Biochemical Principles
Biochemical Test – Enterobacteriaceae
Bacterial spp incapable of fermenting glucose cannot utilize lactose.
2 enzymes necessary for a bacterium to take up lactose:
A. β-galactoside permease – facilitates the entry of lactose molecules to bac cell wall B. β-galactosidase – breaks down lactose into β-D glucose and β-D galacatose
LF possess both enzymes
NLF do not possess β-galactosidase
LLF do not possess β-galactoside permease
Glucose fermenters only (true enteric pathogen)
Glucose and lactose fermenter (most opportunistic enterics)
TRIPLE SUGAR IRON CULTURE MEDIA - TSI MEDIUM
Test whether a gram-negative rod utilizes glucose, and lactose/sucrose and forms H2S
Contents:
1% lactose, 1% sucrose and 0.1% glucose (10:10:1)
Ferrous sulfate and sodium thiosulfate
Bacterium (acid envi.) + Sodium thiosulfate à H2S gas
H2S + Ferric ions à Ferrous sulfide (black precipitate)
Phenol red (ph indicator)
Alkaline slant/Alkaline butt (K/K)
NO FERMENTATION
NOT MEMBERS OF ENTEROBACTERIACEAE
Stab and Streak
GLUCOSE FERMENTATION ONLY
Alkaline slant/ Acid butt (K/A)
18-24 hrs incubation
Acid slant/ Acid butt (A/A)
8-12 hrs incubation
Acid slant/ Acid butt(A/A)
18-24 hrs incubation
LACTOSE (SUCROSE) FERMENTATION
H2S PRODUCTION
Indicators:
Sodium thiosulfate
Ferrous sulfate
(+) black color/black precipitate
*Butt area requires acidic environment.
GAS PRODUCTION
(+) Splitting of media in the butt
(+) Formation of Bubbles
SULFIDE INDOLE MOTILITY CULTURE MEDIA - SIM MEDIUM
H2S Positive: Blackening of the medium
Motile: Diffuse growth extending laterally from line of inoculation
Stab
Indole(pink) H2S Haziness(motility)
Indole Production
Test for the ability of an organism to split tryptophan to form the compound indole.
Positive: Pink to wine colored ring after addition of Kovac’s reagent/Erlich’s
Bacterial spp incapable of fermenting glucose cannot utilize lactose.
2 enzymes necessary for a bacterium to take up lactose:
A. β-galactoside permease – facilitates the entry of lactose molecules to bac cell wall B. β-galactosidase – breaks down lactose into β-D glucose and β-D galacatose
LF possess both enzymes
NLF do not possess β-galactosidase
LLF do not possess β-galactoside permease
Glucose fermenters only (true enteric pathogen)
Glucose and lactose fermenter (most opportunistic enterics)
TRIPLE SUGAR IRON CULTURE MEDIA - TSI MEDIUM
Test whether a gram-negative rod utilizes glucose, and lactose/sucrose and forms H2S
Contents:
1% lactose, 1% sucrose and 0.1% glucose (10:10:1)
Ferrous sulfate and sodium thiosulfate
Bacterium (acid envi.) + Sodium thiosulfate à H2S gas
H2S + Ferric ions à Ferrous sulfide (black precipitate)
Phenol red (ph indicator)
Alkaline slant/Alkaline butt (K/K)
NO FERMENTATION
NOT MEMBERS OF ENTEROBACTERIACEAE
Stab and Streak
GLUCOSE FERMENTATION ONLY
Alkaline slant/ Acid butt (K/A)
18-24 hrs incubation
Acid slant/ Acid butt (A/A)
8-12 hrs incubation
Acid slant/ Acid butt(A/A)
18-24 hrs incubation
LACTOSE (SUCROSE) FERMENTATION
H2S PRODUCTION
Indicators:
Sodium thiosulfate
Ferrous sulfate
(+) black color/black precipitate
*Butt area requires acidic environment.
GAS PRODUCTION
(+) Splitting of media in the butt
(+) Formation of Bubbles
SULFIDE INDOLE MOTILITY CULTURE MEDIA - SIM MEDIUM
H2S Positive: Blackening of the medium
Motile: Diffuse growth extending laterally from line of inoculation
Stab
Indole(pink) H2S Haziness(motility)
Indole Production
Test for the ability of an organism to split tryptophan to form the compound indole.
Positive: Pink to wine colored ring after addition of Kovac’s reagent/Erlich’s