44179573 Isolation Identification of Microbes

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Meenakshi Kashyap M.Phil Botany

Course 2B: Research

Methodology in 2A

ISOLATION, IDENTIFICATION & DETECTION O

MICROBES



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.

MOLECULAR MICROBIOLOGY(Interaction of microbes with their

µenvironment¶ at the molecular level)

Physiology

Interaction with human

Disease Causing

Genetics &

Biotechnology

Energy/EnvironmentImmunology

Industry & Agriculture



MICROBIAL AN ALYSES

CULTURE DEPENDENT CULTURE INDEPENDENT

MEDIA PREP AR ATION

STR AIN ISOLATION

STR AIN PURIFIC ATION

IDENTIFIC ATION

PHENOTYPIC GENOTYPICIMMUNOLOGICAL

EXTR ACT

NUCL

EIC

AC

ID

EXTR ACT

FATT

Y AC

ID

T-RFLP

PCR & DGGEFAME

GC

PHYLOCHIPS



CULTURE DEPENDENT AN ALYSES 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.



CLASSIFIC ATION OF CULTURE MEDIA

Physical Nature Chemical Composition Functional Types

Solid

Liquid

Defined (synthetic)

Complex

Supportive

Enriched

Selective

Differential



CULTURE MEDIA CLASSIFICATIONBased on Physical Nature

SOLID MEDIUM

Semisolid medium prepared by addition of

a solidifying agent such as agar.

LIQUID MEDIUM

No solidifying agent

LB (Luria and Bertani) Broth is for

liquid culture.

The minimal medium is colorless

(left), while the nutrient agar is tan

coloured (right).



CULTURE MEDIA CLASSIFICATIONBased on Chemical Composition

DEFINED / SYNTHETIC MEDIUM

A medium with all known components

Sources of carbon, nitrogen, sulphate,

phosphate & other minerals.

Sustains the growth of photoautotrophs &

chemoorganotrophs.

Used to know what experimental organism is

metabolizing.

Eg: BG-11 medium for cyanobacteria

COMPLEX MEDIUM

Media containing some ingredients of unknown

chemical composition (peptones, yeast extract

etc«)

Source of carbon, nitrogen & energy

Growth of fastidious microbes

Used to find out the nutritional requirement of

microbe.

Eg: Nutrient broth for bacteria



CULTURE MEDIA CLASSIFICATIONBased on Functional Types

SUPPORTIVEMEDIUM

General purpose

medium.

Sustains the growth

of many microbes.

Eg: Tryptic Soy

Broth, Tryptic Soy

Agar etc.

ENRICHEDMEDIUM

Specially fortified

medium.

Special nutrients are

added to general

purpose medium.

Favours growth of

fastidious microbes

SELECTIVEMEDIUM

Medium that inhibits

the growth of certain

microbes as it contain

some agents (bile salts &dyes) that suppress

bacteria.

Favours the growth of

a particular microbe.

MacConkey agar is

used for E .coli detection.

DIFFERENTIALMEDIUM

Medium that displays

visible differences

(colony size, gas bubble

formation and precipitateformation etc) among

different groups of

microbes.

Helps in tentative

identification of microbes

based on biologicalcharacteristics.

Eg: Blood agar,

MacConkey agar



SUPPORTIVE

MEDIUM

E nterobacter sp. growing on

Tryptic Soy Agar medium

ENRICHED

MEDIUM

SELECTIVE

MEDIUMDIFFERENTIAL

MEDIUM

Large, golden colonies of S taphylococcus

aureus growing on a blood-agar plate.



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



STRE AK PLATE METHOD

Petri dish of an agar being streaked

with an Inoculating Loop.

A Commonly used Streaking Pattern.

Organisms that form distinct

colonies on agar plates are

restreaked successive times to

establish a pure culture.



SPRE AD PLATE METHOD

Pipette a small

sample on the

center of an agar

medium plate. Dip a glassspreader into

a beaker of

ethanol. Briefly flame the ethanol

soaked spreader & allow

it to cool.

Spread the sample

evenly over the agar

surface with the

sterlized spreader.



