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Explain the derivation of H2S in amino acid decomposition and the test to detect it
A desulfurase enzyme removes H2S from the sulfur containing amino acids (cysteine and methionine). FeSO4 in the culture medium reacts with the Fe to produce black ppt.
Explain the purpose and results expected for these tests, and what they mean; fermentation -> MR-VP test, urease test, citrate, MIO medium (3 uses), indole test, phenylalanine deaminase test, nitrate reduction, oxidase test, catalase test, OF Glucose deep
- Fermentation: Used to detect acid and gas production from carbohydrates. Tube is red at neutral pH and turns yellow when pH becomes acidic, an inverted tube is used to show if gas was produced. (note: after glucose is exhausted bacteria will use peptone and pH will be restored to neutral/alkaline).
- MRVP: To distinguish organisms that produce large amounts of acid from neutral acetoin. MR - if acid is produce then medium will remain red, if neutral products medium will become yellow. VP - addition of reagant to MR, if acetoin is present the upper part of the medium will turn red. If negative it will turn light brown.
- MIO medium: ability to convert tryptophan to indole (add kovacts reagaent, look for red), motility, and decarboxylation (bromcresol turning purple)
- Citrate: Can bacteria ferment citrate? medium becomes basic when citrate and ammonium are metabolized, indicator will turn blue
- indole test: see above?
- Phenylalanine deaminase test: Has deamination occurred? Deamination results in the creation of an organic acid, forms a complex with iron giving a green color.
- Nitrate reduction: anaerobic respiration resulting in reduction of nitrate to nitrite, nitrous oxide, or nitrogen gas. Phase 1 - red color indicates nitrites present (+), Phase 2 - addition of Zn, if red color appears (-) because nitrates were reduced. If neither is present then either nitrous oxide or nitrogen gas is present (+)
- Oxidase test: test for cytochrome c (respiration). Place on oxidate test strip. Blue/purple indicates positive test.
- Catalase test: test for catalase. Add drops of H2O2 and look for bubbling, release of O2 (+)
- OF Glucose deep: Is the glucose catabolism oxidative, fermentative, or neither? Aerobic tube and anaerobic tube. indicator turns yellow in presence of acids (indicating catabolism of carbohydrate) and dark blue in alkaline (indicating catabolism of peptone). A - yellow/green AN - green indicates oxidizer, A-yellow AN-yellow indicates fermentor, A-blue/green AN-green indicates no glucose use
Examples of selective/differential media
- MSA: selective for gram + bacteria, also must be able to survive at high salt concentration. Differential for mannitol fermentation (yellow +, red -)
- EMB: selective for gram - bacteria, differential for lactose fermentation (+ colonies become black/have metallic sheen [Escherichia and Citrobacter]).