Enterobacteriaceae Biochem Flashcards
OXIDASE TEST(Kovac’s
Method)
To determine the presence of bacterial_______ using the oxidation of the substrate_______ to indophenol, a dark purple-colored end product
cytochrome C oxidase
1% tetramethyl -para-phenylenediamine dihydrochloride
Oxidase
• PRINCIPLE:
1% TMPDD
Oxidase Reagent (Aromatic amines)
Cytochrome C oxidase
INDOPHENOL
(Colored compound - DARK PURPLE)
Oxidase REAGENTS:
• PADAM (dimethyl compound) Pink - Black = para-aminodimethyl aniline monohydrochloride
TMPDD = 1% tetramethyl-para-phenylenediamine dihydrochloride
Oxidase
Colorless → Blue or Dark Purple____sec
_______- delayed
No color change after_____ - negative
5-10 seconds
10-60 seconds
60 seconds
Oxidase
QUALITY CONTROL:
Positive:
Negative:
Pseudomonas aeruginosa
Escherichia coli
Common carbohydrates used in the media:
~ Glucose - monosaccharide
• Lactose - disaccharide
• Sucrose - disaccharide
Carbohydrate Fermentation
2 Enzymes used in Lactose Degradation
• B-galactoside permease (Lactose permease)
• B-galactosidase
- LF:
- NLF:
• LLF:
possess both of the enzymes
do not possess both of the enzymes
possess only B-galactosidase
Carbohydrate Fermentation
Indicator
Color change depending on pH
Bromcresol purple
Andrade’s fuchsin
Phenol red
Purple (alk) yellow (acid)
Pale yellow (alk) reddish pink (acid)
Red (alk) yellow (acid)
CHO fermentation using Broth
positive carbohydrate fermentation media
Media used:
Phenol red Broth: contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (glucose,
lactose, or sucrose)
СНО:
• TSI -
• KIA -
glucose (0.1% or 1 part), lactose (1% or 10 parts), and sucrose (1% or 10 parts)
glucose (0.1% or 1 part), lactose (1% or 10 parts)
CULTURE MEDIA
COMPOSITION
TSI (Triple Sugar Iron)
KIA (Kligler’s Iron Agar)
RDA (Russel’s Double Agar)
glucose, lactose, sucrose, iron
glucose, lactose, iron
glucose, lactose
Triple Sugar Iron
TSI
pH indicator
phenol red
TSI
H2S indicator
: forms H2S upon interaction with the organisms
: binds to H2s = forming a black
precipitate
• Sodium thiosulfate
• Ferrous sulfate
Triple Sugar Iron
• Glucose/Sucrose/Lactose not fermented
• This reaction is typical among the Non-Fermentative organisms
Ex..
K/K
• P. aeruginosa
Triple Sugar Iron
• Glucose/Sucrose/Lactose not fermented
• This reaction is typical among the Non-Fermentative organisms
Ex..
K/K
• P. aeruginosa
Triple Sugar Iron
TSI
• Glucose fermented
• Lactose/sucrose not fermenters
With blackening
Ex..
K/A, H2S (+)
• Salmonella spp.
• Proteus
• Arizonae
• Citrobacter
• Edwardsiella
Triple Sugar Iron
• Glucose fermented
• Lactose/Sucrose not fermented
Ex…
K/A, H2S (-)
• Yersinia
• Escherichia coli
• Serratia
• Shigella
• Providencia
• Morganella
• Citrobacter
Triple Sugar Iron
TSI
• Glucose fermented
• Lactose/Sucrose fermented
With blackening
Ex..
