Elzu's micro notes

  VOGES-PROSKAUER TEST   Aim To differentiate bacteria that produces neutral product acetoin during fermentation of glucose. Principle VP test is a biochemical test that detects the ability of bacteria to metabolize the pyruvate into a neutral intermediate product called ‘acetylmethylcarbinol’ or acetoin .   Pyruvic acid, the important  compound in the fermentative degradation of glucose, is further metabolized through various metabolic pathways, depending on the enzyme systems possessed by different bacteria.   Certain bacteria produce acetoin (acetyl methyl carbinol), a neutral-reacting end product from glucose metabolism.   In the presence of alkali and atmospheric oxygen,  the acetoin reacts with alpha naphthol and oxidised to form diacetyl. Diacetyl condense with  quanidine -containing compounds found in the  peptones of the broth to form a  ruby pink colour.     Materials required · ...

  METHYL RED TEST Aim To differentiate the organism for the production of large amount of acid during fermentation of glucose. Principle The methyl red (MR) test identifies bacteria capable of producing stable acidic end products from glucose fermentation. MR test along with the  VP test is performed simultaneously because they are physiologically related and are performed in MRVP broth. Bacteria which metabolizes pyruvic acid by the mixed acid pathway, produces more acid end products, such as lactic acid and acetic acid, and maintain an acidic environment. The methyl red detects mixed acid fermentation that lowers the pH of the broth. The MR indicator is added after incubation which is red at pH 4.4 and yellow at pH 6.2. If the organism produces a large number of organic acids that include formic acid, acetic acid, lactic acid, and succinic acid from glucose fermentation, the broth medium will remain red after the addition of methyl red, a pH indicator. MR-negative o...

  Aim To test whether the given bacterial culture can produce indole from tryptophan.   Principle The indole test demonstrates the ability of certain bacteria to decompose the amino acid  tryptophan   to   indole. Tryptophanase is an enzyme produced by certain bacteria to degrade the tryptophan in the peptone and other components of media to indole, pyruvate, indole acetic acid and ammonia. Indole  combines p-dimethylaminobenzaldehyde in   Kovac’s reagent   and the solution turns from yellow to   cherry red colour. Kovac’s reagent   also contains  amyl alcohol which is  not water soluble, the red coloration will form in an  oily layer at the top of the broth.   Materials required ·        24-48 hour old bacterial culture ·        Kovac’s reagent ·        Tubes containing 1% Tryptone broth(5 ml in each tub...

  IMViC test consist of four different tests namely in Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test. These tests are mainly for differentiating the various species of Gram negative bacteria based on the biochemical and metabolic activities of these bacteria.   The name IMViC stands for first letter of the name of each test with ‘i’included for ease of pronunciation. IMViC test are used to determine the sanitary quality of water, food etc. The significance of these tests is that when testing food or water for the presence of sewage indicator E. coli, one must be able to rule out Enterobacter aerogenes , which has many of the morphological and physiological characteristics of   E. coli. Since Enterobacter aerogenes is not always associated with sewage, Its presence would not necessarily indicate the sewage contamination.

 Nucleotide Sequence Databases  Nucleotide Sequence Databases are data repositories that accept nucleic acid sequence data and make it freely available to public. The data in these repositories are heterogenous with respect to the source of material, quality, annotation and intended completeness of sequence relative to its biological target.  Nucleotide Sequence Databases are of 2 types 1) Primary Sequence Databases- Genbank, EMBL, DDBJ, TrEMBL  2) Secondary Sequence Databases-Swiss Prot, Prosite, PDB International  Nucleotide Sequence Database Collaboration consist mainly 3 databases; Genbank, EMBL, DDBJ. These 3 databases exchange and update data on a daily basis to achieve optimal synchronization. Genbank GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences .It is a primary database of nucleotide.  GenBank  is accessed and searched through  Entrez gateway at NCBI. User can ...

 SWISS-PROT SWISS-PROT is a secondary databases which provides detailed sequence annotation that includes structure, function, and protein family assignment. It was established in 1986.It is maintained collaboratively by SIB (Swiss Institute of Bioinformatics) and EBI/EMBL. Provides high-level annotations, including description of protein function, structure of protein domains, post-translational modifications, variants, etc. It aims to be minimally redundant. Swiss-Prot is linked to many other resources, including other sequence databases. The sequence data are mainly derived from TrEMBL, a database of translated nucleic acid sequences stored in the EMBL database. The annotation of each entry is carefully curated by human experts and thus is of good quality. The protein annotation includes function, domain structure, catalytic sites, cofactor binding, posttranslational modification, metabolic pathway information, disease association, and similarity with other sequences.