Biochemical Principles

Topics: Hydrogen sulfide, PH indicator, Bacteria Pages: 6 (844 words) Published: October 3, 2013
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 reagent (Escherichia coli, P. mirabilis) Negative: No color change (Klebsiella, Enterobacter, P. vulgaris) CULTURE MEDIA - SIMON CITRATE AGAR (SCA)

CITRATE UTILIZATION TEST
Determine the ability of an organism to use sodium citrate as the sole source of carbon Indicator: Bromthymol blue
(+) BLUE COLOR
Streak

LYSINE IRON AGAR TEST CULTURE MEDIA - LIA MEDIUM
LYSINE IRON AGAR TEST
Used to determine whether a gram (-) rod decarboxylates or deaminates lysine and forms hydrogen sulfide (H2S) Determines the ability of the microorganism to decarboxylate or deaminate lysine and form H2S Lysine, Glucose, H2S Indicator and bromcresol purple

Principle:
When glucose is fermented, the butt of the medium becomes acidic (yellow) If the organism decarboxylates lysine, a purple butt / slant forms; deaminates lysine, a burgundy, plum, reddish-purple color slant forms. Stab and Streak

LIA REACTIONS:
Alkaline slant/ alkaline butt (K/K)
= (-) lysine deamination
(+) decarboxylation

Alkaline slant/ acid butt (K/A)
= (-) lysine deamination
(-) lysine decarboxylation

Red slant/ acid butt (R/A)
= (+) lysine deamination
(-) lysine decarboxylation
Decarboxylase Test
MOELLER’S METHOD
Determines whether an organism is capable of decarboxylating an amino acid to form an diamine Contents: Glucose, bromcresol purple and cresol red, amino acid at 1% concencentration 1. Lysine –Lysine decarboxylaseà Cadaverine

2. Ornithine –Ornithine decarboxylaseà Putrescine 3. Arginine –Arginine dihydrolaseà Citrulline
Positive: Alkaline (purple) color Negative: acid (yellow) color

Decarboxylase Reactions
LDC
ODC
ADH
K. subs. pneumoniae
+


K. subs. oxytoca
+


E. aerogenes
+
+

E. cloacae

+
+
P. vulgaris



P. mirabilis

+


ORTHONITROPHENYL BETA-GALACTOPYRANOSIDE (ONPG)
Test the ability of organism to produce...
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