This document discusses biochemical tests used to identify members of the Enterobacteriaceae family, including lactose fermentation, IMViC (indole, methyl red, Voges-Proskauer, and citrate) tests, urease test, motility test, and hydrogen sulfide production test. It provides details on the principles, methods, expected results and significance of each test for differentiating bacterial species like E. coli, Klebsiella, Proteus, and Salmonella. The document is authored by Dr. Nabil El Aila and focuses on diagnostic microbiology of gram-negative bacteria.

1. Enterobacteriaceae
Biochemical reactions
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University
Dr. Nabil El Aila
DiagnosticMicrobiology

2. 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
Dr. Nabil El Aila
Diagnostic Microbiology

3. Results of Lactose Fermentation
Dr. Nabil El Aila
Diagnostic Microbiology

4. 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
differentiation of the various members of
Enterobacteriaceae.
Dr. Nabil El Aila
Diagnostic Microbiology

5. 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.
Tryptophane
amino acids
Tryptophanase
Indole + Pyurvic acid + NH3
Kovac’s Reagent
Red color in upper Dr. Nabil El Aila organic layer`
Diagnostic Microbiology

6. IMViC: Indole test
Method:
Inoculate the test organism into tryptophane
broth
Incubate at 37°C for 24 hours
After incubation interval, add 1 ml Kovacs
reagent which contain 4 (p) – dimethylamino
benzaldehyde, shake the tube gently and read
immediately
Dr. Nabil El Aila
DiagnosticMicrobiology

7. 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 test
e.g. E. coli
Negative test
e.g. Klebsiella
Dr. Nabil El Aila
DiagnosticMicrobiology

8. IMViC test
Methyl 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 acetoin and 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.

9. IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Glucose
Principle
Acidic pathway
Mixed acids
pH less than 4.4
Methyl Red
indicator
Red color
MR positive
E. coli
Or Neutral pathway
Acety methyl carbinol
(ACETOIN)
Barrit’s A (a-naphthol)
Barrit;s B (40% KOH)
Pink color
VP positive
Klebsiella

10. 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
Dr. Nabil El Aila
Diagnostic Microbiology

11. 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.
Dr. Nabil El Aila
Diagnostic Microbiology

12. 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
espcially E. coli and E. aerogens
Dr. Nabil El Aila
Diagnostic Microbiology

13. IMViC: 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 acetoin
are 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.
Dr. Nabil El Aila
Diagnostic Microbiology

14. IMViC: VOGES-PROSKAUER TEST
• 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.
Dr. Nabil El Aila
Diagnostic Microbiology

15. IMViC: VOGES-PROSKAUER TEST
• Results:
– Positive test: acetoin + alpha-naphthol + KOH = red color
– Negative test: alpha-naphthol +KOH = copper color
• Significance:
This test is used to differentiate
Enterobacteriacaceae species
espcially E. coli and E. aerogens
Dr. Nabil El Aila
Diagnostic Microbiology

16. IMViC: CITRATE TEST
Principle:
Citrate Na2CO3
Alkaline,↑pH
Pyruvate CO2 + Na + H2O
Simmone’s Citrate media
Contains Citrate as a sole of C source
Bromothymol blue
Positive test
Dr. Nabil El Aila
Diagnostic Microbiology

17. IMViC: 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.
Dr. Nabil El Aila
Diagnostic Microbiology

18. IMViC: CITRATE TEST
• 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
Dr. Nabil El Aila Positive Negative
Diagnostic Microbiology

19. Urease Test
Principle
• 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
Urea Urease
CO2 + NH3
H2O
NH4 OH ↑ in pH
Phenol Red
Pink
Positive test
Method
Streak a urea agar tube with the organism
incubate at 37°C for 24 h

20. Urease Test
• Result
• Positive test: production of alkaline
end products = pinkish red color
• Negative test: No color change
• Significance:
• Differentiate salmonella and shigella
which are urease negative from
urease positive Non pathogens.
• Proteus, klebsiella and some
citrobacter species are urease
positive
• Helicobater pylori is also
Urease positive
Negative test Positive test
Dr. Nabil El Aila
DiagnosticMicrobiology

21. 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.
Dr. Nabil El Aila
DiagnosticMicrobiology

22. 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.
Dr. Nabil El Aila
Diagnostic Microbiology

23. H2S Production
• Principle:
• Bacteria use enzyme cysteine desulfurase to 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.
Dr. Nabil El Aila
Diagnostic Microbiology

24. 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
Dr. Nabil El Aila Negative Positive
Diagnostic Microbiology