Enterobacteriacea i biochemical reaction بكتريا عملي

EnterobacteriaceaeBiochemical reactions

ByDr. Nabil El Aila

Assistant Professor of Molecular MicrobiologyMedical Technology Department

Al -Aqsa University

Dr. Nabil El AilaDiagnostic Microbiology

Lactose Fermentation• MacConkey Agar contains bile salts and crystal violet, both

inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as E. Coli.

• It also differentiates lactose-fermenting bacteria, such as E. Coli from non-lactose fermenting bacteria.

• Media and Reagent:MacConkey Agar and neutral red dye

• Method: Streak MAC plate and incubate at 37°C for 2 days.

• Expected results: – Positive test:Lactose fermentation = Growth and color

change to pink

– Negative test:No lactose fermentation = May or may not grow and no color change


Results of Lactose Fermentation


IMViC Test

• Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC ) Tests:– The following four tests comprise a series of

important determinations that are collectively called the IMViC series of reactions

– The IMViC series of reactions allows for the differentiationof the various members of Enterobacteriaceae.


IMViC : Indole test� Principle

� Certain microorganisms can metabolize tryptophan by tryptophanase

� The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia

� The presence of indole is detected by addition of Kovac's reagent.

Tryptophaneamino acids

TryptophanaseIndole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer`Dr. Nabil El AilaDiagnostic Microbiology

IMViC : Indole test

� Method:

� Inoculate the test organism into tryptophanebroth

� Incubate at 37°C for 24 hours

� After incubation interval, add 1 ml Kovacs reagentwhich contain 4 (p) – dimethylamino

benzaldehyde, shake the tube gently and read

immediately


IMViC: Indole test

� Result:

� A bright pink color in the top layer indicates the presence of indole

� The absence of color means that indole was not produced i.e. indole is negative

� Significance:

� Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-).

Positive teste.g. E. coli

Negative teste.g. Klebsiella


IMVi C testMethyl Red-Voges Proskauer (MR-VP) Tests

• Different bacteria convert dextrose and glucose to pyruvate

using different metabolic pathways.

• Some of these pathways produce unstable acidic products

which quickly convert to neutral compounds.

• Some organisms use the butylene glycol pathway, which

produces neutral end products, including acetoinand 2,3-

butanediol.

• Other organisms use the mixed acid pathway, which produces

acidic end products such as lactic, acetic, and formic acid. These

acidic end products are stable and will remain acidic.

IMV iC testMethyl Red-Voges Proskauer (MR-VP) Tests

Glucose

Acidic pathway

Mixed acids �� pH less than 4.4

Methyl Redindicator

Red color

Principle

MR positiveE. coli

Or Neutral pathway

Acety methyl carbinol(ACETOIN)

Barrit’s A (α-naphthol)Barrit;s B (40% KOH)

Pink colorVP positiveKlebsiella

IMViC: Methyl red

Principle:• Methyl red test is used to identify enteric bacteria based on

their pattern of glucose metabolism.

• If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive.

• If they use butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative


IMViC: Methyl red

• Method:• Inoculate 10ml portion of the MR-VP medium and incubate at

37°C for 2-5 days.

• After incubation, transfer 2.5 ml of inoculate to another tube and add five drops of methyl red.

• Roll between the palms of hands to disperse methyl red.


IMViC: Methyl red• Results:

– Positive test:acids + methyl red = red solution

– Negative test:neutral end products + methyl red =

yellow color

• Significance: This test is used to differentiate

Enterobacteriacaceae species

espciallyE. coli andE. aerogens


IM ViC: VOGES-PROSKAUER TEST

Principle:• It is used to identify enteric bacteria based on their pattern of

glucose metabolism.

• The enterics that produce neutral end-products, such as acetoinare detected by VP test.

• Its presence is used as indicator of 2,3 butylene glycol

Fermentation

• The detection of acetoin in alkaline pH is accomplished by

alpha-Naphthol reagent.



• Method:• Inoculate medium and incubate at 37°C for 48 hours.

