Transcript Lab10

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
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:
1. BHIA slant for catalase test: loop
(Exercise 39)
2. Mannitol Salt Agar plate: streak for isolation
(Exercise 67)
3. Rabbit plasma for coagulase: loop
(Exercise 67)
4. 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
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