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

Mixed Sample in Capillary tube

Focus laser beam

Traps a single cell & drags down

the optically trapped cell.

Cell moves away from contaminants

Capillary is severed

Cell is flushed into a tube of sterile medium

U seful for isolating slow growing bacteria

& organisms present in less number.

Cell is isolated from microscopic field & moved away from mixture of cells.

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

device.



LIMIT ATIONS OF CULTURE DEPENDENT

AN ALYSES 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.



PHENOTYPIC

METHODS

OF

IDENTIFIC ATION

(Micro / Macroscopic)

CultureMedia

Microscopic examination

(StainingMethods)

Biochemical Tests

Rapid Tests

Bacteriophage Typing

Flow Cytometry

Fatty acid methyl ester analysis



CULTURE MEDIA

Bacterial Colony MorphologyBacillus subtilis growing on

nutrient poor agar forming

snowflake like colonies.

(Intricate patterns seen in

nonliving systems)



MICROSCOPIC EX AMIN ATION

M icrococcus on agar (x 31,000) Clostridium (x 12,000)

M ycoplasma pneumoniae (x 26,000) E scherichia coli (x 14,000)



MICROSCOPIC EX AMIN ATIONDirect microscopic examination of a stained specimen is the most rapid method

for the identification of characteristics.

Stains include Gram Staining, Endospore Staining, Acid fast Staining, NegativeStaining etc«.

E . coli (white), M icrococcus luteus(yellow), S erratia marcescens (red)

M icrococcus luteus

S erratia marcescens



ST AINING METHODS

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)

Endospore Staining

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.



ST AINING METHODS (contd«)

Negative Staining

M ycobacterium leprae (X 380)

Masses of red bacteria are seen within the host cell.

Klebsiella pneumoniae

Negative Staining with India pink to show

its capsules (X 900)

Acid Fast staining

S pirillum volutans with bipolar tufts of flagella (X 400)

Flagella Staining



BIOCHEMIC AL 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.



BIOCHEMIC AL TESTS

Bacteria produce acidic products whenthey ferment certain carbohydrates.

pH changes if fermentation of thegiven carbohydrate occurs.

Acids lower the pH of the mediumwhich will cause the pH indicator (phenol red) to turn yellow.

Gas production is indicated by thebubble in Durham tube.

The carbohydrate tests can beperformed for Glucose (Dextrose),

Lactose test, Sucrose test

Left tube shows less acid formation than far right

tube, but gas is still made

Center shows no carbohydrate utilization to

produce acid or gas.

Right tube shows acid was produced as

evidenced by the yellow color, and gas

formation.

Carbohydrate Utilization



(BIOCHEMIC AL TESTS contd«)

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 tothe 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.

Citrate Utilization

Positive test indicated by

colourless area around growthNegative test

Starch Hydrolysis

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.



(BIOCHEMIC AL 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«



R APID BIOCHEMIC AL TESTS

A rapid miniaturized system

that can detect for 23characteristics in small 20 lplastic strips.

Contains dehydratedbiochemical substrates whichis inoculated with purecultures and suspended in

physiological saline.

After 5 hrs-overnight the testsare converted to digital profile.

The information from the rapidtest are fed into a computer tohelp in identification of the

organisms.

Useful for the identification of E nterobacteriacae and other Gram ±ve bacteria etc«

ONPG ( galactosidase); ADH (arginine dihydrolase); LDC (lysine

decarboxylase); ODC (ornithine decarboxylase); CIT (citrate

utilization); H2S (hydrogen disulphide production); URE (urease);

TD A ( tryptophan deaminase); IND (indole production); VP (VogesProskauer test for acetoin); GEL ( gelatin liquefaction); the

fermentation of glucose (GLU), mannitol (M AN), inositol (INO),

sorbitol (SOR), rhamnose (RH A), sucrose (S AC); Melibiose

(MEL), amygdalin (AMY), and arabinose (AR A); and OXI

(oxidase).



Identification Key of Enterococcus sp.



FLOW CYTOMETRY

Classical techniques are not successful inidentification of those microorganisms thatcannot be cultured.

Allows single or multiple microorganismsdetection .

