1. MAHATMAGANDHI CENTRALUNIVERSITY
Assignment- E.COLI BACTERIAL TEST
2.LYSINE DECARBOXYLASE TEST
M.Sc (Biotechnology) Semester-4
2. 1.Indole Test
Thistestdemonstrate the abilityof certainbacteriatodecompose the aminoacidtryptophanetoindole,
Indole productiontestisimportantinthe identificationof Enterobacteria.
Most strainsof E. coli,P.vulgaris,P.rettgeri,M.morgani and Providenciaspeciesbreakdownthe amino
acid tryptophanwiththe release of indole.
Thisis performedbyachain of a numberof differentintracellularenzymes,asystemgenerallyreferred
to as “tryptophanase.”
It is usedas part of the IMViCprocedures,atestsdesignedtodistinguishamongmembersof the family
A variationonthistestusingEhrlich’sreagent(usingethyl alcohol inplace of isoamylalcohol,developed
by Paul Ehrlich) isusedwhenperformingthe testonnon-fermentersandanaerobes.
Principle of Indole Test
Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that
express tryptophanase enzyme.
Indole is generated by reductive deamination from tryptophan via the intermediate
molecule indolepyruvic acid.
Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of
the tryptophan molecule is removed.
Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy.
Pyridoxal phosphate is required as a coenzyme.
When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-
dimethylaminobenzaldehydein amyl alcohol) the solution turns from yellow to cherry red.
Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the
top of the broth.
In the spot test, indole combines, in the filter paper matrix, at an acid pH with p-
Dimethylaminocinnamaldehyde (DMACA) to produce a blue to blue-green compound.
3. Indole Spot Reagent has been reported to be useful in detecting indole production by members
of the family Enterobacteriaceaeand certain anaerobicspecies.
Reagents Used in Indole Test
Ingredients per liter:*
Indole Spot Reagent:
p-Dimethylaminocinnamaldehyde(DMACA) 10.0 gm
Hydrochloric Acid, 37% 100.0 ml
Deionized Water 900.0 ml
Indole Kovacs Reagent:
p-Dimethylaminobenzaldehyde 50.0 gm
Hydrochloric Acid, 37% 250.0 ml
Amyl Alcohol 750.0 ml
* Adjusted and/or supplemented as required to meet performance criteria
Indole Spot Reagent (DMACA) Procedure
1. Place several drops of Indole Spot Reagent on a piece of filter paper.
2. With an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour
isolated colony from a non-selective media and rub it onto the reagent saturated area of
the filter paper.
3. Examine immediately
Result Interpretation of Indole Test
Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the
medium within seconds of adding the reagent.
Examples: Aeromonas hydrophila, Aeromonas punctata, Bacillus
alvei,Edwardsiella sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella
4. oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Plesiomonas shigelloides,Pasteurella
multocida, Pasteurella pneumotropica, Enterococcus faecalis, and Vibrio sp.
Negative: No color change even after the addition of appropriate reagent.
Examples: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp.,
most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp.,
most Klebsiella sp., Neisseria sp., Pasteurella haemolytica, Pasteurella ureae, Proteus mirabilis, P.
penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.
Uses of Indole Test
1. To differentiate Proteus mirabilis (indole negative) from all other Proteus species (indole
2. To differentiate Klebssiella pneumoniae (indole negative) from Klebsiella oxytoca (indole
3. To differentiate Citrobacter freundii (indole negative) from Citrobacter koseri (indole
Limitations of Indole Test
1. Indole tests may be used as an aid in the identification and differentiation of gram-
positive and gram-negative organisms. Additional biochemical testing using pure
cultures is recommended for complete identification.
2. The tube test is a more sensitive method of detecting indole than the spot test.
3. When performing a spot test, Kovacs Indole Reagent may be used as a substitute for the
spot test reagent. However, Kovacs Indole Reagent, when used as the spot test reagent,
is less sensitive in detecting indole than the Indole Spot Reagent (DMACA).
4. Kovacs Indole Reagent is not recommended for use with anaerobic bacteria. The Indole
Spot Reagent (DMACA) is suitable for anaerobeuse.
5. Since peptones have been shown to vary with regard to their suitability for use with
indole testing, media selected for indole determination should be tested with known
positive and negative organisms to insure suitability.
2.LYSINE DECARBOXYLASE TEST
Decarboxylase test is a biochemical test performed to differentiate
members of Enterobacteriaceae on the basis of their ability to produce
the enzyme decarboxylase.
