Bacteriology

1. Bacteriology I (MeLS-M3293) for MLS Students
By Temesgen W/medhin (MSc – Medical Microbiology)
Wolaita Sodo University CHSM
Oct. 2023
Biochemical Tests (Chapter Ten)
1

2. Learning objective:
At the end of this chapter the students will be able to:
• List the types of biochemical tests
• Describe the principles of biochemical tests
• List the required materials for the biochemical tests
• Describe the procedures of biochemical tests
• Identify test result and positive and negative control bacteria for the
specific biochemical tests
2

3. INTRODUCTION
• Biochemical tests are used to differentiate different
organisms based on their genus and species characteristics.
• Biochemical tests are performed on pure culture.
• The following are some of the common biochemical tests
used for differentiation of different bacteria.
3

4. Biochemical test & reactions
4

5. 1. Catalase test
• This test is used to differentiate those bacteria that produce the
enzyme catalase such as Staphylococci from non catalase
producing bacteria such as Streptococci.
Principle:
– Catalase acts as a catalyst in the breakdown of H2O2 to O2
& H2O.
– An organism is tested for catalase production by bringing
it into contact with H2O2.
– Bubbles of oxygen are released if the organism is a
catalase producer. The culture should not be more that
24 hour old.
5

6. 1. Catalase test … Cont’d
Material Required
• 3% H2O2
• Test tubes
• Swab
Method
• Pour 2-3 ml of the H2O2 solution into a test tube.
• Using a sterile wooden stick or a glass rod, remove several
colonies of the test organism and immerse in the H2O2 solution.
• Look for immediate bubbling.
• Important: Care must be taken when testing an organism
cultured on a medium containing blood because catalase is
present in red cells
6

7. 1. Catalase test …Cont’d
Results
• Active bubbling ----- Positive test- Catalase produced
• No release of bubbles ----- Negative test - No catalase produced
7

8. 1. Catalase test …Cont’d
Note:
• if the organism has been cultured on an agar slope, pour
about 1ml of the H2O2 solution over a good growth of the
organism, and look for the release of bubbles.
Control
• Positive catalase control – Staphylococcus species
• Negative catalase control – Streptococcus species
Catalase
8

9. 2. Coagulase Test
• This test is used to differentiate Staphylococcus aureus which
produces the enzyme coagulase, from S.epidermidis and
S.saprophyticus which do not produce coagulase.
Principle
• Coagulase causes plasma to clot by converting fibrinogen to
fibrin.
9

10. 2. Coagulase Test
• Two types of coagulase are produced by most strains of S.
aureus.
1. Free Coagulase which converts fibrinogen to fibrin by activating
a coagulase-reacting factor present in plasma.
– Free coagulase is detected by the appearance of a fibrin
clot in the tube test.
2. Bound coagulase (clumping factor) is found on bacterial cell
surface which converts fibrinogen directly to fibrin without
requiring a coagulase reacting factor.
– It can be detected by the clumping of bacterial cells in the
rapid slide test.
10

11. 2. Coagulase Test…Cont’d
• It is usually recommended that a tube test should be
performed on all negative slide tests.
• A tube test must always be performed if the result of the
slide test is not clear, or when the slide test is negative.
• Before performing a coagulase test, examine a Gram stained
smear to confirm that the organism is a Gram positive coccus.
11

12. 2. Coagulase Test…Cont’d
Material Required
EDTA anticogulated human plasma.
The plasma should be allowed to warm to room temperature
before being used.
Oxalate or heparin plasma can also be used.
Do not use citrated plasma because citrate-utilizing bacteria
e.g. Enterococci, Pseudomonas and Serratia may cause clotting
of the plasma (in tube test).
Occasionally, human plasma may contain inhibition substances
which can interfere with coagulase testing. It is therefore
essential to test the plasma using a known coagulase positive S.
aureus.
12

13. 2. Coagulase Test…Cont’d
Slide test method (detects bound coagulase)
1. Place a drop of distilled water on each end of a slide or on two separate
slides
2. Emulsify a colony of the test organism in each of the drops to make two
thick suspensions.
 Note: Colonies from a mannitol salt agar culture are not suitable for
coagulase testing. The organism must first be cultured on nutrient agar
or blood agar.
3. Add a loopful (not more) of plasma to one of the suspensions, and mix
gently.
4. Look for clumping of the organisms within 10 seconds.
5. No plasma is added to the second suspension. This is used to differentiate
any granular appearance of the organism from true coagulase clumping
13

14. 2. Coagulase Test…Cont’d
Results
• Clumping within 10 seconds…...S. aureus
• No clumping within 10 seconds …No bound coagulase
Control
• Positive coagulase control …..S. aureus
• Negative coagulase control … E.coli or S. epidermidIs
14

15. 2. Coagulase Test…Cont’d
Test tube method (detects free coagulase)
1.Take three small test tubes and label as:
T = test organism [18 – 24 hr broth culture]
Pos = positive control. (18 – 24 hr S. aureus broth culture)
Neg = Negative control (sterile broth)
Nutrient broth is suitable. Do not use glucose broth.
2. Pipette 0.2ml of plasma into each tube
3. Add 0.8ml of the test broth culture to tube T.
4. Add 0.8ml of the S.aureus culture to the tube labeled as ‘pos’
5. Add 0.8ml of sterile broth to the tube labeled as ‘Neg’
15

16. 6. After mixing gently, incubate the three tubes at 35 – 37oC for
1 hour.
7. Examine for clotting after 1 hour.
– If no clotting has occurred, examine after 3 hours.
– If the test is still negative, leave the tube at room
temperature over night and examine again by tilting
the tube gently.
2. Coagulase Test…Cont’d
16

