1. 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

2. 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 -)

3. _ + _ + 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

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

5. 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

6. *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

7. 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

8. 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