Elzu's micro notes

  WHATCHECK Aim To check the steriochemical properties of given protein by  WHATCHECK method. Principle Homology modeling is rapidly becoming the method of choice for obtaining three dimensional coordinates for proteins because genome projects produce sequences at a much higher rate than NMR and X ray laboratories can solve the three dimensional structures. However , the quality of protein models will not be immediately clear to invoices and support with the evaluation seems to be needed. Expert users are sometimes interested in evaluating the quality of modeling programs rather than the quality of the model themselves. WHATCHECK has been derived from a subset of protein verification tools from the Whatif program, this does extensive checking of many sterochemical parameters of the residues in the model. The final homology model has to be evaluated to make sure that the structural features of the model are consistent with the physiochemical rules. This involves chec...

  Citrate Utilisation Test Aim To differentiate bacteria on the basis of their ability to utilise citrate as the sole carbon source. Principle Citrate utilisation test identifies the ability of an organism to utilize citrate as a source of energy. Certain bacteria possess citrate permease and citrate lyase enzymes, enabling them to utilize sodium citrate as the sole carbon source and ammonium salts as the nitrogen source for growth, with production of CO₂. Simmons citrate agar is formulated with sodium citrate (carbon source), ammonium dihydrogen phosphate (nitrogen source), buffers, and bromothymol blue (pH indicator; green at pH ≈6.9). When the bacteria metabolize  citrate , the  ammonium salts  are broken down to  ammonia , which increases  alkalinity .  The shift in pH turns the bromthymol blue indicator in the medium from green to Prussian blue above pH 7.6   Materials required Simmon citrate agar 24-48 hour old bacteri...

  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.