1. The document discusses various biochemical tests used to identify bacteria, including enzymatic tests, metabolic pathway tests, and specific tests. 2. Metabolic pathway tests include carbohydrate oxidation/fermentation tests like the oxidative fermentation test, carbohydrate fermentation in TSI agar, and methyl red and Voges-Proskauer tests. They also include amino acid degradation tests and single substrate utilization tests. 3. Examples of specific tests discussed are the citrate utilization test and acetate utilization test to determine if bacteria can use certain compounds as the sole carbon source.

1. Medical Microbiology Laboratory
(Biochemical tests - II)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. 2
BIOCHEMICAL TESTS
 Group of tests, used to differentiate between bacterial
genera/species.
 Biochemical tests classified as:
1. Enzymatic tests
2. Metabolic pathways tests
a) Carbohydrate oxidation and fermentation tests
b) Amino acids degradation tests
c) Single substrate utilization tests
3. Antibiotic inhibition tests
4. Specific tests

3. 3
METABOLIC PATHWAYS TESTING
a) Carbohydrate oxidation and fermentation tests
1. Oxidation fermentation test
2. Carbohydrate fermentation in TSI agar
3. Methyl red test
4. Voges-Proskauer test
b) Amino acids degradation tests
1. Decarboxylase-dihydrolase reactions
2. Deamination reaction
3. Decarboxylation and deamination reactions in LIA
c) Single substrate utilization tests
1. Citrate utilization test
2. Acetate utilization test
3. Acetamide utilization test

4. 4
OXIDATIVE FERMENTATIVE (O-F) TEST
 Developed by Hugh and Leifson in 1953.
 They developed OF media to differentiate between
oxidative bacteria (that produces acid from carbohydrates
under aerobic condition only) and fermentative bacteria
(that produces acid both under aerobic and anaerobic
conditions).
 Used to separate organisms into two major groups:
 Enterobacteriaceae – fermentative
 Pseudomonas – oxidative

5. 5
TOOLS, CONSUMABLES AND REAGENTS
Straight needle
loop for stabbing O-F medium
(Two tubes)
Liquid paraffin
Bacteria to be tested

6. 6
PROCEDURE
1. Inoculate two tubes of O-F test medium with the test
organism using a straight wire by stabbing “half way to the
bottom” of the tube.
2. Cover one tube of each pair with 1 cm layer of sterile mineral
oil or liquid paraffin (it creates anaerobic condition in the tube
by preventing diffusion of oxygen), leaving the other tube
open to the air.
3. Incubate both tubes at 35 ºC for 48 hours (Slow growing
bacteria may take 3 to 4 days before results can be
observed).

7. 7
RESULTS AND INTERPRETATION
 Glucose fermenter: when acid production is detected on
both tubes since fermentation can occur with or without
oxygen.
 Glucose oxidizer: acid is detected by the open aerobic tube.
 Nonutilizer: some bacteria do not use glucose as a substrate
Open (aerobic) tubeCovered (anaerobic) tubeMetabolism
Acid (Yellow)Alkaline (Green)Oxidative
Acid (Yellow)Acid (Yellow)Fermentative
Alkaline (Green)Alkaline (Green)Glucose not metabolized

8. 8
RESULTS

9. 9
RESULTS

10. 10
TRIPLE SUGAR AGAR (TSI)
 Triple Sugar Iron (TSI) or Kligler Iron Agar: is a semi solid
media having slant and butt, consists of:
1. 0.1% glucose: If only glucose is fermented, only enough acid is
produced to turn the butt yellow. The slant will remain red.
2. 1% lactose / 1% sucrose: a large amount of acid turns both butt
and slant yellow, thus indicating the ability of the culture to ferment
either lactose or sucrose.
3. Iron (FeSO4): Indicator of H2S formation (black color).
4. Phenol red: pH indicator (yellow in acidic, red in alkaline).
5. Peptone: which acts as source of nitrogen.

