1. BIOCHEMICAL TESTING
PART TWO

2. Distinguishing Enterobacteriaceae
•Many found in the intestines of human or other mammals
•Varrying pathogenicity:
•Commensals, opportunists or pathogens
•Some found in the environment
Example species:
Escherichia coli
Klebsiella pneumoniae
Citrobacter freundii
Enterobacter aerogenes
Proteus mirabilis
Salmonella typhi
Shigella dysenteriae
Yersinia enterocolitica
•Large family of bacteria
•Gram negative rods
•Capable of fermenting various sugars

3.  Large amounts of acid from
lactose fermentation cause the
dyes to precipitate on the
colony surface, producing a
black center or a “green
metallic sheen” (E. coli)
 Smaller amounts of acid
production result in pink
coloration of the growth (E.
aerogenes)
 Nonfermenting enterics do not
produce acid so their colonies
remain colorless or take on the
color of the media (P. vulgaris)
Previous Experience with
Enterobacteriaceae: EMB Media

4. Triple Sugar Iron (TSI): fermentation of sugars, sulfur reduction
IMViC:
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end products
Citrate: Citrate fermentation
Urease: Hydrolyzation of Urea
Phenylalanine Deaminase: converts the amino acid
phenylalanine to phenylpyruvic acid
Nitrate Reductase: Reduction of nitrate (NO3) to nitrite (NO2)
Biochemical Testing

5. TRIPLE SUGAR IRON TEST
(TSI)
 Used to differentiate among the different
groups of Enterobacteriaceae
based on their ability to ferment glucose,
lactose and/or sucrose
 Also differentiates between groups capable of
reducing sulfur to hydrogen sulfide
(Sodium Thiosulfate -> Hydrogen sulfide)

6. TSI Results:
 Red slant/Red butt = no fermentation
 Red slant/Yellow butt = only glucose
fermentation
 Yellow slant/yellow butt = lactose
and/or
sucrose
fermentation
Dark color: Hydrogen Sulfide produced
Sodium thiosulfate reduced
P 190

7. IMViC TESTS
 A series of four tests consisting of:
 Used to differentiate the Enterobacteriaceae
 We will look at each test individually
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end
products
Citrate: Citrate fermentation

8. Indole Test
(SIM: Sulfide,Indole,Motility)
 Identifies bacteria capable of producing indole
 Some bacteria are capable of converting tryptophan
(an amino acid) to indole and pyruvic acid by using the
enzyme tryptophanase
 Pyruvic acid can be converted to energy or used to
synthesize other compounds required by the cell
Tryptophan Indole Ring Pyruvic Acid Ammonia

9. Procedure:
 Obtain 4 SIM Deep tubes
 Inoculate by the stab method with the
following organisms:
E.coli, P.vulgaris,
E. aerogenes K. pneumoniae

10. Indole Test Results:
 Motility (if present) can be seen as
growth of the bacteria away from the
stab line
 Sulfur in the media may be reduced
to hydrogen sulfide (H2S); this
appears as a “blackening” within the
media
If indole is produced, upon addition of
Kovac’s Reagent (10 drops), a “cherry-red”
band forms on the surface of the media

11. Methyl Red Test
 Used to determine the ability of a bacteria to
oxidize glucose and produce stable acid end
products
 Methyl red is a pH indicator (red at pH less
than 4.4 and yellow at a pH greater than 6)
 The combination medium used for this test is
the MR-VP (methyl red/Voges-Proskauer)
broth
Acid production: positive methyl red
End products of neutral pH : positive Voges-Proskauer

12. Procedure:
 Obtain 3 MR-VP broth tubes
 Inoculate (using a loop) with the
following organisms:
E.coli K.pneumoniae E.aerogenes

13. Results:
 From the 3 MR-VP broths that you inoculated,
transfer 2 mLs from each and place into 3
separate clean tubes (set aside these
aliquots for the VP test)
 To the remaining, original tubes that you
inoculated add 5 drops of methyl red indicator
 A red color indicates that glucose has been
oxidized

14. Methyl Red Test Results:
 Methyl red positive
tube on the right
 Methyl red negative
tube on the left
A red color indicates that
glucose has been oxidized.

15. Voges-Proskauer Test
 Used to determine the ability of microbes to
produce nonacidic or neutral end products
 Remember that the MR-VP broth is a
combined medium used for two tests—
Methyl Red and Voges-Proskauer
 You have already inoculated the 3 MR-VP
broth tubes from the previous procedure
(Methyl Red Test) with
E.coli K.pneumoniae E.aerogenes

16.  To the aliquots of each broth culture
separated during the methyl red test, add:
10 drops of Barritt’s Reagent A; shake
10 drops of Barritt’s Reagent B; shake
Reshake the culture every 3 to 4 min.
It can take as long as 15 min. for a color
change to occur
Voges-Proskauer Procedure:

17. Voges-Proskauer Results:
 Voges-Proskauer
positive on the right
 Voges-Proskauer
negative on the left
The presence of a deep rose color after
15 minutes is indicative of non-acidic /
neutral metabolic end products and a
positive VP test result.

