Medical

Hippurate  Test  Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse substrate hippurate into glycine and benzoic acid by action of hippuricase enzyme present in bacteria. Hippuricase is a constitutive enzyme that hydrolyzes the substrate hippurate to produce the amino acid glycine. Glycine is detected by oxidation with Ninhydrin reagent, which results in the production of a deep purple color.  Objective To detect the production of the enzyme hippuricase for the presumptive identification of a variety of microorganisms. Principle Hippurate is the glycine conjugate of benzoic acid.  When hippurate is hydrolysed by an organism glycine and benzoic acid are formed. Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during the process. The end products of the ninhydrin oxidation react to form a purple-colored product. Reagent and material Hippurate disk Ninhydrin reagent  Normal saline  Bl...

Kligler’s Iron Agar Test  The Kligler’s Iron Agar test employs a medium for the identification of Enterobacteriaceae, based on double sugar fermentation and hydrogen sulphide production.   Principle Hydrogen sulfide determinations using Kligler Iron Agar should be limited to members of Enterobacteriaceae. Media: Composition of Kligler’s Iron Agar Beef extract: 3 gm Yeast extract: 3 gm Peptone: 15 gm Proteose peptone: 5 gm Lactose 10 gm Glucose: 1 gm Ferrous sulfate: 0.2 gm Sodium chloride: 5 gm Sodium thiosulfate: 0.3 gm Agar: 12 gm Phenol red: 0.024 gm Distilled water to equal 1 L Final pH : 7.4 Method 1.     Stab the center of the medium into the deep of the tube to within 3-5mm from the bottom. 2.     Withdraw the inoculating Loop and streak the surface of the slant. 3.     Loosen closure on the tube before incubating. 4.     Incubate aerobically at 35ºC. ...

SIM (SULFIDE, INDOLE, MOTILITY) MEDIUM  SIM Medium is designed to allow the detection of sulfide production, indole formation and motility. Principle The medium contains ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide. Hydrogen sulfide production is detected when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with H 2  S gas. Casein peptone, another component of SIM Medium, is rich in tryptophan. Organisms possessing the enzyme tryptophanase degrade tryptophan to indole. Indole is detected upon the addition of Kovacs Reagent  following incubation of the inoculated medium. Indole combines with p-dimethylaminobenzaldehyde and produces a red band at the top of the medium. A negative indole test produces no color change upon the addition of Kovacs Reagent. The small amount of agar added to the medium provides a semi-solid structure allo...

Bacitracin Susceptibility Test  The test is used for presumptive identification and differentiation of beta-hemolytic group A streptococci ( Streptococcus pyogenes – susceptible) from other beta-hemolytic streptococci. It is also used to distinguish staphylococci species (resistant) from micrococci (susceptible). Method 1.     Using an inoculating loop, streak two or three suspected colonies of a pure culture onto a blood agar plate. 2.     Using heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Gently tap the disk to ensure adequate contact with the agar surface. 3.     Incubate the plate for 18 to 24 hours at 35°-37°C in ambient air for staphylococci and in 5% to 10% carbon dioxide (CO2) for streptococci differentiation. 4.     Look for a zone of inhibition around the disk. Medium Used: Mostly, blood agar plate is used (trypticase soy agar...

Bile Esculin Test  Bile esculin agar  is a selective and differential medium which is used to presumptively identify enterococci and group D streptococci based on the ability of an organism to hydrolyze esculin. Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin). The two moieties of the molecule (glucose and 7-hydroxycoumarin) are linked together by an ester bond through oxygen. Many bacteria can hydrolyze esculin, but few can do so in the presence of bile. Thus the bile esculin test is based on the ability of certain bacteria, notably the group D streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile). Bacteria that are bile-esculin positive are able to grow in the presence of bile salts and the hydrolysis of the esculin in the medium results in the format...

Novobiocin Susceptibility Test   for differentiating of  Staphylococcus spp . which included a novobiocin disk test. Principle  Susceptibility to novobiocin is determined by placing a novobiocin-impregnated paper disk on a agar plate seeded with the organism under investigation. As the organism multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the antibiotic diffusing into the agar from the paper disk. If the bacteria are susceptible to novobiocin, there will be a formation of visible zone of inhibition around the disk, representing an area where the antibiotic concentration has prevented bacterial growth. No zone of inhibition around the disk represents that organism is resistant to the anitibiotic.   Novobiocin Disc: Disk is prepared by impregnating 5ug of novobiocin onto high quality 6mm diameter filter paper disks (commercially available). Method 1.  ...