Services at NCMR Pune

PART-II Microbial identification and characterization services

NCMR Pune offers multiple services for microbial identification and characterization. All of these services are listed below:

A. rRNA gene/ ITS sequencing for microbial identification: rRNA gene sequence of bacteria/ Internal Transcribed Sequence (ITS) of fungi is one of the most reliable molecular methods for microbial identification. We offer this sequencing service for identifying bacteria, archaea and fungi using universal primers for each type of organism.
Once the organism is received at NCMR, it goes through the following procedure: DNA isolation, PCR using universal primers for the type of organism, purification of the PCR amplicons, cycle sequencing reactions, purification and run them on an automated capillary-based Sanger DNA Sequencing system. At every step, there is in-house quality check to ensure success of the sequencing reactions. Post sequencing, fragments are manually checked and only good quality sequences are used to form contigs, which are then matched in well-curated databases for assigning closest neighbor as the tentative identification of your submitted organism.

B. Phylogenetic analysis: Phylogenetic analysis based on 16S rRNA gene is a powerful tool to study the evolutionary relationships among micro-organisms. It provides insights of genes or sequences which include relationships, origins, and closest taxonomic identification. These relationships are described by a branching of the tree.

C. Determination of Polar lipids: Lipids are one of the most verdiagram, or tree, with branches joined by nodes and leading to the terminal at the tips of satile compounds present in living system. Bacterial cell membrane is mostly composed of polar lipids like phospholipids, amino lipids and glycolipids etc. Lipids profile of a bacterial species is well suited to be used as a taxonomic character and is required to be included for description of novel taxa.

D. Determination of G+C mol% in DNA: G+C content in DNA of a species of bacteria is a characteristic feature and in bacterial world G+C mol% varies from 24% to 76%. This value is essential for description of a novel species.
There is a linear correlation between the melting temperature of DNA and G+C mol %. NCMR uses fluorimeteric method using double strand specific fluorescent dye and real-time PCR thermocyclers for determination of melting temperature (Tm) and G+C mol % is calculated from the Tm. This service includes isolation of high quality DNA from microbial culture(s), melting temperature experiment (in triplicate) and calculation of the G+C mol% content.

E. DNA-DNA hybridization: DNA-DNA hybridization (DDH) values determine relatedness between bacteria and is one of the minimum criteria for the description of novel taxon when 16S rRNA gene sequence similarity is more than 97%. Since 1960s DDH values have been used for species delineation with 70% as a recommended cut-off. At high temperature DNA gets denatured and when it cooled down gradually, it starts reassociating with the complementary sequences and results into the formation of double stranded DNA again. But when the DNA of two organisms mixed together and denatured, while reassociation of these hybrid DNA the degree of binding is directly proportional to the sequence similarity between the two DNAs. This degree of binding is converted into the percentage hybridisation. NCMR uses fluorimeteric method for the calculation of DDH.
This service includes isolation of high quality DNA from related species, hybridisation experiment (in triplicate) and calculation of the DDH value.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterisation includes: Colony morphology (such as size, shape, colour, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterization includes: Colony morphology (such as size, shape, color, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.
G. Phenotypic characterization by API: The Analytical Profile Index (API) is a miniaturized panel of biochemical tests compiled for identification of groups of closely related bacteria. Different test panels are available in dehydrated forms which are then reconstituted at the time of use by addition of bacterial suspensions of desired cell density. After proper incubation, test results are scored (after 24 and some time 48 hr) as +/- to generate a seven-digit code (profile). Identity of the bacterium is then derived from the database with the relevant cumulative profile code book or software (apiweb™).
NCMR offers phenotypic characterization by four type of API methods using either one or a combination of strips depending on the users’ requirements.
API 20E: API 20 E is a standardized characterization/identification system for Enterobacteriaceae and other non-fastidious, Gram-negative rods. It consists of 20 microtubes containing dehydrated substrates.
API 20NE: A standardized system characterization/identification of non-fastidious, non-enteric Gram-negative rods (e.g. Pseudomonas, Acinetobacter, Flavobacterium, Moraxella, Vibrio, Aeromonas, etc.), combining 8 conventional and 12 assimilation tests.
API 50 CH: Used for characterization/identification Bacillus and related genera as well as gram-negative rods belonging to Enterobacteriaceae and Vibrionaceae families based on fermentation of 49 carbohydrates.
API ZYM is a semi-quantitative micro-method designed for detection of enzymatic activities.

For more details on these services visit: http://210.212.161.138/services

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