Microbes are identified on the basis of thecytometry parameters or by means of certain

dyes called fluorochromes that can be usedindependently or bound to specific antibodies.

The cytometer forces a cell suspension througha laser beam and measures the light theyscatter or the fluorescence the cell emits asthey pass through the beam.

The cytometer can also measure the cell¶sshape, size and the content of the DN A or RN A



FATTY ACID METHYL ESTER AN ALYSIS

Fatty acids present in cytoplasmicmembrane 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 specifictemperature in order to compare its

profile in database.

FAME analyses is limited to some

organisms.

CHROM ATOGR AM

showing types & amount

of fatty acid from

unknown bacterium



LIMIT ATIONS OF PHENOTYPIC METHODS

OF IDENTIFIC ATION

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.



IMMUNOLOGIC AL

METHODS OFIDENTIFIC ATION

(Serological)

Precipitation Reactions

Agglutinations Reactions

Fluorescent Antibodies



PRECIPIT ATION RE ACTIONS

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.



AGGLUTIN ATIONS RE ACTIONS

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

Direct agglutination: When a soluble Ab results in clumping by interaction with an Agwhich is part of a surface of a cell. Eg: Detection of M ycoplasma pneumonia.

Indirect agglutination. Ab/Ag is adsorbed or chemically coupled to the cell. Latexbeads or charcoal particles serve as an inert carrier & detect for surface Ag. Eg:Commercial suspension of latex beads are available for the detection of S taphylococcus aureus, S treptococcus pyogenes etc

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

Benefits :

Simple to perform.

Highly specific.

Inexpensive and rapid.



Direct method

Fluorescent Ab is directed to surface Ag of the organism.

Indirect method

A non-fluorescent Ab reacts with the

organism's Ag and a fluorescent Ab reacts with

the non-fluorescent Ag.

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

FLUORESCENT ANTIBODIES



Cells of S ulfolobus

acidocaldarius attached to

soil particle visualized by

Fluorescent antibody

technique.

Fluorescent Staining usingD API (4¶,6-diamido-2-

phenylindole) or Acridine

Orange

Viability Staining

differentiates between

live cells (green) &

dead cells (red) of

M icrococcus luteus &

Bacillus cereus.



GENOTYPIC

METHODS OFIDENTIFIC ATION

(Genetic)

Nucleic Acid Probes

Nucleic Acid Sequencing

Fluorescent in situ hybridisation

DN A-DN A hybridization

Polymerase Chain Reaction

Restriction fragment lengthPolymorphism

Random Amplified Polymorphic DN A

Plasmids Fingerprinting

Ribotyping

Multilocus Sequence Typing

Linking specific genes to specific

organism using PCR

Environmental genomics



NUCLEIC ACID PROBES

A Probe is a ssDN A sequence that can be used to identify an organism by forming a

³hybrid´ with a unique complementary sequence on the DN A or r RN A 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 DN A sequence can be used for microbial identification.

Sequence based identification requires the recognition of a molecular target that allowsdiscrimination between the microbes.

In bacteria such molecular target is r DN A gene complex.

r DN A gene complex has both:

Highly variable sequence (Internal Transcribed Spacer) called Signaturesequences, short oligonucleotides unique to certain groups of organisms.

Conserved Regions (16S rRNA) that contain the Genomic code.

16S r RN A is small subunit ribosomal RN A 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

rDNA gene complex in bacteria



16 S r RN A GENE SEQUENCING

Secondary Structure

of 16S rRNA gene

Basic Local Alignment Search Tool

(BLAST) is a computational method for

sequence comparison alignment on NCBI

GenBank database.



Use of SSU 16 S r RN A gene

was pionered by Carl Woese

at University of Illinois for phylogenetic studies in early

1970.

Database of r RN A 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.S

equence diversity between strains is more accurately measured by aDN

A±DN A Hybridization assay.

Sequencing of the entire 16S r RN A gene (1500bp) is required for establishing anovel isolate. While Automated sequencers can generate approximately 500 bpof sequence data i.e. sufficient for species identification (Heterogeneity of first500 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.



substitution per sequence position

Rooted universal phylogenetic tree

showing the three domains based upon

16S r RN A sequences.

