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Introduction to Lab Ex. 16 Diagnostic Media Urea broth Sulfide Indole Motility Citrate Introduction to Lab Ex. 16 – Diagnostic Media – Urea, SIM, Citrate Urease: Urea is a protein compound. Bacteria that are able to break down the molecule have access to a lot of protein nutrients. Bacteria that have the enzyme urease are able to use the urea. Urea------------- CO2 +NH3 The reaction is made observable by including a pH indicator in the medium. Phenol red is the indicator (is peach color at neutral pH, turns yellow below 6.8 and pink above 8.4). The release of ammonia by the breakdown of urea results in the pH of the medium becoming alkaline turning the medium pink. Thus pink color indicates production of urease by the organisms and no pink color indicates a negative test for urease. S.I.M. Agar: This test tests 3 different characteristics of the bacterium (combination test). The 3 characteristics tested are: hydrogen sulfide production, indole production and motility Hydrogen sulfide gas may be produced by bacteria that are able to metabolize certain sulfur containing compounds (amino acids/ thiosulfate compounds). There are at least 2 different enzymes bacteria may use for sulfide production: The amino acid Cysteine contains sulfur in its structure and may be broken down by bacteria that are able to produce the enzyme cysteine desulfurase. This results in the production of hydrogen sulfide gas, which will get trapped in the agar. The bacteria that are able to produce another enzyme thiosulfate reductase metabolize thiosulfates with the production of hydrogen sulfide gas. In order to visualize the presence of H2S in the medium an indicator is included: iron salts will react with H2S and form Fe2S, which is a black precipitate. Hence the black discoloration of the medium indicates H2S production by the bacterium. Indole is a compound that is produced by bacteria when they metabolize the amino acid tryptophan. Those bacteria able to produce the enzyme tryptophanase break down the tryptophan and produce indole. Indole can be visualized by its reaction with a reagent Kovac’s reagent forming a red color. After incubation, a few drops of the reagent is added to look for red coloration. Motility is a characteristic seen in some bacteria that have flagella (motility organelle- prokaryotic flagella are very different in structure and mode of action from the eukaryotic flagella). Presence of motility is tested by the use of a semi-solid medium (reduced concentration of agar in the medium – 0.9%). Motile bacteria show dispersed growth (seen as turbidity or cloudiness) in contrast to non-motile bacteria that are restricted in their growth to the stab line. Citrate: The Simmon’s Citrate medium tests the ability of the bacteria culture to be able to use citrate as the sole C source. Bacteria that are able to produce the enzyme citrase are able to transport the citrate into the cell and use it as a source of C. Since the medium does not contain any other source for C, only those bacteria that can produce citrase are able to grow in this medium. When cultures are able to use the citrate they break it down, producing sodium bicarbonate, which changes the pH of the medium to alkaline. The pH indicator in the medium (bromothymol blue) changes to a blue color from its original green color.