Staphylococcus

Tryptic Soy Agar medium aureus growing on a blood-agar plate.

SELECTIVE DIFFERENTIAL
MEDIUM MEDIUM
 ISOLATION IN PURE CULTURES

 Pure Culture : Contain a single kind of microorganism.

OR
A population of cells arising from a single cell, to characterize an individual
species.

 Plating Techniques (Classical Methods)

• Spread Plate Method
• Streak Plate Method
• Pour Plate Method (Serial Dilution Method)

 Microscopic Tool (New Technology)

• Laser Tweezers
 STREAK PLATE METHOD

Petri dish of an agar being streaked

with an Inoculating Loop.

Organisms that form distinct

colonies on agar plates are
restreaked successive times to
establish a pure culture.
A Commonly used Streaking Pattern.
 SPREAD PLATE METHOD

Pipette a small Spread the sample

sample on the evenly over the agar
center of an agar surface with the
medium plate. Dip a glass sterlized spreader.
spreader into
a beaker of
ethanol. Briefly flame the ethanol
soaked spreader & allow
it to cool.
 POUR PLATE METHOD

 Serial Dilution Method

Original sample is diluted
several times to thin out the
population sufficiently.

 Most diluted samples are

then mixed with warm agar
and poured into petri dishes.

 Isolated cells grow into

colonies.

 Used to establish Pure

Culture.
 THE LASER TWEEZERS
 Cell is isolated from microscopic field & moved away from mixture of cells.

 Consists of an inverted light microscope equipped with infrared laser & micromanipulation
device.

 Mixed Sample in Capillary tube

Focus laser beam

Traps a single cell & drags down

the optically trapped cell.

Cell moves away from contaminants

Useful for isolating slow growing bacteria
Capillary is severed & organisms present in less number.

Cell is flushed into a tube of sterile medium

LIMITATIONS OF CULTURE DEPENDENT
ANALYSES OF MICROBES
 Time consuming.

 Failure to isolate viable but non culturable organisms.

 Does not provide comprehensive information on the composition of

microbial communities.

 Predominant species that cannot be cultivated by standard techniques

cannot be detected.
  Culture Media

 Microscopic examination
PHENOTYPIC (Staining Methods)

METHODS  Biochemical Tests

OF  Rapid Tests

IDENTIFICATION  Bacteriophage Typing

(Micro / Macroscopic)
 Flow Cytometry

 Fatty acid methyl ester analysis

 CULTURE MEDIA

Bacillus subtilis growing on

Bacterial Colony Morphology nutrient poor agar forming
snowflake like colonies.
(Intricate patterns seen in
nonliving systems)
 MICROSCOPIC EXAMINATION

Micrococcus on agar (x 31,000) Clostridium (x 12,000)

Mycoplasma pneumoniae (x 26,000) Escherichia coli (x 14,000)

 MICROSCOPIC EXAMINATION
Direct microscopic examination of a stained specimen is the most rapid method for
the identification of characteristics.

Micrococcus luteus

E. coli (white), Micrococcus luteus

(yellow), Serratia marcescens (red)

Serratia marcescens

Stains include Gram Staining, Endospore Staining, Acid fast Staining, Negative
Staining etc….
 STAINING METHODS

Gram Staining

Discovered by Hans Christian Gram

Differentiates bacterial species between Gram positive & Gram Negative

based on the physical & chemical properties of their cell wall.

Endospore Staining
Endospore is a dormant, tough & non-reproductive structure
produced by Gram positive bacteria. Eg: Bacillus, Clostridium

Position of endospore (terminal, sub terminal or centrally

placed) differs from bacterial species & is useful in identification.

Molecular details of endospore formation is widely studied in

Bacillus subtilis (Model for Cellular differentiation)
 STAINING METHODS (contd…)
Acid Fast staining

Negative Staining

Mycobacterium leprae (X 380)

Masses of red bacteria are seen within the host cell.

Klebsiella pneumoniae
Negative Staining with India pink to show Flagella Staining
its capsules (X 900)

Spirillum volutans with bipolar tufts of flagella (X 400)

 BIOCHEMICAL TESTS

 Microbe is cultured in a media with a special substrate and tested

for an end product.