Rooted phylogenetic tree for the

Bacteria based on 16S r RN A

sequences.



FLUORESCENT IN SITU HYBRIDIZ ATION

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. r RN A (16S r RN A in prokaryotes).

Cells become uniformly fluorescent & can be observed by fluorescent microscope.

Important tool in microbial ecology & clinical diagnostics.

Phase-contrast photomicrograph of microbes

stained with fluorescently labeled r RN A probes.

Confocal laser scanning micrograph of a

sewage sludge sample. Multiple FISH probes

each containing a different dye targeted

different bacteria to give different colours.



DN A-DN A

HYBRIDIZ ATION

A genome wide comparison of sequencesimilarity.

Useful to distinguish species within a genus.

Genomic DN A is isolated from test organisms.

One of the DN A is radioactively labeled with32P or 3H, sheared to small extent &

denatured.

Mixed with an excess of unlabeled DN A (toprevent labeled DN A from reannealing toitself) prepared in same way from other organism.

Cool DN A mixture so that ssDN A canreanneal.

Separate hybridized dsDN A from unhybridizedDN A

Measure radioactivity in hybridized DN A &compare with control.



STEPS:

Two nucleic acid primers arehybridized to a complementarysequence in a target gene.

DN A polymerase copies the target

gene. Multiple copies of the target gene aremade by repeated melting of complementary strands, hybridizationof primers and new synthesis.

Allows for the detection even if only a fewcells are present

Presence of the appropriate amplified PCRproducts confirms the presence of theorganisms.

POLYMER ASE CH AIN RE ACTION

In vitro amplification of the target DN A used for the identification of microbes

Primers are available for the identification of microbes such as S almonella and

S taphylococcus to monitor food.



RESTRICTION FR AGMENT LENGTH

POLYMORPHISM

RFLP involves digestion of the genomic DN A of the organism with

restriction enzymes.

The restricted fragments are separated by agarose gel electrophoresis.

The DN A fragments are transferred to a membrane and probed with

probes specific for the desired organisms.

A DN A profile emerges which can be used for microbe identification.



R ANDOM AMPLIFIED POLYMORPHIC DN A

R APD uses a random primer (10-mer) to generate a DN A profile.

Primer anneals to several places on the DN A template and generates a DN A profilewhich is used for microbe identification.

R APD has many advantages: Pure DN A is not needed

Less labour intensive than RFLP.

No need for prior DN A sequence data.

R APD 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,

S almonella, Campylobacter and Pseudomonas

has demonstrated that this method is moreaccurate 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.



DN A PROFILING METHOD : RIBOTYPING

It is a r RN A 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 DN A

with one or more restrictionenzymes.

Separation of DN A fragments

by gel electrophoresis.

Transfer of fragments

onto nylon membranes.

Hybridization with

16 S r RN A gene

probe

DN A banding pattern or

RIBOTYPE generated is

digitized.

Ribotype results from 4 different lactic acid bacteria.

Position & Intensity of band is important in identification.

Computer compares the

pattern with patterns of

reference organisms

present in database.



MULTI LOCUS SEQUENCE TYPING

Characterizes strains within species & thusdistinguishes closely related strains.

Involves sequencing of several

³housekeeping genes´ from an organism

and comparing their sequences with

sequences of the same genes from different

strains of the same organism.

MLST : Strains with identical sequences for

a given gene will have the same allele

number for that gene & two strains for

identical sequences for all the genes have

same allelic profile

Expressed by Linkage distances inDendrogram.

0 indicates Strains are identical

1 indicates Strains are distantly related.

Compare each nucleotidealong the sequence & note

the variant i.e. allele & assign

a series of numbers.

Allelic profile

Multilocus

Sequence Type

Widely used in Clinical microbiology, Epidemiology, Environmental Studies:



LINKING SPECIFIC GENES TO SPECIFIC

ORG ANISMS USING PCR

Specific genes are used asmeasures of biodiversity.

Genes are linked to specific

organisms.

Detection of genes implies that thespecific organism linked to this

gene is present.