5. Decarboxylation occurs in the presence of a decarboxylase
enzyme that catalyzes the breaking of the bond that binds the
carboxylic group to the rest of the amino acid.
Objectives of Decarboxylase Test
To test the ability of an organism to produce a decarboxylase
To differentiate the members of the Enterobacteriaceae family on
the basis of their ability to produce decarboxylase enzyme.
Arginine, lysine, and ornithine decarboxylase media are used to detect an
organism’s ability to decarboxylate or hydrolyze an amino acid, forming an amine
that produces an alkaline Ph
The decarboxylation of lysine yields cadaverine, decarboxylation of ornithine
produces putrescine, and decarboxylation of arginine results in agmatine, which is
hydrolyzed by a Dihydrolase to form putrescine
Enteric Gram-negative rods and Vibrio, Plesiomonas, and Aeromonas for
identification to the species level
Probable Stenotrophomonas and Burkholderia (lysine and arginine)
Fluorescent Pseudomonas (arginine)
Viridans group streptococci (arginine)
Miscellaneous non-glucose-fermenting, Gram-negative rods (arginine)
Spreading indole-negative Proteus (ornithine)
Procedure of decarboxylase test
A. Preparation of the media
B. Decarboxylase test
1.For Glucose-Fermenting Organisms
Uses of decarboxylase test
Decarboxylase test is used to differentiate the members of the
Enterobacteriaceae family with closely related physiological
Arginine decarboxylase is useful in the identification
of Enterococcus to the species level; Enterococcus
6. faecalis and Enterococcus faecium are arginine positive
but, Enterococcus avium is arginine negative.
Lysine decarboxylase is used to differentiate
between Salmonella (+) and Shigella (-).
Limitation of decarboxylase test
Mineral oil or a similar barrier to gas release must be applied to
the surface of each inoculated broth medium. Oil on the surface
reduces the possibility of an alkaline shift occurring in the medium
due to oxidation.
Result interpretation should not be made prior to 18 to 24 hours of
incubation. Earlier interpretation may lead to erroneous results.
Glucose fermentation occurs within the first 10 to 12 hours of
incubation. The acidic environment from fermentation is necessary
for the production of decarboxylase
The purpose is to see if the microbe can ferment the carbohydrate (sugar) mannitol as a
If mannitol is fermented to produce acid end products, the pH of the medium will drop.
The principle of carbohydrate fermentation states that the action of organism on a carbohydrate
substrate results in acidification of the medium, detected by a pH indicator dye. Carbohydrate
fermentation is the process microorganisms use to produce energy. Most microorganisms
convert glucose to pyruvate during glycolysis; however, some organisms use alternate pathways.
A fermentation medium consists of a basal medium containing a single carbohydrate (glucose,
lactose, sucrose, mannitol etc.) for fermentation. However, the medium also contains various pH
indicators. In addition to a pH indicator to detect the production of acid from fermentation, a
Durham tube is placed in each tube to capture gas produced by metabolism. The carbohydrate
fermentation patterns shown by different organisms are useful in differentiating among bacterial
groups or species.
The purple broth consists of peptone with the pH indicator bromcresol purple. Specific
carbohydrates are added in a concentration of 0.5-1%.
1. Allow medium to warm to room temperature prior to inoculation.
7. 2. Inoculate the Purple Broth (with carbohydrate of choice) with isolated colonies from an
18-24 hour pure culture of the organism.
3. Inoculate a control tube of Purple Broth Base in parallel with the carbohydrate based
4. Incubate inoculated media aerobically at 35-37ºC. for 3-5 days.
Note: Increased incubation up to 30 days may be necessary for some microorganisms.
5.Observe daily for development of a yellow color in the medium.
Positive: The development of a yellow color in the medium is indicative of a positive
carbohydrate fermentation reaction.
Negative: Lack of yellow color development is indicativeof a negative carbohydrate
Gas formation is indicated by the appearance of gas bubbles in the Durham tube.
It is recommended for the determination of fermentation reactions of microorganisms,
especially enteric bacilli and Enterococcus
They are used primarily for the differentiation and presumptive identification of gram-
negative enteric bacilli basedon patterns of carbohydrate fermentation.
It is recommended that biochemical, immunological, molecular, or mass spectrometry
testing be performed on colonies from pure culture for complete identification.
It may be necessary to invert the tube prior to inoculation if bubbles are trapped in the
durham tube. Trapped bubbles that are not released may lead to false-positiveresults