 The information from the biochemical tests are input into a

computer to generate a biochemical profile.

 Biochemical tests can target a specific reaction Eg. nitrate

reduction, protein degradation, growth at high temperatures etc…

 Give a comprehensive description of the organism's properties

which can be important in differentiation at the strain level.
 BIOCHEMICAL TESTS
Carbohydrate Utilization
• Bacteria produce acidic products when
they ferment certain carbohydrates.

• pH changes if fermentation of the

given carbohydrate occurs.

• Acids lower the pH of the medium

which will cause the pH indicator
(phenol red) to turn yellow.

• Gas production is indicated by the

bubble in Durham tube. Left tube shows less acid formation than far right
tube, but gas is still made

• The carbohydrate tests can be Center shows no carbohydrate utilization to

performed for Glucose (Dextrose), produce acid or gas.
Lactose test, Sucrose test Right tube shows acid was produced as
evidenced by the yellow color, and gas
formation.
 (BIOCHEMICAL TESTS contd…)
Citrate Utilization Starch Hydrolysis

Positive test indicated by Negative test

• Tests for the ability of bacteria to convert
citrate (an intermediate of the Kreb’s cycle) into
oxaloacetate (another intermediate of the
Kreb’s cycle).
• Citrate is the only carbon source available to
the bacteria.
• If citrate is not used : No growth
• If citrate is utilized : Bacteria will grow and the
media will turn a bright blue as a result of an
increase in the pH of the media.
colourless area around growth
• Test is used to detect the enzyme amylase,
which breaks down starch.
• After incubation the plate is treated with
Gram’s iodine.
• Hydrolysis of starch is indicated by reddish
color or a clear zone around the bacterial
growth.
• No hydrolysis : Blue/Black area indicating
the presence of starch.
 (BIOCHEMICAL TESTS contd…)
Other biochemical tests include:

1. H2S Production
2. Indole Test
3. Catalase Test
4. Nitrate Reduction
5. Urea Test
6. Gelatin Utilization
7. Oxidase Test
8. MRVP (Methyl Red-Vogues Proskauer)
9. Oxidation Fermentation
10. Motility Test
11. Phenylalanine Deaminase Test
12. Antibiotic Susceptibility Tests etc…
 RAPID BIOCHEMICAL TESTS
 A rapid miniaturized system
that can detect for 23
characteristics in small 20 μl
plastic strips.

 Contains dehydrated
biochemical substrates which
is inoculated with pure
cultures and suspended in
physiological saline.

 After 5 hrs-overnight the tests

are converted to digital profile.

 The information from the rapid
test are fed into a computer to
help in identification of the
organisms.
 Useful for the identification of
Enterobacteriacae and other
Gram –ve bacteria etc…
ONPG (β galactosidase); ADH (arginine dihydrolase); LDC (lysine
decarboxylase); ODC (ornithine decarboxylase); CIT (citrate
utilization); H2S (hydrogen disulphide production); URE (urease);
TDA ( tryptophan deaminase); IND (indole production); VP
(Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the
fermentation of glucose (GLU), mannitol (MAN), inositol (INO),
sorbitol (SOR), rhamnose (RHA), sucrose (SAC); Melibiose
(MEL), amygdalin (AMY), and arabinose (ARA); and OXI
(oxidase).
Identification Key of Enterococcus sp.
 BACTERIOPHAGE TYPING
 Based on the specificity of phage surface receptor for the cell surface receptor.

 Only those phages that can attach to the surface receptors can cause lysis.

The phage type is reported as a

specific genus and species
followed by the types that can
infect the bacterium.

 E.g. 10/16/24 means that the bacteria is sensitive to phages 10, 16 and 24.

Phage tying is a tool for research and reference labs.

 FLOW CYTOMETRY
 Classical techniques are not successful in
identification of those microorganisms that
cannot be cultured.

 Allows single or multiple microorganisms

detection .

 Microbes are identified on the basis of the

cytometry parameters or by means of certain
dyes called fluorochromes that can be used
independently or bound to specific antibodies.

