This document discusses various biochemical tests used to identify bacteria, including: 1. Enzymatic and metabolic pathway tests like carbohydrate fermentation and amino acid degradation. 2. Inhibitor profile tests using antibiotics like bacitracin, novobiocin, and vancomycin to differentiate bacterial species. 3. Other specific tests including growth in salt concentrations, optochin susceptibility, bile solubility, and the CAMP test. The document provides details on procedures, interpretations, and applications of these biochemical tests for medical microbiology purposes.

1. Medical Microbiology Laboratory
(Biochemical tests - III)
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. Establishing inhibitor profiles
4. Specific tests

3. 3
METABOLIC PATHWAYS TESTING
3) Establishing Inhibitor Profiles
1. Bacitracin susceptibility test
2. Bacitracin and sulfamethoxazole-trimethoprim susceptibility test
3. Novobiocin susceptibility test
4. Vancomycin susceptibility test
5. Antibiotic disks for presumptive identification of anaerobes
4) Other more specific tests
1. Growth in various NaCl concentrations
2. Susceptibility to optochin and solubility in bile
3. Ability to hydrolyze esculin in the presence of
4. CAMP

4. 4
BACITRACIN SUSCEPTIBILITY TESTING
 To differentiate Micrococcus and Stomatococcus from
Staphylococcus when combined with other procedures
such as the modified oxidase test.
Principle
 Bacitracin (0.04 units) inhibits the growth of Micrococcus
and Stomatococcus and Group A Streptococcus while
having no effect on Staphylococcus which is resistant.
Results
 Susceptible (S): zones of inhibition ≥ 10 mm.
 Resistant (R): zones of inhibition ≤ 9 mm.

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. 10
BACITRACIN AND SULFAMETHOXAZOLE-
TRIMETHOPRIM SUSCEPTIBILITY TEST
 To identify the different species of Streptococcus especially Group A
and Group B beta hemolytic streptococci.
Principle:
 Group A beta hemolytic streptococci (Streptococcus pyogenes) are
susceptible to 0.04 units bacitracin but resistant to 1.25 ug
sulfamethoxazole-trimethoprim (SXT).
 Group B beta hemolytic streptococci (Streptococcus agalactiae)
resistant to both bacitracin and SXT.
Results
 Susceptible (S): any zone of inhibition around either disk.
 Resistant (R): growth up to the disk (no zone of inhibition).

9. 11
TOOLS, CONSUMABLES AND REAGENTS
TSI agar (slant)Bacteria to be tested
Straight needle
loop for stabbing

10. 15
RESULTS

11. 14
INTERPRETATION
SXTBacitracinStreptococcus spp.
ResistantSusceptibleGroup A
ResistantResistantGroup B
SusceptibleResistantGroup C, F, G

12. 16
NOVOBIOCIN SUSCEPTIBILITY TESTING
 To differentiate the different species of coagulase negative
staphylococci.
Principle:
 After incubation with 5 ug of novobiocin, Staphylococcus
saprophyticus is not inhibited (resistant) by the antibiotic
whereas Staphylococcus epidermidis are susceptible to
novobiocin.
Results:
 Susceptible (S): zone greater than 16 mm
 Resistant (R): zone diameter ≤ 16 mm

13. 17
TOOLS, CONSUMABLES AND REAGENTS
Wire loop MR-VP broth
Bacteria to be tested
Methyl red
indicator

14. 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.

15. 19
RESULTS
Negative Positive

16. 20
VANCOMYCIN SUSCEPTIBILITY TEST
 To differentiate Pediococcus from other alpha hemolytic
streptococcus.
Principle:
 After incubation with 5 ug of vancomycin, Pediococcus is not
inhibited (resistant) by the antibiotic whereas Viridans
streptococcus is susceptible to vancomycin.
Results:
 Susceptible: zone of inhibition
 Resistant: no zone of inhibition

17. 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%)

18. 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.

19. 23
RESULTS

20. 24
ANTIBIOTIC DISKS FOR PRESUMPTIVE
IDENTIFICATION OF ANAEROBES
 To determine an anaerobe’s inhibition that can be used for
presumptive identification based on its characteristic
susceptibility pattern to colistin (10 ug), vancomycin (5 ug) and
kanamycin (1 mg).
Principle:
 Most anaerobes have a characteristic susceptibility pattern to the
above mentioned antibiotics.
1. Kanamycin: inhibits facultative gram (– ve) bacilli.
2. Vancomycin: inhibits facultative & obligate gram (+ve) bacteria
3. Colistin: inhibits facultative gram (– ve) bacilli.

