Medical

Simmons Citrate Reaction It  used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae. Principle  Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains  citrate  as the sole  carbon source  and  inorganic ammonium salts (NH4H2PO4)  as the sole source of  nitrogen . Bacteria that can grow on this medium produce an enzyme,  citrate-permease , capable of converting  citrate  to  pyruvate .  Pyruvate  can then enter the organism’s metabolic cycle for the production of  energy . Growth is indicative of utilization of citrate, an intermediate metabolite in the  Krebs cycle . 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 blue  above pH 7.6. Christensen  developed an alternative citrate test medium that does not require the organism

Urease Test The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. Principle  Urea  is the product of decarboxylation of  amino acids . Hydrolysis of  urea  produces  ammonia  and  CO2 . The formation of  ammonia  alkalinizes the medium, and the pH shift is detected by the color change of  phenol red  from  light orange  at pH 6.8 to  magenta (pink)  at pH 8.1. Rapid urease-positive organisms turn the entire medium  pink  within 24 hours. Weakly positive organisms may take several days, and negative organisms produce  no color change  or  yellow  as a result of  acid production . Uses  1.     This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease. 2.     This test can be used as part of the identification of several genera and species of Enterobacteriaceae, including  Proteus, Klebsiella ,

Bile Solubility Test Bile Solubility Test is the test which differentiate  Streptococcus pneumoniae  (positive- soluble) from alpha-hemolytic streptococci (negative- insoluble).  Streptococcus pneumoniae  is bile soluble whereas all other alpha-hemolytic streptococci are bile resistant. Principle  S. pneumoniae  has an autolytic enzyme which can be demonstrated by allowing a broth culture to age in the incubator; at 24 hours the broth is turbid; after a few days the medium will become clear. Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process. Bile salts activate the autolytic enzyme which induces clearing of the culture. Reagents  Bile salt preparation 1-Prepare 10% bile salt solution for plate m

PYR Test  (Pyrrolidonyl-β-naphthylamide) PYR is a rapid method for presumptive identification of bacteria based on the pyrrolidonyl arylamidase enzyme.  Principle The enzyme  L- pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl- β-naphthylamide substrate to produce a β-naphthylamine. The β-naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde reagent by the production of a bright red precipitate. Following hydrolysis of the substrate by the peptidase, the resulting b-naphthylamide produces a red color upon the addition of 0.01% cinnamaldehyde reagent. When a visible inoculum of microorganism is rubbed onto a small area of a disk impregnated with the substrate, the hydrolysis occurs within 2 min, at which time the cinnamaldehyde reagent is added to detect the reaction by a color change to purple. Uses of PYR Test 1.     It is used for the presumptive identification of group A streptococci ( Streptococcus pyogenes ). 2.     It is used for