 The cytometer forces a cell suspension through

a laser beam and measures the light they
scatter or the fluorescence the cell emits as they
pass through the beam.

 The cytometer can also measure the cell’s

shape, size and the content of the DNA or RNA
 FATTY ACID METHYL ESTER ANALYSIS
 Fatty acids present in cytoplasmic
membrane of bacteria is highly variable
( in terms of chain length, presence or
absence of double bonds, rings, branched
chain, hydroxy groups etc.)

 Fatty acid profile can identify the species.

 Drawbacks:
• Requires rigid standardization as
fatty acid profiles can vary according
to temperature, growth medium etc.

• Unknown organism should be grown

on specific medium & at a specific
temperature in order to compare its
profile in database.
CHROMATOGRAM
showing types & amount
• FAME analyses is limited to some
of fatty acid from
organisms.
unknown bacterium
LIMITATIONS OF PHENOTYPIC METHODS
OF IDENTIFICATION

 Difficult & time consuming for slow growing organisms.

 Not all strains within a given species may exhibit a common characteristic.

 The same strain may give different results upon repeated testing.

 The corresponding databases does not include newly or not yet described
species.

 The test result relies on individual interpretation and expertise.

  Precipitation Reactions
IMMUNOLOGICAL
METHODS OF  Agglutinations Reactions

IDENTIFICATION  Fluorescent Antibodies

(Serological)
 PRECIPITATION REACTIONS
 Precipitation (ppt) is the interaction of a soluble Ag with an soluble Ab to form an
insoluble complex.

The complex formed is an aggregate of Ag and Ab.

Ppt rxns occurs maximally only when the optimal proportions of Ag and Ab are
present.

Ppt can also be done in agar referred to as immunodiffusion.

Ppt test uses antibodies to detect for streptococcal group antigens.

 AGGLUTINATIONS REACTIONS
 Visible clumping of an Ag when mixed with a specific Ab.

 Direct agglutination: When a soluble Ab results in clumping by interaction with an Ag

which is part of a surface of a cell. Eg: Detection of Mycoplasma pneumonia.

 Indirect agglutination. Ab/Ag is adsorbed or chemically coupled to the cell. Latex

beads or charcoal particles serve as an inert carrier & detect for surface Ag. Eg:
Commercial suspension of latex beads are available for the detection of
Staphylococcus aureus, Streptococcus pyogenes etc

 Standardized tests are available for the determination of blood groups and identification
of pathogens and their products.

 Benefits :
Simple to perform.
Highly specific.
Inexpensive and rapid.
 FLUORESCENT ANTIBODIES
 Abs can be chemically modified with fluorescent dyes such as rhodamine B, fluorescent red.

 Cells with bound fluorescent Ab emit a bright red, orange, yellow or green light depending on the dye
used.

 Fluorescent Ab can be used to detect suspected pathogen such as Bacillus anthracis and HIV virus

Indirect method
Direct method
Fluorescent Ab is directed to surface Ag of A non-fluorescent Ab reacts with the
the organism. organism's Ag and a fluorescent Ab reacts
with the non-fluorescent Ag.
 Fluorescent Staining using
DAPI (4’,6-diamido-2-
phenylindole) or Acridine
Orange

Cells of Sulfolobus Viability Staining

acidocaldarius attached to differentiates between
soil particle visualized by live cells (green) &
Fluorescent antibody dead cells (red) of
technique. Micrococcus luteus &
Bacillus cereus.
  Nucleic Acid Probes

 Nucleic Acid Sequencing

 Fluorescent in situ hybridisation

 DNA-DNA hybridization

 Polymerase Chain Reaction

GENOTYPIC  Restriction fragment length

Polymorphism

METHODS OF  Random Amplified Polymorphic DNA

IDENTIFICATION  Plasmids Fingerprinting

(Genetic)  Ribotyping

 Multilocus Sequence Typing

 Linking specific genes to specific

organism using PCR

 Environmental genomics
 NUCLEIC ACID PROBES
 A Probe is a ssDNA sequence that can be used to identify an organism by forming a
“hybrid” with a unique complementary sequence on the DNA or rRNA of that organism.

