Wednesday, 18 February 2015

Gelatin Hydrolysis and Urease Test for Bacteria


Urease test:
About Test For Bacteria

 Urea is a nitrogen containing compound that is produced during the decarboxylation process of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is thus it is an efficient way for the human body to excess nitrogen. This excess urea is then taken away from the body with the help of the kidneys as a part of urine.
Certain bacteria produce the enzyme urease during its metabolism process and that will break down the urea in the medium to ammonia and carbon dioxide:

Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. The urease test is useful in identifying the genera Proteus, Providentia, and Morganella, which liberate this enzyme.       
                                          
Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Therefore this test serves to rapidly distinguish members of this genus from other lactose non fermenting enteric micro organisms.

Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia. The presence of urease is detectable when the organisms are grown in a urea broth medium containing the pH indicator phenol red. As the substrate urea is split into its products, the presence of ammonia creates an alkaline environment that causes the phenol red to turn to deep pink. This is a positive reaction for the presence of urease. Failure of deep pink colour to develop is evidence of a negative reaction.
The ability of an organism to move by itself is called motility. Motility is closely linked with chemotaxis, the ability to orientate along certain chemical gradients. Eucaryotic cells can move by means of different  locomotor organelles such as cilia, flagella, or pseudopods. Prokaryotes move by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Almost all spiral bacteria and about half of the bacilli are motile, whereas essentially none of the cocci are motile.
The medium mainly used for this purpose is  SIM medium ( Sulphide Indole Motility medium) which is a combination differential medium that tests three different parameters, sulphur reduction, indole production and motility. This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness. In soft agar tubes non-motile bacteria will only grow on the inoculated region. Motile bacteria will grow along the stab line and will tend to swim out away from the stabbed area. Therefore, a negative result is represented by growth in a distinct zone directly along the stab. A positive result is indicated by diffuse or cloudy growth mainly at the top and bottom of the stabbed region.
SIM agar may also be used to detect the presence of  H2S production. The SIM medium contains peptones and sodium thiosulfate  as  substrates,  and  ferrous  ammonium  sulfate, Fe(NH4)SO4,  as  the H2S  indicator. Cysteine  is  a component of the peptones used in SIM medium. Sufficient agar  is present  to make  the medium semisolid. Once H2S  is produced,  it  combines with  the  ferrous ammonium sulfate, forming an insoluble, black ferrous sulfide precipitate that can be seen along the line of the stab inoculation. If the organism is also motile, the entire  tube may  turn black. This black line or tube indicates a positive H2S  reaction; absence of a black precipitate indicates a negative reaction.

Gelatin Hydrolysis Test:
About Test For Bacteria

Gelatin, a protein derived from the animal protein collagen. It has been used as a solidifying agent in food for a long time. Robert Koch used nutrient gelatin as an early type of solid growth medium. One problem is that many bacteria have the ability to hydrolyze (liquefy) gelatin. This gelatin liquefaction ability (or inability) forms the basis for this test. Some microorganisms possess an enzyme called gelatinase, which breaks down gelatin into amino acids. Gelatin deeps contain the substrate gelatin, which is a protein produced by the hydrolysis of collagen. Organisms which hydrolyze gelatin will cause the gelatin to liquefy.
The gelatin hydrolysis tests for an organism's ability to break down the protein gelatin which is derived from collagen.  Gelatin causes the media to thicken, especially at cooler (below 28oC)  temperatures.  If the organism can release gelatinase enzymes the gelatin is broken down or liquefied.  The media is checked over a period of about a week after inoculation and incubation at room temperature, for gelatinase activity.  The tube is placed on ice for a few minutes and if the media fails to solidify it is considered a positive test.   The gelatinase reaction may be slow or incomplete.

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