BIOCHEMICAL TESTS GROUP 3 AND 7

BIOCHEMICAL TESTS FOR IDENTIFICATION OF ENTERIC ORGANISMS

Basis:

metabolic action of microorganisms on the culture media Used for the identification of enteric organisms/ gram negative bacilli

IMViC TEST
I

= INDOLE M = METHYL RED Vi = VOGES PROSKAUER TEST C =CITRATE AGAR

IMViC
One

of the earliest sets of test used for the identification of enteric bacilli includes such organisms as Klebsiella, Enterobacter, Citrobacter and Escherichia coli
This

acronym stands for I - Indole M- Methyl red V - Voges Proskauer ( i ) is inserted for euphony C - Citrate

-Enterobacteriaeae

(enterics) are Gram-negative bacteria that grow in the intestinal tract of humans and other animals.
-These

IMViC tests are useful for differentiating the family Enterobacteriaceae, especially when used alongside the Urease test.

-Enterobacteriaceae,

especially when used alongside the Urease test. used alone, the IMViC tests are particularly useful for differentiating Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae (although colonial morphology and the presence of capsules can also be used to differentiate

-When

INDOLE TEST

DESCRIPTION
Tryptophan hydrolysis -Some bacteria split tryptophan into indole and pyruvic acid using the hydrolase called tryptophanase. Indole can be detected with Kovac's reagent (Indole reagent). This test is very important in differentiating E. coli (indole positive) from some closely related enteric bacteria. It also differentiates Proteus mirabilis (indole negative) from all other Proteus species (indole positive). Tryptone broth is used for this test as it contains a large amount of tryptophan.

INDOLE
Indole,

a benzyl pyrrole, is one of the metabolic degradation product of amino acid tryptophan

Indole

positive bacteria produce tryptophanase, an enzyme that is capable of hydrolyzing and diaminating tryptophan, thus producing: - indole - pyruvic acid - ammonia Materials: 2% Peptone broth tube Test organisms Ether indicators: Erlich/Kovac's reagent (para-dimethyl-aminobenzaldehyde)

 Procedure:

1.

Inoculate 1 loopful of the test organism into the tube of

peptone broth.

2. Incubate at 370C for 24-48 hours. 3. Next laboratory period, add 1 ml. of ether. 4. Shake well and allow to stand for a few minutes until the ether rises to the surface. 5. Gently add about, 1mL. of Kovacs or Erlichs reagent down the side of the tube so that it forms a ring between the medium and the ether layer.

INTERPRETATION:
After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Add a few drops of Indole reagent to the broth culture (tryptone broth). DO NOT SHAKE THE TUBE. A positive result has a red layer at the top. A negative result has a yellow or brown layer.

Observation:

Positive result Bright red or purple ring If indole has been produced by the organism it will, being soluble in ether, it will be concentrated in the ether layer and upon the addition of Erlichs reagent, a positive result is the production of a purple ring at the junction of the medium and the ether layer

Negative result Yellow color - no red or purple ring

METHYL RED TEST

DESCRIPTION
Mixed acid fermentation - Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter.

METHYL RED TEST

All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose

MR test is a quantitative test for acid production, requiring positive organism to produce strong acids (lactic, acetic, formic) from glucose via the mixed acid fermentation pathway

End result is based on the final pH reached only those organism that can maintain low ph of about ph 4-4.5 can be called methyl red positive organisms that are MR (+) are always VP (-)

 Purpose:

The Methyl Red (MR) test is used to identify mixed acid fermenting bacteria that yield a stable acid end product.
Principle:

MRVP media contains glucose, peptone and phosphate buffer. Many enteric organisms can overcome the buffering capacity of the media by producing large quantities of a stable acid end product, thus lowering the pH. Acid produciton is detected using the pH indicator methyl red (red pH<4.4, yellow pH > 6). In order to insure that the acid is stable this test should be conducted a minimum of 48 hrs. post inoculation.

Materials: MR-VP broth medium

glucose)

(contains 10%

Test organisms (E. Coli & Aerobacter) Methyl red pH indicator

Procedure:

1. Inoculate loopful of the test organism into a tube MR-VP medium. 2. Incubate for 24-48 hours at 370C. 3. Next laboratory period, add 5 to 10 drops of methyl red reagent. 4. Mix thoroughly and observe the

INTERPRETATION
After

incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test if you are going to use the same broth for the VP test. Add 3-4drops of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer.

Observation:
Positive

result cherry red/bright red color ph 4-4.5 Ex. Salmonella, Escherichia, Citrobacter, Proteus, Morganella and Providencia result Yellow color At neutral pH the growth of the bacteria is not inhibited The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 4.5 At this pH, methyl red indicator produce a yellow color Ex. Enterobacter and Klebsiella

Negative

VOGES-PROSKAUER TEST

DESCRIPTION
Organisms

that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do. Serratia and some species of Bacillus produce these substances. is no satisfactory test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.

Enterobacter,

There

VOGES-PROSKAUER TEST

is a test for the detection of acetyl-methyl carbinol (acetoin) which is also a degradation product of glucose Materials:

MR-VP medium (contains 10% glucose) Test organism Potassium Hydroxide Alpha-napthanol reagent

When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a burgundy color/crimson red color (a positive VP test)

organisms that are VP (+) are always MR (-)

Reaction process
acetyl-methyl carbinol + Potassium Hydroxide
0xidized

dimethyl-carbinol

React with

guanidine compounds Crimson red

Procedure:
1.

Inoculate MR-VP medium with 1 loopful of the test

organism
2.Incubate

for 48 hours at 370C.

3.Next

laboratory period add 0.6 ml. 5% alpha-napthol

reagent. Mix and shake the mixture lightly.

4.Add

0.2 ml (5drops). of 40% potassium hydroxide reagent Mix and shake the mixture lightly. the tube gently to expose the medium to

(KOH).
5.

6.Shake

INTERPRETATION

After incubation: Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and cannot be tested. Barritt's reagent A (VP A) contains naphthol and Barritt's B (VP B) contains KOH. Add the entire contents of the VP A reagent (15drops) and 5 drops of the VP B reagent to the 1 ml of your broth culture. SHAKE WELL.

SHAKE the tube every few minutes for best results.

With a positive reaction the medium will

change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change color will be copper colored. Wait at least 15 minutes for color.

Observation:
Positive

result

Crimson Red color Presence of Acety methyl carbinol Ex. Enterobacter and Klebsiella

Negative
Ex. E. coli

result

Remains Yellow to Amber; no change in color

CITRATE TEST

DESCRIPTION
Simmon's

citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH.

CITRATE TEST

a test depends upon the ability of the organism, to utilize citrate as the sole source of carbon and energy growth

Materials: Solid media : Simmons Citrate Agar Liquid media: Koshers Media Test Organism

Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6 uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color (neutral pH) Growth of bacteria in the media leads to development of a Prussian blue color at more alkaline pH's (around 7.6) (positive citrate)

Procedure:
1. Inoculate the test organism on the medium by stab streaking. 2. Incubate at 370C for 24 - 48 hours. 3.Observe.

INTERPRETATION
After

incubation: A positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green.

Observation
Positive

result

Deep blue/ Prussian blue color indicating that the test organism has been able to utilize citrate for energy source Ex. Enterobacter, Klebsiella, Salmonella, Citrobacter and Providencia

Negative

result

Retains its original color (Green) Ex. Escherichia, Shigella and Morganella