 Hybridization is detected by a reporter molecule (radioactive, fluorescent,

chemiluminescent) which is attached to the probe.

 Disadvantages:
• Limited Selectivity
• Lack Sensitivity when testing from direct specimens.

 Nucleic acid probes have been marketed for the identification of many pathogens such as
N. gonorrhoeae.
 NUCLEIC ACID SEQUENCING
 Small amount of DNA sequence can be used for microbial identification.

 Sequence based identification requires the recognition of a molecular target that allows
discrimination between the microbes.

 In bacteria such molecular target is rDNA gene complex.

rDNA gene complex in bacteria

 rDNA gene complex has both:

• Highly variable sequence (Internal Transcribed Spacer) called Signature sequences,
short oligonucleotides unique to certain groups of organisms.
• Conserved Regions (16S rRNA) that contain the Genomic code.

 16S rRNA is small subunit ribosomal RNA gene (approx 1,500 bp) used extensively for
sequence based evolutionary analysis because they are:
• Universally distributed (i.e. found among a wide range of bacteria)
• Functionally constant
• Sufficiently conserved (i.e. slow changing)
• Adequate length
16 S rRNA GENE SEQUENCING

Basic Local Alignment Search Tool

(BLAST) is a computational method for
sequence comparison alignment on NCBI
Secondary Structure
GenBank database.
of 16S rRNA gene
Use of SSU 16 S rRNA gene
was pionered by Carl Woese
at University of Illinois for
phylogenetic studies in early
1970.

Database of rRNA gene

sequence is Ribosomal
Database Project –II (RDP-
II; http:/rdp.cme.msu.edu)
has collection of sequences
& analytical programs.
 NUCLEIC ACID SEQUENCING
 Advantages:
• High degree of confidence & accuracy due to robustness of genetic identification.
• Rapid recognition.

 Drawbacks:
• Expensive Technology.
• Not a perfect measure of overall sequence divergence between bacteria.
Sequence diversity between strains is more accurately measured by a DNA–
DNA Hybridization assay.
• Sequencing of the entire 16S rRNA gene (1500bp) is required for establishing a
novel isolate. While Automated sequencers can generate approximately 500 bp
of sequence data i.e. sufficient for species identification (Heterogeneity of first
500 bp from 5’ end is sufficient).

 Applications:
• Identification of bacterial isolates.
• Clinical diagnosis of microbial infections.
• Construction of Phylogenetic trees such as three domain classification &
Bacterial classification.
 Rooted phylogenetic tree for the
Bacteria based on 16S rRNA
sequences.

Rooted universal phylogenetic tree

showing the three domains based upon
16S rRNA sequences.

substitution per sequence position

 FLUORESCENT IN SITU HYBRIDIZATION
 FISH uses a fluorescent probe (fluorescent dyes attached to nucleic acid probes) to detect
microbes that contain nucleic acid sequence complementary to the probe.

 Cells are treated with reagents that make cells permeable to probe dye mixture.
Fluorescent probes hybridize directly to cellular ribosomes i.e. rRNA (16S rRNA in prokaryotes).
Cells become uniformly fluorescent & can be observed by fluorescent microscope.

Phase-contrast photomicrograph of microbes Confocal laser scanning micrograph of a

sewage sludge sample. Multiple FISH probes
stained with fluorescently labeled rRNA probes .
each containing a different dye targeted
different bacteria to give different colours.
 Important tool in microbial ecology & clinical diagnostics.
 DNA-DNA
HYBRIDIZATION
 A genome wide comparison of sequence
similarity.

 Useful to distinguish species within a genus.

 Genomic DNA is isolated from test organisms.

 One of the DNA is radioactively labeled with

32P or 3H, sheared to small extent &
denatured.

 Mixed with an excess of unlabeled DNA (to

prevent labeled DNA from reannealing to
itself) prepared in same way from other
organism.

 Cool DNA mixture so that ssDNA can

reanneal.