Techniques:

1. Polymerase Chain Reaction

2.

Denaturing

Gradient

GelElectrophoresis

3. Molecular Cloning



Polymerase Chain

Reaction

Target genes can be :-

Genes encoding small subunitribosomal RN A

Metabolic genes that encode proteinsunique to to specific organism

Group of related organism

If target gene is widely distributed (Eg:r RN A gene), PCR will amplify eachphylotype (Multiple copies of each genevariant).

Sort out the Phylotypes ««.

Denaturing Gradient GelElectrophoresis

Separated genes of same size differ in

their melting (denaturing) profiles because of

differences in their base sequence.

DGGE employs a gradient of DN A

denaturant (mixture of urea & formamide)

ds DN A 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 DN A

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

Six PCR products, all yielded a

single band on gel but actually

they consisted of six distinct 16S

r RN A gene sequences (b; lane 1

and 8).

1.Purify six bands.

2.Reamplify by PCR

(a; lanes 2-7)3.Run on DGGE

(b; lanes 2-7)



Terminal Restriction Fragment Length

Polymorphism

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

Steps :

Amplification of target gene (usually r RN A 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 r RN A sequence variation in the

community sampled.



Phylochips

Phylogenetic microarrays constructed for rapid analyses in biodiversity studies.

Constructed by affixing r RN A- or r RN A gene targeted oligonucleotide probes to the

chip surface in known pattern.

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

Arrange phylochips in known pattern

(Oligonucleotide complementary to 16S r RN A genes)

Isolation of total community DN A

PCR amplification

Fluorescence Labeling of 16S r RN A genes

Hybridization between DN A & probe

Observe presence or absence of fluorescence

Less time consuming than DGGE, Cloning,

Sequencing etc«.

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)



ENVIRONMENT AL GENOMICS

µMetagenomics¶ 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 ascompared 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 r RN A)

Applications:

Detection of new genes.

Assessment of Phylogenetic &

Metabolic Diversity of microbial

communities



CONCLUSION

C urrent 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 ´Nanobiotechnologyµ. 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.



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Edition Vol.2

Bhattacharya, S. e t a l . 2002. Uncultivable bacteria: implications and recent trendstowards identification. Indian journal of medical microbiology, Vol. 20 (4):174-177

Bosshard, P.P. et al may 2004. Comparison of conventional and molecular methods foridentification of aerobic catalase-negative gram-positive cocci in the clinicallaboratory. Journal of clinical microbiology. Vol 42: no.5 p. 2065²2073

Drancourt et al. 2000. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates. Journal of clinicalmicrobiology Vol. 38: No. 10 p. 3623²3630

Kenzaka, et al . 2005. rRNA sequence-based scanning electron microscopic detection.

Applied and environmental microbiology, Vol. 71: no. 9 p. 5523²5531 Madigan, M. T. et al BROCK Biology of Microorganisms. Twelfth Edition Pearson

International

Manero, A. et al. 1999. Identification of E nterococcus spp. With a biochemical key.Applied and environmental microbiology Vol. 65: no. 10 p. 4425²4430

Muyzer et al . 1993 .Profiling of Complex Microbial Populations by Denaturing GradientGel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Codingfor 16S rRNA. Applied and environmental microbiology, Vol. 59, No. P. 695-700

Patel, J. B. 2001. 16S rRNA Gene Sequencing for Bacterial Pathogen Identification in

the Clinical Laboratory. Molecular Diagnosis Vol. 6 No. 4 Prescott, L . et al MICROBIOLOGY Sixth Edition The McGrawïHill Companies, 2002

Pereira,F. et al 2008. Identification of species with DNA-based technology: currentprogress and challenges. Vol. 2, 187-200

Persing D. H. & Kolbert, C.P.1999. Ribosomal DNA sequencing as a tool foridentification of bacterial pathogens Current Opinion in Microbiology, Vol 2:299-305

Woese, C. & Olsen, G. J.1993.Ribosornal RNA: a key to phylogeny FASEB Journal7:113-l 23.

Website for Biochemical Tests : http://web.fccj.edu



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44179573 Isolation Identification of Microbes

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