Exercise 38: Cultural Characteristics (Gelatin) put on ice!!! Lab 10 Goals and Objectives: Exercise 38: Cultural Characteristics (Gelatin) put on ice!!! Exercise 40: Hydrolytic and Degradative Reactions (Urea) Exercise 41: Multiple Test Media (SIM) Exercise 69: Gram Negative Intestinal Pathogens (MacConkey) Read results: some tubes will require additional reagents *Share and record all class results* Exercise 39: Oxidation and Fermentation Tests Each person inoculate one BHIA slant for catalase test Exercise 67: Staphylococci Identification Each person inoculate one MSA plate and one rabbit plasma Exercise 68: Streptococci & Enterococci Identification Each person inoculate one BAP and add Bacitracin Each pair will need: 2 BHIA Slants Kovac’s Reagent 2 Mannitol Salt Agar plates 2 Rabbit Plasma/Coagulase Test tubes 2 Blood Agar Plates 2 TaxoA/Bacitracin discs

Nutrient Gelatin Stab Inoculation method: stab with needle Contains: beef extract, peptone, high gelatin concentration to gel (no agar) Must be set on ice 5-10 min before reading Discriminates organisms that can produce gelatinases to hydrolyze the gelatin into amino acids Results: liquid = gelatin hydrolysis, positive for gelatinase production solid = negative for gelatinase production (Liquifaction patterns discussed on page 269 & Figure 38.3 are not important, we only score + or -)

_ + _ + Urea Broth Inoculation method: loop transfer Contains: yeast extract, urea, buffer, Phenol red pH indicator: acidic/neutral pH = yellow/orange, alkaline pH = bright pink Discriminates organisms that can produce urease to hydrolyze urea into carbon dioxide and ammonia Results: Bright pink = cleavage of urea into ammonia, positive for urease production Yellow/orange = no ammonia present, negative for urease production _ + _ + Figure 40.4

SIM Medium Inoculation method: stab deep with needle Contains: casein (source of tryptophan and cysteine), ferrous salts (reacts with H2S to produce black ferrous sulfide), 0.7% agar (semisolid) Additional reagents added: Kovac’s reagent, reacts with indole to produce a red product Discriminates three characteristics: S = “sulfide”, discriminates organisms that can produce cysteine desulfurase to hydrolyze the amino acid cysteine into pyruvic acid, ammonia and hydrogen sulfide I = “indole”, discriminates organisms that can produce tryptophanase to hydrolyze the amino acid tryptophan into indole, ammonia and pyruvic acid M = “motility” discriminates motility (presence of flagella), ability to “swim” through media

SIM Medium Results: S Black = formation of ferrous sulfide, hydrolysis of cysteine into hydrogen sulfide, positive for cysteine desulfurase production Colorless = negative for cysteine desulfurase production I Red with Kovac’s = cleavage of tryptophan into indole, positive for tryptophanase production Colorless = no indole present, negative for tryptophanase production M Organism growing only in line of inoculation = non-motile Organism appears as haze beyond line of inoculation = motile Figure 41.2

*Dead organisms cannot be scored for lactose fermentation!* MacConkey Agar Inoculation method: surface streak with loop Contains: bile salts, crystal violet and sodium desoxycholate to inhibit Gram positive growth, lactose, Neutral Red pH indicator: neutral pH = red, acidic pH = bright hot pink Selective and differential medium: selects for growth of Gram negative organisms by inhibiting growth of Gram positives. Of those that grow, differentiates ability to ferment lactose Results: Growth = Gram negative Bright pink = positive for lactose fermentation Pale pink = negative for lactose fermentation No growth = Gram positive, inconclusive for lactose fermentation lactose + lactose - No growth = Gram positive *Dead organisms cannot be scored for lactose fermentation!* Figure 69.2 Growth = Gram negative

New Inoculations from single colonies on BHIA: BHIA slant for catalase test: loop (Exercise 39) Mannitol Salt Agar plate: streak for isolation (Exercise 67) Rabbit plasma for coagulase: loop Blood Agar Plate: heavy & swoosh, stab in swoosh with loop, add Bacitracin disc (Exercise 68) Discard previous plates and tubes Share all results with class

Exercise 38: Cultural Characteristics (Gelatin) put on ice!!! Lab 10 Goals and Objectives: Exercise 38: Cultural Characteristics (Gelatin) put on ice!!! Exercise 40: Hydrolytic and Degradative Reactions (Urea) Exercise 41: Multiple Test Media (SIM) Exercise 69: Gram Negative Intestinal Pathogens (MacConkey) Read results: some tubes will require additional reagents *Share and record all class results* Exercise 39: Oxidation and Fermentation Tests Each person inoculate one BHIA slant for catalase test Exercise 67: Staphylococci Identification Each person inoculate one MSA plate and one rabbit plasma Exercise 68: Streptococci & Enterococci Identification Each person inoculate one BAP and add Bacitracin Each pair will need: 2 BHIA Slants Kovac’s Reagent 2 Mannitol Salt Agar plates 2 Rabbit Plasma/Coagulase Test tubes 2 Blood Agar Plates 2 TaxoA/Bacitracin discs