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TOOLS, CONSUMABLES AND REAGENTS
TSI agar (slant)Bacteria to be tested
Straight needle
loop for stabbing

12. 12
PROCEDURE
1. With a sterilized straight inoculation
needle touch the top of a well-isolated
colony.
2. Inoculate TSI Agar by first stabbing
through the center of the medium to the
bottom of the tube and then streaking on
the surface of the agar slant.
3. Leave the cap on loosely and incubate the
tube at 35°C in ambient air for 18 to 24
hours.
Inoculation in TSI Agar
slant
butt

13. 13
EXPECTED RESULTS IN TSI TEST

14. 14
EXAMPLES
H2SGasButtSlant
Name of the
organisms
Negative (-)Positive (+)Acid (A)Acid (A)
Escherichia,
Klebsiella,
Enterobacter
Negative (-)Negative (-)Acid (A)Alkaline (K)
Shigella,
Serratia
Positive (+)Positive (+)Acid (A)Alkaline (K)
Salmonella,
Proteus
Negative (-)Negative (-)Alkaline (K)Alkaline (K)Pseudomonas

15. 15
RESULTS

16. 16
METHYL RED (MR) TEST
 MR test is a test for acid production, it is positive when the
organism is able to produce strong acids (lactic, acetic,
formic) from glucose via the mixed acid fermentation pathway.
 Only those organism that can maintain low pH of about 4-4.5
can be called methyl red positive

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TOOLS, CONSUMABLES AND REAGENTS
Wire loop MR-VP broth
Bacteria to be tested
Methyl red
indicator

18. 18
PROCEDURE
1. Inoculate two tubes containing MR-VP broth with a pure
culture of the microorganisms under investigation.
2. Incubate at 35 °C for up to 4 days.
3. Add about 5 drops of the methyl red indicator solution to the
first tube.
4. A positive reaction is indicated, if the colour of the medium
changes to red within a few minutes.
MR Positive: When the culture medium turns red after addition of methyl red,
because of a pH at or below 4.4 from the fermentation of glucose.
MR Negative: When the culture medium remains yellow, which occurs when
less acid is produced (pH is higher) from the fermentation of glucose.

19. 19
RESULTS
Negative Positive

20. 20
VOGES-PROSKAUER (VP) TEST
 Is a test used to detect acetoin in a bacterial broth culture.
 Performed by adding alpha-naphthol and potassium hydroxide to
the MR-VP broth which has been inoculated with bacteria. A cherry
red color indicates a positive result, while a yellow-brown color
indicates a negative result.
 The test depends on the digestion of glucose to acetylmethyl-
carbinol.
 In the presence of oxygen and strong base, the acetylmethylcarbinol
is oxidized to diacetyl, which then reacts with guanidine compounds
commonly found in the peptone medium of the broth.
 Alpha-naphthol acts as a color enhancer, but the color change to red
can occur without it.

21. 21
TOOLS, CONSUMABLES AND REAGENTS
Wire loop MR-VP broth
Bacteria to be tested
Barritt’s
reagents A & B
Barritt’s reagents:
A) Alpha-naphthol (5%)
B) Potassium Hydroxide (40%)

22. 22
PROCEDURE
1. Inoculate a tube of MR/VP broth with a pure culture of the test
organism.
2. Incubate for 24 hours at 35 ºC.
3. At the end of this time, aliquot 1 mL of broth to clean tube.
4. Add 0.6 mL of 5% alpha naphthol, followed by 0.2 mL of 40%
KOH. (Note: It is essential that the reagents be added in
this order).
5. Shake the tube gently to expose the medium to atmospheric
oxygen and allow the tube to remain undisturbed for 10 to 15
minutes.

23. 23
RESULTS

24. 24
DECARBOXYLASE-DIHYDROLASE REACTIONS
 To determine the production of decarboxylase by bacteria
(Enterobacteriaceae).
 Decarboxylase enzyme: removes carboxyl groups from the amino
acids lysine and ornithine.
 Dihydrolase enzyme: removes a carboxyl group from arginine.
 Glucose base without the amino acid and tubes containing
glucose plus the amino acid substrates are inoculated.
 Decarboxylation and dihydrolation are anaerobic reactions so
overlay the inoculated tubes with mineral oil (or paraffin) to
exclude air.

