Identification of micro organisms

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1. Akash Mahadev Iyer 2 nd semester M.Sc Biochemistry University of Kerala Methods to Identify Microorganisms

2. Microscopic methods Cultural methods Physiological methods Immunological methods Molecular methods (Nucleic acid based methods) Methods to Identify Microorganisms in Wastewater

3. Bright Field Dark Field Phase contrast Fluorescence

4. Gram’s Staining Flagella Staining Endospore Staining Negative Staining

5. MFT Anaerobic Cultivation Cultural Methods

6. Media Organism XLD agar Salmonella S1 medium (Sucrose, SLS, casamino acids, Trimethoprim ) Fluorescesnt Pseudomonads Agar medium containing K + buffers, Mg 2+ salts, Trace elements, NaNO 2 , Bromothymol blue Nitrosomonas and Nitrobacter BG11 medium BGA Bold’s Basa medium ([phone] lux light) Green and yellow-green Algae PDA, MEA (+Rose Bengal) Fungi

7. This medium supports growth of photoautotrophic blue green algae . They require light as source of energy. Synthetic nitrogen and carbon sources and other inorganic salts comprise this medium . Exposure to light intensity of 2,000 to 3,000 lux is optimal for cultivation of blue green algae. Neon light source is found to be sufficient to provide this illumination. For maintenance of blue green algae exposure for period of 24 hours a day is optimal .Often the flasks kept for incubation may be covered with grease proof paper. They grow optimally at room temperature between range of 20-25°C.

8. Physiological Methods Provides information about what activities are carried out in microbial communities- Contribution of microbe in the environment Detection and characterization of unknown compounds- MS Distinguish metals- ICP-MS , eg ; Measures oxidation of S to sulphate . Estimates sulphate as a precipitate with Barium chloride Winkler method- DO- Based on precipitation of dissolved oxygen using Manganous sulphate and a KOH-KI mixture The oxygen precipitate, MnO 2 reacts with sulfuric acid to form manganic sulphate which inturn reacts with KI to liberate iodine No. of moles of Iodine = No. of moles of oxygen present

9. Cell mass- Turbidimetric method Measure protein- Folin lowry , BCA, Bradford assay

10. Enzymes- Dehydrogenase (TTC), Esterase (FDA)

11. Immunological methods

12. Immunoaffinity Chromatography Immobilisation of antibody onto a matrix, normally beads, which are then placed into a chromatography column. The columns are made by reacting highly purified antibody (monoclonal or polyclonal) with the chromatography beads to form the affinity matrix.

13. Immunofluorescence

14. Phage Typing Determining which phages a bacterium is susceptible to. The tested strain was grown over entire plate; known phages are placed in different squares; plaques (areas of lysis ) appear dark indicating sensitivity to a specific phage

15. Flow cytometry FACS

16. Molecular methods DNA base composition- Guanine + cytosine moles% (GC) DNA fingerprinting- Electrophoresis of restriction enzyme digests rRNA sequencing Gene Probing- ssDNA fragments to identify particular nucleic acid sequence Hybridization Microarray Polymerase chain reaction (PCR) Metagenomics

17. Fingerprinting- Hybridization

18. Gene probe based detection of DNA- Colorimetric, Fluorescence, Chemiluminesence

19. Microarray A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene. Often, these slides are referred to as gene chips or DNA chips . The DNA molecules attached to each slide act as probes to detect gene expression.

20. FISH Analysis of Sewage Sludge

21. PCR

22. 16s rRNA sequence analysis 16S rRNA most conserved (least variable) in all cells. Portions of the sequence from distantly related organisms are remarkably similar . Sequences from distantly related organisms can be precisely aligned, making the true differences easy to measure. Genes that encode the rRNA ( rDNA ) have been used extensively to determine taxonomy, phylogeny (evolutionary relationships), and to estimate rates of species divergence among bacteria . Thus, the comparison of 16s rRNA sequence can show evolutionary relatedness among microorganisms .

23. RIBOSOMAL RNA • To infer relationships that span the diversity of known life, it is necessary to look at genes conserved through the billions of years of evolutionary divergence. Examples of genes in this category are those that define the ribosomal RNAs ( rRNAs ). In Bacteria, Archaea , Mitochondria, and Chloroplasts, the small ribosomal subunit contains the 16S rRNA (where the S in 16S represents Svedberg units). The large ribosomal subunit contains two rRNA species (the 5S and 23S rRNAs ). Most prokaryotes have three rRNAs , called the 5S, 16S and 23S rRNA . Bacterial 16S, 23S , and 5S rRNA genes are typically organized as a co-transcribed operon . There may be one or more copies of the operon dispersed in the genome (for example, E coli has seven ). The Archaea contains either a single rDNA operon or multiple copies of the operon rRNA targets were studied originally, most researchers now target the corresponding ribosomal DNA ( rDNA ) because DNA is more stable and easier to analyse

24. Conventional microbiology techniques -to identify most of the bacteria, fungi and other pathogens- about 8 to 20 hours for an accurate result. The presence of hyper variable regions in the 16S rRNA gene provides a species specific signature sequence which is useful for bacterial identification process.

25. Metagenomics - Environmental genomics Metagenomics is the study of the metagenome —the collective genome of microorganisms from an environmental sample—to provide information on the microbial diversity and ecology of a specific environment. DNA is cloned from microbial community and sequenced Detects as many genes as possible Yields picture of gene pool in environment Powerful tool for assessing the phylogenetic and metabolic diversity of an environment

26. Metagenomics