21. 25

22. 26
RESULTS
Susceptible (S): zone greater than 10 mm
Resistant (R): zone of 10 mm or less

23. 27
SALT TOLERANCE (6.5% NACL) TEST
 To classify bacteria based on their ability to grow in the
presence of 6.5% NaCl, a characteristic of certain species
of gram positive and gram negative bacilli.
 To differentiate the Group D (salt tolerant) from the
nonenterococci (intolerant).
 Results:
Growth in 6.5% NaClGrowth in nutrient broth
EqualEqualPositive
Very weak growthGood growthNegative

24. 28
RESULTS AND INTERPRETATION
 Positive: if growth is
equivalent in both media
(salt tolerant)
 Negative: growth on the
salt containing medium
is very weak or absent &
growth in the salt free
medium is good
(intolerant).
 Indicator: bromcresol purple, when positive the medium
turns yellow from purple or the appearance of growth.
E. faecalis S. bovis
A) Positive B) negative

25. 29
GROWTH IN BILE ESCULIN AGAR
 To distinguish Group D streptococci and Enterococcus species from
other Lancefield group of streptococci.
Principle:
 Based on the organisms ability to grow in 40% bile and to hydrolyze
esculin to produce esculetin.
 Esculin reacts with ferric citrate to form a brown black precipitate.
Results:
 Positive: growth indicates tolerance to grow in 40% bile, blackening
indicates hydrolysis of esculin.
 Negative: lack of growth indicates inability to grow in 40% bile, no
color change indicates inability to hydrolyze esculin

26. 30
RESULTS AND INTERPRETATION
A. Positive: Enterococcus faecalis
B. Negative: Streptococcus viridans

27. 31
OPTOCHIN SUSCEPTIBILITY TESTING
 To differentiate Streptococcus pneumoniae from other
alpha hemolytic streptococci.
Principle:
 In the presence of optochin, colonies of Streptococcus
pneumoniae are selectively lysed indicated by a zone of
inhibition after incubation under increased CO2.
 Other alpha hemolytic streptococci are resistant to
optochin.

28. 34
RESULTS AND INTERPRETATION
 Positive: zone of inhibition at least 14 mm in diameter
using (10 µg, P disk) and at least 10 mm when using (6
µg, P disk).
 Negative: growth up to the disk or a zone of inhibition
less than 14 mm with (10, µg, P disk) or less than 10 mm
with (6 µg, P disk).
A. Positive – Streptococcus pneumoniae
B. Negative – Viridans streptococci

29. 35
BILE SOLUBILITY TEST
 To differentiate Streptococcus pneumoniae (which is
positive for bile solubility) from other alpha hemolytic
streptococci.
Principle:
 Pneumococcal colonies are rapidly lysed by bile or a
solution of a bile salt such as sodium deoxycholate.
 Lysis depends on the presence of an intracellular autolytic
enzyme.
 Bile salts lower the surface tension between the bacterial
cell membrane and the medium thus accelerating the
organism’s natural autolytic process

30. 36
RESULTS
 Positive: colony disintegrates; an imprint of the lysed
colony may remain within the zone.
 Negative: intact colonies.

31. 37
CAMP TEST
 To demonstrate the phenomena of synergistic hemolysis
between group B streptococcus and beta hemolytic
Staphylococcus aureus.
Principle:
 A characteristic “arrowhead” hemolytic pattern results
when the organism is streaked perpendicular to beta
hemolytic Staphylococcus aureus.

32. 37
RESULTS AND INTERPRETATION
 Positive: a zone of enhanced hemolysis given by an
arrowhead appearance at the junction of the
Staphylococcus and Streptococcus, indicative of Group
B streptococcus.
 Negative: no zone of enhanced hemolysis not indicative
of Group B streptococci.

33. 37
Api test
 Positive: a zone of enhanced hemolysis given by an
arrowhead appearance at the junction of the
Staphylococcus and Streptococcus, indicative of Group
B streptococcus.
 Negative: no zone of enhanced hemolysis not indicative
of Group B streptococci.