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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. MOLECULAR MICROBIOLOGY (Interaction of microbes with their ‘environment’ at the molecular level) Physiology Interaction with human Disease Causing Genetics & Biotechnology Energy/Environment Immunology Industry & Agriculture

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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 Liquid Defined (synthetic) Complex Supportive Enriched Selective Differential


CULTURE MEDIA CLASSIFICATION Based 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 CLASSIFICATION Based on Chemical Composition: 

CULTURE MEDIA CLASSIFICATION Based 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 CLASSIFICATION Based on Functional Types SUPPORTIVE MEDIUM General purpose medium. Sustains the growth of many microbes. Eg: Tryptic Soy Broth, Tryptic Soy Agar etc. ENRICHED MEDIUM Specially fortified medium. Special nutrients are added to general purpose medium. Favours growth of fastidious microbes SELECTIVE MEDIUM 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. DIFFERENTIAL MEDIUM Medium that displays visible differences (colony size, gas bubble formation and precipitate formation etc ) among different groups of microbes. Helps in tentative identification of microbes based on biological characteristics. Eg : Blood agar, MacConkey agar

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SUPPORTIVE MEDIUM Enterobacter sp. growing on Tryptic Soy Agar medium ENRICHED MEDIUM SELECTIVE MEDIUM DIFFERENTIAL MEDIUM Large, golden colonies of Staphylococcus 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


STREAK 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.


SPREAD PLATE METHOD Pipette a small sample on the center of an agar medium plate. Dip a glass spreader 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 Useful 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.


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.

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PHENOTYPIC METHODS OF IDENTIFICATION (Micro / Macroscopic) Culture Media Microscopic examination (Staining Methods) Biochemical Tests Rapid Tests Bacteriophage Typing Flow Cytometry Fatty acid methyl ester analysis


CULTURE MEDIA Bacterial Colony Morphology Bacillus subtilis growing on 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. Stains include Gram Staining, Endospore Staining, Acid fast Staining, Negative Staining etc…. E. coli (white), Micrococcus luteus (yellow), Serratia marcescens (red) Micrococcus luteus Serratia marcescens


STAINING 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.


STAINING METHODS (contd…) 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 its capsules (X 900) Acid Fast staining Spirillum volutans with bipolar tufts of flagella (X 400) Flagella Staining


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 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. The carbohydrate tests can be performed 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


(BIOCHEMICAL 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 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. Citrate Utilization Positive test indicated by colourless area around growth Negative 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.


(BIOCHEMICAL TESTS contd…) Other biochemical tests include: H 2 S Production Indole Test Catalase Test Nitrate Reduction Urea Test Gelatin Utilization Oxidase Test MRVP (Methyl Red-Vogues Proskauer) Oxidation Fermentation Motility Test Phenylalanine Deaminase Test 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).

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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. 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 . The phage type is reported as a specific genus and species followed by the types that can infect the bacterium.


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. FAME analyses is limited to some organisms. CHROMATOGRAM showing types & amount of fatty acid from 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.

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IMMUNOLOGICAL METHODS OF IDENTIFICATION (Serological) Precipitation Reactions Agglutinations Reactions Fluorescent Antibodies


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.


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

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Cells of Sulfolobus acidocaldarius attached to soil particle visualized by Fluorescent antibody technique. Fluorescent Staining using DAPI (4’,6-diamido-2-phenylindole) or Acridine Orange Viability Staining differentiates between live cells (green) & dead cells (red) of Micrococcus luteus & Bacillus cereus.

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GENOTYPIC METHODS OF IDENTIFICATION (Genetic) Nucleic Acid Probes Nucleic Acid Sequencing Fluorescent in situ hybridisation DNA-DNA hybridization Polymerase Chain Reaction Restriction fragment length Polymorphism Random Amplified Polymorphic DNA Plasmids Fingerprinting 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 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 rDNA gene complex in bacteria


16 S rRNA 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.

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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:/ ) 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.

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substitution per sequence position Rooted universal phylogenetic tree showing the three domains based upon 16S rRNA sequences. Rooted phylogenetic tree for the Bacteria based on 16S rRNA sequences.


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. Important tool in microbial ecology & clinical diagnostics. Phase-contrast photomicrograph of microbes stained with fluorescently labeled rRNA 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.


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 32 P or 3 H, 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.


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. POLYMERASE CHAIN REACTION In vitro amplification of the target DNA used for the identification of microbes 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 with one or more restriction enzymes. Separation of DNA fragments by gel electrophoresis. Transfer of fragments onto nylon membranes. Hybridization with 16 S rRNA gene probe DNA 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 & 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. 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 in Dendrogram. 0 indicates Strains are identical 1 indicates Strains are distantly related. Compare each nucleotide along 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 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: Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis Molecular Cloning

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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: 

PCR & DGGE Gels Microbial community Extract Bulk DNA Amplify by PCR using primers for 16S rRNA 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 rRNA 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: 

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 Fluorescence Labeling of 16S rRNA genes Hybridization between DNA & 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)


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.


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