 Separate hybridized dsDNA from

unhybridized DNA

 Measure radioactivity in hybridized DNA &

compare with control.
 POLYMERASE CHAIN REACTION
 In vitro amplification of the target DNA used for the identification of microbes

 STEPS:
• Two nucleic acid primers are
hybridized to a complementary
sequence in a target gene.
• DNA polymerase copies the target
gene.
• Multiple copies of the target gene are
made by repeated melting of
complementary strands, hybridization
of primers and new synthesis.

 Allows for the detection even if only a few

cells are present

 Presence of the appropriate amplified PCR

products confirms the presence of the
organisms.

 Primers are available for the identification of microbes such as Salmonella and
Staphylococcus to monitor food.
 RESTRICTION FRAGMENT LENGTH
POLYMORPHISM
 RFLP involves digestion of the genomic DNA of the organism with
restriction enzymes.

 The restricted fragments are separated by agarose gel electrophoresis.

 The DNA fragments are transferred to a membrane and probed with

probes specific for the desired organisms.

 A DNA profile emerges which can be used for microbe identification.

RANDOM AMPLIFIED POLYMORPHIC DNA
 RAPD uses a random primer (10-mer) to generate a DNA profile.

 Primer anneals to several places on the DNA template and generates a DNA profile
which is used for microbe identification.

 RAPD has many advantages:

• Pure DNA is not needed
• Less labour intensive than RFLP.
• No need for prior DNA sequence data.

 RAPD has been used to fingerprint the outbreak of Listeria monocytogenes from milk.
 PLASMID FINGERPRINTING
 Identifies microbial species or similar strains as
related strains as they contain the same number of
plasmids with the same molecular weight.

 Plasmid of many strains and species of E. coli,

Salmonella, Campylobacter and Pseudomonas
has demonstrated that this method is more
accurate than phenotypic methods such as phage
typing.

 Procedure involves:
• Bacterial strains are grown, the cells lysed
and harvested.
• Plasmids are separated by agarose gel
electrophoresis.
• Gels are stained with EtBr and the plasmids
located and compared.
 DNA PROFILING METHOD : RIBOTYPING
 It is a rRNA based bacterial identification technique that distinguishes between
species & strains within a species.
 Highly specific and rapid.
 Finds application in clinical diagnostics & microbial analyses of food, water etc……

Digestion of bacterium’s DNA Separation of DNA fragments

with one or more restriction by gel electrophoresis.
enzymes.

Transfer of fragments
onto nylon membranes.

Ribotype results from 4 different lactic acid bacteria.

Position & Intensity of band is important in
identification.
Computer compares the Hybridization with
pattern with patterns of DNA banding pattern or 16 S rRNA gene
reference organisms RIBOTYPE generated is probe
present in database. digitized.
 MULTI LOCUS SEQUENCE TYPING
 Characterizes strains within species & thus
distinguishes closely related strains.

 Involves sequencing of several

“housekeeping genes” from an organism
and comparing their sequences with
sequences of the same genes from different
strains of the same organism.
Allelic profile
 MLST : Strains with identical sequences for Multilocus
a given gene will have the same allele Sequence Type
number for that gene & two strains for
identical sequences for all the genes have
same allelic profile

Compare each nucleotide

 Expressed by Linkage distances in
along the sequence & note
Dendrogram.
the variant i.e. allele & assign
0 indicates Strains are identical a series of numbers.
1 indicates Strains are distantly related.

 Widely used in Clinical microbiology, Epidemiology, Environmental Studies:

LINKING SPECIFIC GENES TO SPECIFIC
ORGANISMS USING PCR
 Specific genes are used as
measures of biodiversity.

 Genes are linked to specific

organisms.

 Detection of genes implies that the

specific organism linked to this
gene is present.