A/ A H2S (+)
Salmonella
Proteus
Citrobacter
Triple Sugar Iron
TSI
• All sugars are fermented
A/A, H2S (-)
• Escherichia coli
• Klebsiella
• Enterobacter
• Serratia
ONPG
O-nitrophenyl-beta-D-galactopyranoside
• Determines the presence of late or slow lactose fermenting strains
ONPG (O-nitrophenyl-beta-D-galactopyranoside)
- structurally similar with lactose
-Can enter the bacterial cell without the Lactose permease
• ONG molecule
-In the presence of Beta-galactosidase, ONPG (colorless), is converted into_____ and _____ which is a yellow chromogen
galactose and o-nitrophenyl
KOVAC’S METHOD
• Medium:
•
• Indole indicator:
•
• Positive Result:
• Development of a
Tryptone broth or trypticase broth
Kovac’s reagent (p-dimethylaminobenzal-dehyde)
pink to deep red color
EHRLICH’S METHOD
• Medium:
• Tryptone broth or trypticase broth
• With ______
• Indole indicator:
*______
• Positive Result:
•_____
Xylene or Ether
Ehrlich’s reagent (p-dimethylaminobenzal-dehyde)
Brilliant red ring between the solvent and the medium.
• This is a rapid method for the detection of indole
Positive Result:
SPOT INDOLE TEST
blue or blue green color
• Principle
‹ Some organisms produce large amounts of acid from dextrose while others produce less.
This test is based upon the final hydrogen ion concentration (acidity) reached by a culture.
The test will be positive if the pH is 4.5 or lower, and negative if above pH 4.5.
Methyl Red Test (Mixed Acid Fermentation Pathway)
MR
• Medium:
•
• pH indicator:
*
• Results
• Positive:
• Negative:
MR-VP or the Clark and Lubb’s dextrose broth
Methyl Red
Red color (pH 4.5 or below)
Yellow color (above 4.5)
• Principle
• Some bacteria have the ability to produce acetoin (acetylmethylcarbinol) from glucose.
In an alkaline pH, acetoin is oxidized to diacetyl (dimethylcarbinol), which reacts with guanidine compounds present in the broth to give a red-colored complex.
Voges-Proskauer Test (Butylene glycol pathway)
Voges-Proskauer Test (Butylene glycol pathway)
Reagents:
•______ in 95% ethyl alcohol
•_____ in distilled H2O (provides alkalinity)
‹______ in distilled H20 (prevents false negative results)
5% alpha-naphthol
40% KOH
0.5% creatine
• Principle
• Some organisms can utilize CITRATE as their sole source of carbon producing acetate and other alkaline carbonate end products in the process.
CITRATE
Citrate test
Medium
Simmons citrate agar
Christensen citrate medium
Citrate
pH indicator:
•
Bromthymol blue
Citrate
• acid: pH 6 ()
‹ uninoculated: pH 6.9 ()
• alkaline: pH 7.6 ()
Yellow
Green
Blue
Citrate
Results:
• Positive:
-Growth with an intense prussian blue color on the slant
-Ex.
Enterobacter and Klebsiella
Citrate
~ Negative:
-Absence of growth and no color change in the medium (remains green)
-Ex.
Escherichia coli
++–
IMViC
E. coli
Morganella
Proteus
Edwardsiella
IMViC
–++
Klebsiella
Enterobacter
Serratia
Hafnia
IMViC
-+-+
-+-+
Salmonella
Arizonae
Citrobacter
Principle:
• This is based upon the ability of some bacteria to hydrolyze Urea into Ammonia, water and CO2 by means of the enzyme, Urease.
If urea is split to form ammonia, the medium becomes alkaline and the indicator turns from Yellow to Pink
Urease
Urease
• Medium:
‹ _____ or _____
Original color:____
• pH indicator:
•______
• Positive result:
Color yellow changes to____
Christensen’s Urea Agar tubes, or Stuart Urea broth
yellow
Phenol red
PINK
Urease
- Useful in the identification of RAPID UREASE PRODUCERS (w/in 2-4 hours)
• WEAK UREASE PRODUCERS within 4 hours
- Proteus, Providencia, Morganella (PPM)
- Citrobacter, Klebsiella, Enterobacter, Yersinia, and Serratia (CKEYS)
Motility test
• Medium:
* SIM medium or any semisolid motility medium
-Can be added with______ to aide visualization (optional)
SIM
1% triphenyltetrazolium chloride
SIM
• Results:
v (+) motile organisms spread out/ grow away from inoculation line
v (-) nonmotile organisms grow only along the stab line
SIM
Nonmotile @ 37C:
Shigella, Klebsiella, Yersinia
