Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Lab 11 Goals and Objectives:Exercise 39: Oxidation and Fermentation TestsRead results: some tubes will require additional reagents (share reagents across the bench)Do controls first so you have something to compare to!Exercise 40: Hydrolytic and Degradative ReactionsSet up according to Fig 40.1 except both of your two unknowns well separated on each type of plate and control on a different plate (one control plate per pair)One set of controls per pair using broth cultures:Bacillus subtilis Staphylococcus aureusEscherichia coli Proteus vulgarisEach pair needs:3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants3 spirit blue plates 5 tryptone broths***Save streak plates of unknowns for use next class***Durham Sugar Tube Fermentation (Glucose, Lactose, Mannitol)Inoculation method: loop transferContains: single carbohydrate peptone broth with durham tube for gas collection, Phenol red pH indicator: alkaline pH = red, acidic pH = yellowDiscriminates the ability to ferment a single carbohydrate (glucose, lactose, or mannitol) into acid products (e.g. pyruvic acid) or acid plus gas Results: Red = inert, negative for fermentation of specified carbohydrateYellow = positive for fermentation of carbohydrate to acid productsYellow with bubble = positive for fermentation of carbohydrate to acid + gasNegativeAcid plus gasAcidMR-VP Medium: Methyl Red TestInoculation method: loop transferContains: peptone, glucose, and buffer (buffer will neutralize weak acids so only strong stable acids will be detected by methyl red)Additional reagents added: methyl red pH indicator: acid pH = red, neutral or alkaline pH = yellow Distinguishes ability to catabolize glucose into stable mixed acids (lactic, acetic, and formic acids) in the mixed acid pathwayResults: Red = positive for mixed acid formationYellow = negative for mixed acid formation+_+_+_MR-VP Medium: Voges-Proskauer TestInoculation method: loop transferContains: peptone and glucoseAdditional reagents added: Barritt’s A (alpha napthol) and Barritt’s B (KOH) (will react with acetoin to produce a red product, alone produce a copper colored product) Distinguishes the ability to catabolize glucose into the neutral end product butanediol (the oxidized product is acetoin) in the butylene glycol pathwayResults: Red = positive for acetoin and thus for 2,3-butanediol productionYellow/Orange = no acetoin, negative for 2,3-butanediol productionSimmon’s Citrate AgarInoculation method: streak and stab slant with needleContains: citrate as sole carbon source, ammonium salts as sole nitrogen source, bromthymol blue pH indicator: neutral pH = green, alkaline = prussian blueDiscriminates organisms that can produce citrase to metabolize citrate into oxaloacetate and pyruvate. These organisms are forced to utilize ammonium salts as the nitrogen source producing alkaline ammonia waste.Results: Prussian blue slant and or butt = positive for citrase productionGreen = negative for citrase production+_Oxidase TestDiscriminates organisms that can produce cytochrome oxidase which catalyzes the transfer of electrons from reduced cytochrome c in the electron transport chain to molecular oxygen.Test uses NNNN-tetramethyl-p-phenylenediamine (Oxidase Reagent) as an artificial electron acceptor: when oxidized it is colorless, when reduced it turns purple*Look for color change on the bacteria, not on the cotton swab! (The reagent will turn light purple when exposed to oxygen in the air)Catalase TestDiscriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygenhttp://ftp.ccccd.edu/dcain/CCCCD%20Micro/Catalase.jpg+_12 Possible UnknownsGram PositiveGram NegativeGelatinase + Gelatinase - Gelatinase + Gelatinase -Bacillus subtilisPseudomonas aeruginosaCatalase +Catalase -Nitrate Reduction BrothInoculation method: loop transferContains: beef extract, peptone, KNO3 as nitrate source, durham tube for gas collectionAdditional reagents added: sulfanilic acid (reagent A), dimethyl-alpha-naphthylamine (reagent B), (together form a complex with nitrite creating a red product), zinc (reduces nitrate to nitrite allowing reaction with reagent A and B)Discriminates organisms that can produce nitrate reductases to utilize nitrate as a final electron acceptor resulting in the production of either nitrite (partial reduction) or to NH4, N2O or N2 gas (complete reduction).Results: Red with reagents A and B = positive for nitrate to nitrite reductionRed only after zinc = negative for nitrate reduction, negative for nitrate reductasesClear with/wo gas = positive for complete reduction of nitrate to nitrogen gas (or nongaseous N2O or NH4)sulfanilic acid (reagent A) + dimethyl-alpha-naphthylamine (reagent B)Nitrate to nitriteadd zinc to negative tubesNo reductionComplete reductionA + B + nitrite = redZinc converts nitrate to nitriteFig. 40.1ControlUnknownsSeparate Plates!brothbrothLab 11 Goals and Objectives:Exercise 39: Oxidation and Fermentation TestsRead results: some tubes will require additional reagents (share reagents across the bench)Do controls first so you have something to compare to!Exercise 40: Hydrolytic and Degradative ReactionsSet up according to Fig 40.1 except both of your two unknowns well separated on each type of plate and control on a different plate (one control plate per pair)One set of controls per pair using broth cultures:Bacillus subtilis Staphylococcus aureusEscherichia coli Proteus vulgarisEach pair needs:3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants3 spirit blue plates 5 tryptone broths***Save streak plates of unknowns for use next