3 starch plates 5 urea broths (replaces urea slant) Lab 11 Goals and Objectives: Exercise 39: Oxidation and Fermentation Tests Read 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 Reactions Set 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 aureus Escherichia coli Proteus vulgaris Each pair needs: 3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants 3 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 transfer Contains: single carbohydrate peptone broth with durham tube for gas collection, Phenol red pH indicator: alkaline pH = red, acidic pH = yellow Discriminates 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 carbohydrate Yellow = positive for fermentation of carbohydrate to acid products Yellow with bubble = positive for fermentation of carbohydrate to acid + gas

Acid plus gas Acid Negative

_ + MR-VP Medium: Methyl Red Test Inoculation method: loop transfer Contains: 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 pathway Results: Red = positive for mixed acid formation Yellow = negative for mixed acid formation _ +

_ _ + + MR-VP Medium: Voges-Proskauer Test Inoculation method: loop transfer Contains: peptone and glucose Additional 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 pathway Results: Red = positive for acetoin and thus for 2,3-butanediol production Yellow/Orange = no acetoin, negative for 2,3-butanediol production _ _ + +

_ + Simmon’s Citrate Agar Inoculation method: streak and stab slant with needle Contains: citrate as sole carbon source, ammonium salts as sole nitrogen source, bromthymol blue pH indicator: neutral pH = green, alkaline = prussian blue Discriminates 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 production Green = negative for citrase production _ +

Oxidase Test Discriminates 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 Test Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen _ + http://ftp.ccccd.edu/dcain/CCCCD%20Micro/Catalase.jpg

12 Possible Unknowns Gram Positive Gram Negative Gelatinase + Catalase + Catalase - Pseudomonas aeruginosa Bacillus subtilis

Nitrate Reduction Broth Inoculation method: loop transfer Contains: beef extract, peptone, KNO3 as nitrate source, durham tube for gas collection Additional 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 reduction Red only after zinc = negative for nitrate reduction, negative for nitrate reductases Clear 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) A + B + nitrite = red Zinc converts nitrate to nitrite sulfanilic acid (reagent A) + dimethyl-alpha-naphthylamine (reagent B) add zinc to negative tubes Nitrate to nitrite Complete reduction No reduction

Fig. 40.1 Unknowns Separate Plates! broth Control broth

3 starch plates 5 urea broths (replaces urea slant) Lab 11 Goals and Objectives: Exercise 39: Oxidation and Fermentation Tests Read 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 Reactions Set 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 aureus Escherichia coli Proteus vulgaris Each pair needs: 3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants 3 spirit blue plates 5 tryptone broths ***Save streak plates of unknowns for use next class***