495040421-Tools-for-Systematic-Study-of-Bacteria.pptx

Tools for the systematic study of bacteria

1. TOOLS FOR SYSTEMATIC STUDY OF BACTERIA ANJANA JOSEPH WHO R U?

2. Classification and identification CLASSIFICATION :- placing organisms in groups of related species. lists of characteristics of known organisms. IDENTIFICATION:- Matching characteristics of an unknown to lists of known organism .

3. TECHNIQUES Methods used for identification of unknown bacterium . Approaches used for classifying and identifying micro organism are divided into classical :- Classical approaches to taxonomy make use of phenotypic characteristics of bacteria molecular:- use modern molecular techniques to identify phylogenetic relationships .

4. Classical approaches Morphological characteristics Biochemical test Differential staining Ecological analysis Genetic analysis Serolgy Phage typing Flow cytometry

5. 1.Morphological characteristics Morphology is easy to study and analyze. Morphological comparisons are valuable because structural features depend on the expression of many genes, are usually genetically stable, and normally do not vary greatly with environmental changes. Transmission and Scanning electron microscopes can be used.

6. 1.Morphological characteristics

7. 2.Biochemical test set of chemical tests that are made to the microorganisms present in a sample in order to identify them. Presence of bacterial enzymes. Each species of bacteria has a well-defined set of metabolic activities different from all other species. These biochemical "fingerprints“ are properties controlled by bacterial enzymes . Provides insight to species niche in the ecosystem. Specific series of biochemical test have been developed for fast identification. Rapid identification methods (6-24 hrs, several tests at a time) .also called numerical identification

8. 3.Differential staining Gram staining to differentiate gram positive and gram negative bacteria Acid fast stain Staining is based on composition of cell wall Staining cannot done for wall-less

9. 3.Differential staining

10. 4.Ecological Analysis The ability of a microorganism to colonize a specifi c environment is of taxonomic value. Some microbes may be very similar in many other respects but inhabit different ecological niches, suggesting they may not be as closely related . Some examples of taxonomically important ecological properties are life cycle patterns; the nature of symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as requirements for temperature, pH, oxygen, and osmotic concentration

11. 5.Genetic analysis study of chromosomal gene exchange through transformation, conjugation, and transduction is sometimes useful in their classification. Transformation can occur between different prokaryotic species but only rarely between genera. The demonstration of transformation between two strains therefore provides evidence of a close relationship. disadvantage:- an absence of transformation may result from factors other than major differences in DNA sequence.

12. 6.Serology Serology is the science that deals with serum and immune responses within serum Solutions of antibodies used for identification of medically important microorganisms are called antisera . Combine known antiserum with unknown bacterium . Serological test can differentiate not only among microbial species , but also among strains within species. Tests:-slide agglutination test,ELISA,Western blotting.

13. 7.PHAGETYPING It is a test for determining which phages a bacterium is susceptible to Bacteriophages (phages) are bacterial viruses and they usually cause lysis of bacteria . They are highly specialised,they usually infect only members of particular species, or even particular strain within species.

14. PHAGE TYPING

15. 8.Fatty acid profiles Bacteria synthesize a wide variety of fatty acids These fattyacids are constant for a particular species Fatty acid profiles [FAME] Fatty acid methyl esters. Bacterial cultures are raised onto specific media , esterified using alkali catalyzed reaction and analyzed by gas chromatography using a microbial identification system.

16. 9.Flow cytometry It is used to identify a bacterium without culturing the bacterium. Moving fluid containing bacterium is allowed to pass through small pore. Detects the presence of bacteria by detecting the difference in electrical conductivity between the cell and the surrounding medium. If the fluid is passed with laser ,scattering of light provides cell size, shape,density,mass

17. FLOW CYTOMETRY

18. Molecular characteristics Modern techniques based on phyolgenetic relationships.

19. 1.DNA base composition The base composition of a single species is theoretically a fixed property. Base composition is expressed as percentage of guanine plus cytosine content. Comparison of G+C content can reveal species relatedness. Two organisms that are closely related and hence have many identical or similar genes will have similar amount of various bases in their genome More than 10% ,species are un related. Mol%G+C = G+C/(G+C+A+T) *100

20. Methods to find G+C content Hydrolysis of DNA and HPLC. From melting temperature. Animals and plants =30 % Mo=25-80 %

21. 2.Nucleic acid hybridisation Similarity between genomes are compared. ds dna -> ss dna ->adding probes-> adding radio labelled ss dna -> radiography The degree of similarity or homology is expressed as the percent of experimental DNA radioactivity retained on the filter compared with the percent of homologous DNA radioactivity bound under the same conditions . Two strains whose DNAs show at least 70% relatedness under optimal hybridization conditions and less than a 5% difference in T m often, but not always, are considered members of the same species.

22. NA hybridisation If DNA molecules are very different in sequence, they will not form a stable, detectable hybrid. Therefore DNA-DNA hybridization is used to study only closely related microorganisms. More distantly related organisms can be compared by carrying out DNA RNA hybridization experiments using radioactive ribosomal or transfer RNA . Southern hybridisation,DNA chips,FISH

23. 3.Nucleic acid sequencing small subunit rRNAs are almost ideal for studies of microbial evolution and relatedness because they play the same role in all microorganisms. because the ribosome is absolutely necessary for survival and the rRNAs are part of the complex ribosome structure, the genes encoding these rRNAs cannot tolerate large mutations. Thus these genes change very slowly with time and do not appear to be subject to horizontal gene transfer, an important factor in comparing sequences . Oligo nucleotide signature sequences variable region –closely related species Stable region-distantly related species.

24. 4.Genomic finerprinting capacity of restriction endonucleases to recognize specific nucleotide sequences. Thus the pattern of DNA fragments generated by endonuclease cleavage (called restriction fragments) as examined by gel electrophoresis is a direct representation of nucleotide sequence. The comparison of restriction fragments of specifi c genes or DNA fragments is the basis of restriction fragment length polymorphism (RFLP) analysis. Rrna - ribotyping

25. 5.Aminoacid sequencing Determination of the amino acid sequence of proteins with the same function. If these sequences are similar, the organisms possessing them may be closely related. The sequences of cytochromes and other electron transport proteins, histones and heat-shock proteins, transcription and translation proteins, and a variety of metabolic enzymes have been used in taxonomic and phylogenetic studies.

26. Relative taxonomic resolution of various techniques.

27. REFERENCES 1.Presscott’s principles of microbiology 2.Microbiology an introduction-Gerald J tortora . THANKYOU…