 Techniques:
1. Polymerase Chain Reaction
2. Denaturing Gradient Gel
Electrophoresis
3. Molecular Cloning
 Polymerase Chain
Reaction
 Target genes can be :-
• Genes encoding small subunit
ribosomal RNA
• Metabolic genes that encode proteins
unique to to specific organism
• Group of related organism
 If target gene is widely distributed (Eg:
rRNA gene), PCR will amplify each
phylotype (Multiple copies of each gene
variant).
 Sort out the Phylotypes …….
Denaturing Gradient Gel
Electrophoresis
 Separated genes of same size differ in
their melting (denaturing) profiles because of
differences in their base sequence.
 DGGE employs a gradient of DNA
denaturant (mixture of urea & formamide)
 ds DNA moves through a gel, reaches a
region containing sufficient denaturant, the
helical strand begins to melt at this point and
migration stops.
 Different bands in DGGE gel are
phylotypes that can differ in base sequence.
 Individual bands are excised, then
sequenced & species identification is done
by phylogenetic analyses.
 PCR & DGGE Gels

Microbial community

Extract Bulk DNA

Amplify by PCR using primers for 16S rRNA genes of bacteria (a; lane 1 and 8)

1.Purify six bands.

2.Reamplify by PCR
(a; lanes 2-7)
Six PCR products, all yielded a 3.Run on DGGE
single band on gel but actually (b; lanes 2-7)
they consisted of six distinct 16S
rRNA gene sequences (b; lane
1 and 8).
 Terminal Restriction Fragment Length
Polymorphism

 T- RFLP measures single gene diversity in a microbial community.

 Steps :

• Amplification of target gene (usually rRNA genes) by PCR using a primer set
(One primer is end labeled with fluorecent dye)

• Restriction Digestion of the PCR products.

• Separation of fluorescently labeled terminal fragments on a gel.

• Pattern of bands obtained indicates rRNA sequence variation in the

community sampled.
 Phylochips
 Phylogenetic microarrays constructed for rapid analyses in biodiversity studies.

 Constructed by affixing rRNA- or rRNA gene targeted oligonucleotide probes to the

chip surface in known pattern.

 Depending upon the study phylochips can be made specific or broad & several
thousand different probes can be added to a single phylochip.

Arrange phylochips in known pattern

(Oligonucleotide complementary to 16S rRNA
genes)
Isolation of total community DNA

PCR amplification
• Less time consuming than DGGE, Cloning,
Fluorescence Labeling of 16S rRNA genes Sequencing etc….

Hybridization between DNA & probe
Observe presence or absence of fluorescence
• Chips are used to carry probes targeting
genes that encode a key metabolic function
(Eg: Nitrogen fixation to find out whether
nitrogen fixing organisms are present in the
sample)
 ENVIRONMENTAL GENOMICS

 ‘Metagenomics’ or the molecular study of

microbial communities.

 Based on cloning, sequencing & analysis of

the collective genomes of the organisms
present in the community.

 Sampling of all genes in community as

compared to single gene diversity in
community sampling approach.

 Determination of phylogenetic group can be

achieved by sequencing overlaps to the
genes (incl. Phylogenetic marker 16S rRNA)

 Applications:
• Detection of new genes.
• Assessment of Phylogenetic &
Metabolic Diversity of microbial
communities
 CONCLUSION
Current developments in nucleic acid detection technologies are guided by two
general trends: miniaturization of genotyping instruments and high-throughput
sample analysis. A broad spectrum of highly innovative automated assays have
been devised based on conventional genotyping techniques (DNA hybridization
or sequencing) to provide reliable, rapid and low-cost DNA screenings.

Molecular-based methods are complementary to traditional

methods and are revolutionizing microbial diversity, and taxonomy research and
applied fields.
FUTURE DEVELOPMENTS
A number of recent biotechnological achievements offer great potentials for the
development of a portable DNA diagnostic device for the rapid identification of
species from low amount of biological material. The most promising area of
emerging technologies that might permit a high-throughput analysis from single
DNA molecule and resolve the technical challenges related with species
identification is “Nanobiotechnology”. For instance, an approach to directly read
multiple polymorphic sites on single DNA molecules has been recently proposed,
using atomic force microscopy with a high-resolution single-walled carbon
nanotube probe .
Any future method will only be possible under a coherent scientific
understanding of population genetics, evolution, systematics, ecology and
molecular biology.
 REFERENCES
• Brenner , D. J. et al . BERGEY’S manual of Systematic Bacteriology. Springer 2nd Edition
Vol.2
• Bhattacharya, S. et al. 2002. Uncultivable bacteria: implications and recent trends
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