• After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritt’s Reagent A (contains alpha-naphthol) and two drops of Barritt’s Reagent B(contains KOH).

• Gently mix and let it sit for 10-15 minutes to allow time for color development.



• Results:– Positive test: acetoin + alpha-naphthol + KOH = red color

– Negative test:alpha-naphthol +KOH = copper color

• Significance: This test is used todifferentiate

Enterobacteriacaceaespecies

espciallyE. coli andE. aerogens


Principle:

Citrate Na2CO3

Alkaline,↑pH

Bromothymol blue

Simmone’s Citrate media

CO2 + Na + H2OPyruvate

Positive test

Contains Citrate as a sole of C source

IMVi C: CITRATE TEST



• Principle:• Citrate is an organic molecule that can be utilized by bacteria

that produce the enzyme citrase. • Citrase is produced by some bacteria such as E. aerogenes but

not by others like E. Coli• Method:• Inoculate the test organism onto a slant containing Simmon

Citrate agar.• Simmon’s Citrate Agar contains sodium citrate (carbon

source), & pH indicator—bromthymol blue.• Incubate at 37°C for 24 hours.



• Results:– Positive test:Growth and color changes to blue– Negative test:No growth and color remains green

• Significance:• This test is used to help differentiate

species of the family Enterobacteriaceae.

• It is selective for bacteria that has

the ability to consume citrate as

its sole source of carbon

NegativePositiveDr. Nabil El AilaDiagnostic Microbiology

UreaseTest

• Urea agar contains urea and phenol red• Urease is an enzyme that catalyzes the conversion of urea

to CO2 and NH3• Ammonia combines with water to produce ammonium

hydroxide, a strong base which ↑ pH of the medium. • ↑ in the pH causes phenol red r to turn a deep pink. This is

indicative of a positive reaction for urease

UreaUrease

CO2 + NH3H2O

NH4 OH ↑ in pH

Phenol Red

PinkPositive test

� Streak a urea agartube with the organism

� incubate at 37°C for 24 h

Method

Principle

Urease Test• Result

• Positive test: production of alkaline end products = pinkish red color

• Negative test:No color change

• Significance:

• Differentiatesalmonella and shigellawhich are urease negative fromurease positive Non pathogens.

• Proteus, klebsiella and somecitrobacter species are ureasepositive

• Helicobater pylori is also

Urease positive

Positive testNegative test


Motility Test• Principle:

• Motility Test Media is a semi-solid agar designed to

demonstrate motility by diffusion.

• This is not a biochemical test, but it can distinguish bacteria. It

determines presence of flagella.

• Method:

• Inoculate a semi-solid nutrient medium by stabbing 2 cm

into the center of the medium

• Inoculate at 37C° for 24-48 hours.


Motility Test• Expected results:

– Positive test:Growth spread away from the line of

inoculation = motile

– Negative test:Growth only occurred at the line of

inoculation = Non-motile

• Significance:• This test is used for the differentiation

of microorganisms on the basis of

motility.


H2S Production

• Principle:

• Bacteria use enzyme cysteine desulfuraseto hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product.

• To test for hydrogen sulfide production, a medium with a sulfur-containing compound and iron salts is inoculated and incubated. If the sulfur is reduced and hydrogen sulfide is produced, it will combine with the iron salt to form a visible black ferric sulfide (FeS) in the tube

• Media and Reagent: SIM tube (sulphide, Indole and Motility) with cysteine and ferrous sulfate (detects H2S)

• Method: Inoculate the media and incubate at 37°C for 24-48 hours.


H2S Production

• Expected Results:

– Positive Test:H2S production = Black

– Negative Test:No H2S production = No blackening of medium

• Significance:

• This test is used to determine the ability

to reduce sulfur into H2S.

• Differentiate species of the family

Enterobacteriaceae.

• Identifying unknown organisms such as

certain Proteus and salomenella

Negative PositiveDr. Nabil El AilaDiagnostic Microbiology

Health & Medicine

Enterobacteriacea i biochemical reaction بكتريا عملي