Biochemical Rxn PDF

Fermentation rxns Sugars Sugar fermentation tubes= sugar, media with pH indicator, Durham tube Fermentation: sugar (oxidized then reduced) → organic compounds (lactic acid, alcohol) Aerobic respiration: sugar (oxidized) → CO2 gas + H2O Ferments sugar=color change ○ Posi...

Fermentation rxns Sugars Sugar fermentation tubes= sugar, media with pH indicator, Durham tube Fermentation: sugar (oxidized then reduced) → organic compounds (lactic acid, alcohol) Aerobic respiration: sugar (oxidized) → CO2 gas + H2O Ferments sugar=color change ○ Positive: red/purple to yellow, below pH7 (from acidic products) Produces gas=gas bubble MR-VP MR-VP: methyl red and voges proskauer test Used to differentiate between intestinal bacteria coliforms Ferment dextrose to acid products called mixed acid fermentation Methyl red: detects mixed acid fermentation (changes from yellow to red) Butanediol formation by Voges Proskauer test measures presence of acetoin (precursor to butanediol) Voges Proskauer test: uses Barritt’s reagents A and B to detect acetoin in Butanediol fermentation pathway (tube shaken) ○ pink/red is positive for acetoin Enzyme Production Exoenzymes: enzymes that are excreted to degrade large polymers Endoenzymes: produced in cell and catalyze intracellular rxns Amylase Production Amylase (exoenzyme) degrades stretch ○ Detected with Lugol’s Iodine on starch agar Lugol’s iodine binds to starch ○ Amylase Present: starch degraded, clear zone  ○ Amylase absent: no clear zone Catalase production Catalase (endoenzyme) detoxifies hydrogen peroxide (lethal byproduct of aerobic respiration) ○ TSA plate ○ 2H2O2→ 2H2O + O2 Catalase present: oxygen bubbles Oxidase Production Oxidase (endoenzyme) reduce oxygen during respiration ○ Cytochrome oxidase is final component in ETC for bacteria, reducing O2 to H2O Detected with oxidase reagent ○ Positive is pink/maroon/dark blue/black color ○ Negative is colorless Indole Production (Tryptophan hydrolysis) Tests for tryptophanase Tryptone broth Tryptophan (hydrolyzed) → indole + pyruvic acid + ammonia Indole detected with Kovac’s reagent ○ Positive: red top layer ○ Negative: brown top layer Citrate Utilization Citrate metabolism distinguishes between enterobacteriaceae Simmons citrate agar Citrate utilization produces alkaline carbonate which increases pH Bromothymol blue is indicator dye ○ Negative: green, neutral pH ○ Positive: dark blue, citrate utilized Single Media, Multiple tests SIM tubes Sulfide-indole-motility Distinguishes among gram - found in gut Motility observed if bacteria migrate from stab line Sulfide production if medium blackens ○ H2S reacts with ferrous ammonium sulfate to form black precipitate Indole presence is red color after Kovac’s reagent added TSI (triple sugar iron agar) Triple sugar iron ○ Dextrose, lactose, sucrose Anaerobic aerobic environment Results ○ nonfermenter= slant/butt red ○ Ferment dextrose only= slant/butt yellow first, slant red after 24 hrs ○ Ferment dextrose, lactose and/or sucrose= slant/butt yellow ○ Gas produced= cracks in media ○ Sulfide produced= black precipitate