25. 25
DECARBOXYLASE-DIHYDROLASE REACTIONS
 Early incubation, both tubes yellow due to acidification of the
indicator (bromcresol purple) by the acid end products of glucose
fermentation.
 If amino acid is decarboxylated, alkaline amines are formed and
cause the indicator to revert to an alkaline pH.
A. Positive (purple); decarboxylation
B. Negative (yellow); no decarboxylation;
only glucose fermentation
Moeller decarboxylase medium

26. 26
RESULTS

27. 27
DEAMINATION REACTIONS
 To determine the deaminase activity using the amino
acids phenylalanine or tryptophan.
 Only Proteus, Providencia and Morganella spp. possess
the deaminase enzyme.
 Deamination of the amino acid results in a colored
compound with the addition of 10% ferric chloride (FeCl3).
 Principle:
Phenylalanine
phenylalanine deaminase
PPA + 10% FeCl3
Tryptophan
tryptophan deaminase
indol−pyruvic acid + 10% FeCl3

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RESULTS AND INTERPRETATION
1. Positive deamination for phenylalanine, intense green color.
2. Positive deamination for tryptophan, brown color.
3. Negative, slant retains its original color after addition of FeCl3.
Phenylalanine deamination test
A. Negative, Escherichia coli
B. Positive, Proteus vulgaris

29. 29
LYSINE IRON AGAR (LIA) TEST
 To determine the ability of the organism to deaminate lysine,
decarboxylate lysine and produce H2S.
 To identify Salmonella, Proteus, Providencia and Morganella.
 Lysine decarboxylation – butt
 Positive – purple
 Negative – yellow
 Lysine deamination – slant
 Positive – red
 Negative – purple

30. K/K, H2S (+)
• Negative deamination
• Positive decarboxylation
• With black precipitate in
the butt
Salmonella typhimurium
K/ K, H2S (-)
• Negative deamination
• Positive decarboxylation
• No blackening of the butt
Escherichia coli
K/A, H2S (-)
• Positive deamination
• Negative decarboxylation
• No black precipitate in
the butt
Proteus vulgaris
K/A, H2S (-)
• Negative deamination
• Negative decarboxylation
• No black precipitate in
the butt
Shigella flexneri
30
RESULTS AND INTERPRETATION
A = acid (yellow), K = alkaline (purple), K/K = alkaline slant/alkaline butt

31. 31
CITRATE UTILIZATION TEST
 Also called Simmons citrate test.
 To determine if a member of the Enterobacteriaceae is capable
of utilizing citrate as the sole source of carbon.
 Useful in the identification of the lactose fermenting
Enterobacteriaceae, Escherichia coli is citrate negative;
Enterobacter and Klebsiella are positive.
Results
 Positive: growth with an intense blue color on the slant or
solely the presence of growth
 Negative: absence of growth and no color change in the
medium (remains green)

32. 32
RESULTS AND INTERPRETATION
The agar slants inoculated as the same of TSI.

33. 33
ACETATE UTILIZATION TEST
 Is used to determine if an organism can use acetate as the
sole source of carbon. (Another similar test in which organisms
ability to utilize citrate as a sole source of carbon is useful for
differentiating the members of Enterobacteriaceae family, this
test is called Citrate Utilization test or Simmons citrate test).
 If so, breakdown of the sodium acetate causes the pH of the
medium to shift toward the alkaline range, turning the indicator
from green to blue.

34. 34
PROCEDURE
1. With a straight inoculating needle, inoculate
acetate slant lightly from an 18-24 hour
culture. (Do not inoculate from a broth
culture, because the growth will be too
heavy).
2. Incubate at 35 ºC for up to 7 days.
Positive Negative

35. 35
ACETAMIDE UTILIZATION TEST
 To determine the ability of an organism to use acetamide as
the sole source of carbon.
 Bacteria that can grow on this medium deaminate acetamide to
release ammonia.
 The production of ammonia results in a pH-driven color change
of the medium from green to royal blue.
Results
 Positive: deamination of the acetamide resulting in a blue
color.
 Negative: no color change.

36. 36
PROCEDURE
1. Using a sterile inoculating loop or straight
wire, pick a well isolated colony from18-24
hour old culture.
2. Inoculate acetamide agar slant by streaking
the surface of the slant.
3. Incubate the tubes aerobically at 33-37 ºC
for up to with 7 days with loosened caps to
ensure adequate aeration.
4. Examine the tubes daily for 4 days and
again at 7 days before discarding it as
negative.
Positive Negative

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LIMITATIONS
 Acetamide utilization test is not reliable test for
identification of pyocyanin producing Pseudomonas
aeruginosa.
 Only 38% of non pyocyanin producing strains of
Pseudomonas aeruginosa will give positive result.