Exercise 39: Oxidation and Fermentation Tests Lab 9 Goals and Objectives: Exercise 39: Oxidation and Fermentation Tests Read results: some tubes will require additional reagents *Share and record all class results* Exercise 38: Cultural Characteristics Each person inoculate one nutrient gelatin deep Exercise 40: Hydrolytic and Degradative Reactions Each person inoculate one urea broth Exercise 41: Multiple Test Media Each person inoculate one SIM medium deep Exercise 69: Gram Negative Intestinal Pathogens Each person inoculate one MacConkey agar plate Make new streak for isolation on BHIA Each pair will need: 2 Nutrient Gelatin Deeps Methyl Red 2 Urea Broths Alpha Naphthol 2 SIM Medium Deeps KOH 2 MacConkey Agar Plates 2 BHIA plates

Durham Sugar Tube Fermentation (Lactose or Mannitol) Inoculation method: loop transfer Contains: single carbohydrate (lactose or mannitol) 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 (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 Figure 39.7

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 _ + Figure 39.8

_ _ + + 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 will 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 _ _ + + Figure 39.8

_ + 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 _ + Figure 39.9

New Inoculations from single colonies on BHIA: Nutrient Gelatin: stab with needle (Exercise 38) Urea Broth: loop (Exercise 40) SIM Medium: stab with needle (Exercise 41) MacConkey Agar: streak for isolation with loop (Exercise 69) BHIA: streak for isolation with loop Discard previous plates and tubes Share all results with class

Exercise 39: Oxidation and Fermentation Tests Lab 9 Goals and Objectives: Exercise 39: Oxidation and Fermentation Tests Read results: some tubes will require additional reagents *Share and record all class results* Exercise 38: Cultural Characteristics Each person inoculate one nutrient gelatin deep Exercise 40: Hydrolytic and Degradative Reactions Each person inoculate one urea broth Exercise 41: Multiple Test Media Each person inoculate one SIM medium deep Exercise 69: Gram Negative Intestinal Pathogens Each person inoculate one MacConkey agar plate Make new streak for isolation on BHIA Each pair will need: 2 Nutrient Gelatin Deeps Methyl Red 2 Urea Broths Alpha Naphthol 2 SIM Medium Deeps KOH 2 MacConkey Agar Plates 2 BHIA plates