18. Citrate Utilization Test
 Used to determine if an organism is
capable of fermenting citrate and using
that citrate as its sole carbon source
 The ability of an organism to utilize
citrate occurs via the enzyme citrase

19. Procedure:
 Obtain 3 Simmons Citrate agar slants
 Inoculate these slants using the stab
and streak method (the same way you
inoculated the TSI media using a
needle) with the following organisms:
E.coli K.pneumoniae E.aerogenes

20. Citrate Test Results:
 Simmon’s Citrate agar utilizes
sodium citrate as its sole carbon
source
 Bromthymol blue is included as a
pH indicator; the medium initially is
green
 Organisms capable of using citrate
as a carbon source turn the media
“Prussian blue”.
- + - +

21. Page 199
Summary of IMViC Reactions

22. Urease Test
 Used to differentiate organisms based on
their ability to hydrolyze urea with the enzyme
urease
 The pH indicator, phenol red, is used to
detect the breakdown of urea and the
production of ammonia which is used by
bacteria to produce amino acids and
nucleotides

23. Procedure:
 Obtain 2 urea broth tubes
 Inoculate with the following organisms:
E.coli P.vulgaris

24. Urease Test Results:
 Urinary tract pathogens from the genus
Proteus may be distinguished from other
enterics
urease
 Urea + H2O CO2 + H20 + NH3
 As the alkaline end products build, phenol red
turns from yellowish gold to pink—a positive
result

25. Urease Test Results
 Urease positive
organism on the
right
 Urease negative
organism on the left
As the alkaline end products build, phenol red turns
from yellowish gold to pink—a positive result

26. Phenylalanine Deaminase
Test
 Used to identify bacteria possessing the
enzyme phenylalanine deaminase
 Phenylalanine deaminase converts the amino
acid phenylalanine to phenylpyruvic acid + NH3

27. Procedure:
 Obtain 2 phenylalanine agar slants
 Inoculate (with a loop on the surface)
with the following organisms:
E.coli P.vulgaris

28. Results
 Phenylpyruvic acid produced by some
organisms is colorless
 After inoculation and incubation, 10% ferric
chloride, an oxidizing agent, is added to the
surface of the slants
 Ferric chloride (FeCl3) reacts with the
phenylpyruvic acid (if present) and changes
color from yellow to green—a positive result

29. Phenylalanine Deaminase Results:
Ferric chloride (FeCl3) reacts with the
phenylpyruvic acid (if present) and
changes color from yellow to
green — a positive result
Positive Negative
Positive Negative

30. Nitrate Reductase Test
 Used to detect the ability of an
organism to reduce nitrate (NO3) to
nitrite (NO2) or some other nitrogenous
compound, such as molecular nitrogen
(N3) using the enzyme nitrate reductase
NO3 NO2
Nitrate Reductase
- - Forms red color once
solutions A and B are added.

31. Procedure:
 Obtain 3 Nitrate broth tubes
 Inoculate (with a loop) those tubes with
the following organisms:
E.coli A.faecalis P.aeruginosa

32. Results: (Pay close attention to this
test; its one of the hardest test to read)
 After inoculation and incubation, the ability of
an organism to reduce nitrate to nitrite (or
molecular nitrogen) is detected by adding two
reagents:
Solution A (sulfanilic acid)
Solution B (α-naphthylamine)
 If a red color appears after addition of solution
A and B, this is considered a positive result
NO3 NO2
Nitrate Reductase
- - Forms red color once
solutions A and B are added.

33. Results continued:
 If there is no color change occurs after
additions of solutions A & B, two
possibilities must be considered:
1) nitrates were not reduced by the organism
2) the organism possessed such potent nitrate
reductase enzymes that nitrates were
reduced beyond nitrites to ammonia or even
molecular nitrogen
NO3 NO2
Nitrate Reductase
- -
NH3
+
(Ammonia)
N2 (Nitrogen Gas)
P 219
NO3 NO3
- -

34. Results Continued:
 Next, solution C (zinc) is added to the tubes that showed no
color change after addition of Nitrate A & B
 Zinc, a reducing agent, is capable of converting nitrate to
nitrite; the development of a red color after addition of
Nitrate C means that nitrates were present and reduced by
the zinc not the organism; this is a negative result
 If the addition of zinc does not produce a color change, the
nitrates were reduced beyond nitrites to ammonia or
nitrogen gas; this is a positive reaction
NO3 NO3
- -
NO3 NO2
Nitrate Reductase
- -
NH3
+
(Ammonia)
N2 (Nitrogen Gas)

35.  Solutions A and B
have been added to
these tubes
 Solution C has been
added to these tubes
E. coli - Reductase Positive
NO3 NO2
Nitrate Reductase
- -
P. aeruginosa
Reductase Positive
C. xerosis - Reductase Negative
NO3 NO3
- -
NO3 NO2
Nitrate
Reductase
- -
NH3
+
(Ammonia)
N2 (Nitrogen Gas)
Unreactive
tubes