17. 2. Coagulase Test…Cont’d
Results
• Clotting in the tube……….S.aureus
• No clotting ……………… Negative test
Note: There should be no clotting in the negative control tube.
17

18. 3. DNase (Deoxyribonuclease) test
• This test is used to identify S. aureus which produces DNase enzymes.
• The DNase test is particularly useful when plasma is not available to
perform coagulase test or when the results of a coagulase test are difficult
to interpret.
Principle
• The enzyme DNase hydrolyzes DNA when the test organism is cultured on a
medium which contains DNA.
• After overnight incubation, the colonies are tested for DNase production
by flooding the plate with a weak HCl solution.
• The acid precipitates unhydrolyzed DNA.
• DNase producing colonies are therefore surrounded by clear areas due to
DNA hydrolysis.
18

19. Material Required
• DNase agar plate
– Up to six organisms may be tested on the same plate.
• Hydrochloric acid, 1 mol/L (1 N)
3. DNase test…Cont’d
19

20. 3. DNase test…Cont’d
Method
1.Divide a DNase plate into the required number of strips
by marking the underside of the plate.
2.Using a sterile loop or swab, spot-inoculate the test and
control organisms.
3.Make sure each test area is labeled clearly.
4.Incubate the plate at 35-370C overnight.
5.Cover the surface of the plate with 1 mol/L HCl solution.
6.Tip off the excess acid.
7.Look for clearing around the colonies within 5 minutes of
adding the acid.
20

21. 3. DNase test…Cont’d
Results
• Clearing around the colonies………….DNase positive strain
• No clearing around the colonies………DNase negative strain
Controls
• Controls positive ……………Staphylococcus aureus
• Negative control: ……………Staphylococcus epidermidis
Dnase test 21

22. 4. Bile solubility test
• This helps to differentiate S.pneumoniae, which is soluble in
bile and bile salts, from other alpha-haemolytic streptococci
(viridans streptococci) which are insoluble.
Principle
• A heavy inoculum of the test organism is emulsified in
physiological saline and the bile salt sodium deoxycholate is
added.
• This dissolves S. pneumoniae as shown by a clearing of the
turbidity within 10-15 minutes.
• Viridans and other streptococci are not dissolved and therefore
there is no clearing of the turbidity.
22

23. 4. Bile solubility test…Cont’d
Material Required
• Sodium deoxycholate, 100 g/l (10% w/v)
• Physiological saline (sodium chloride, 8.5 g/l)
Method
• Although the bile solubility test can be performed by testing
colonies directly on a culture plate or on a slide, a tube
technique is recommended because the results are easier to
read.
23

24. 4. Bile solubility test…Cont’d
Tube method
1. Emulsify several colonies of the test organism in a tube
containing 2 ml sterile physiological saline, to give a turbid
suspension.
2. Divide the organism suspension between two tubes
3. To one tube, add 2 drops of the sodium deoxycholate reagent
and mix
4. To the other tube (negative control), add 2 drops of sterile
distilled water and mix
5. Leave both tubes for 10-15 minutes at 35-37OC.
6. Look for a clearing of turbidity in the tube containing the
sodium deoxycholate.
24

25. 4. Bile solubility test…Cont’d
Results
• Clearing of turbidity ………………probable S. pneumoniae
• No clearing of turbidity…………… probable not S. pneumoniae
• There should be no clearing of turbidity in the negative control
tube to which distilled water was added.
Controls
• Bile solubility positive control:
…. S. pneumonia
• Bile solubility negative control:
… Enterococcus faecalis
25

26. 5. Litmus milk decolorization test
• This test is a rapid inexpensive technique to assist in the
identification of Enterococci.
• It is based on the ability of most strains of Enterococcus species to
reduce litmus milk by enzyme action as shown by decolorization
of the litmus.
Principle
• A heavy inoculum of the test organism is incubated for up to 4
hours in a tube containing litmus milk.
• Reduction of the litmus milk is indicated by a change in colour of
the medium from mauve to white or pale yellow.
26

27. 5. Litmus milk decolorization test…Cont’d
Material required
• Litmus milk medium
Method
• Using a sterile loop, inoculate 0.5 ml of sterile litmus milk
medium with the test organism.
– Important: A heavy inoculum of the test organism must
be used.
• Incubate at 35-370C for up to 4 hours,
• Examining at half hour intervals for a reduction reaction as
shown by a change in colour from mauve to white or pale
yellow
– compare with the positive control. 27

28. Results
• White or pale yellow……………….Suggestive of Enterococcus
• No change or pink colour………………………. Probably not
Enterococcus
5. Litmus milk decolorization test…Cont’d
28
In this figure a) represents reduction b) represents acidic reaction c) alkaline reaction

29. 5. Litmus milk decolorization test…Cont’d
Controls
• Positive control: Enterococcus species
• Negative control: Viridans Streptococci
Note:
• A negative result can be checked by culturing the organism in
aesculin broth and examining daily for up to 7 days for aesculin
hydrolysis as shown by a blackening in the medium
• Enterococci hydrolyze aesculin
Litmus milk
29

30. 6. Aesculin hydrolysis test (using blood agar)
• Enterococci can also be identified using an aesculin hydrolysis
test
• This test can be economically performed using a Rosco bile
aesculin tablet
 The test can be performed by placing a tablet on a blood agar
plate inoculated with the test organism and incubating it at 35-
370C overnight.
 A positive test is indicated by the tablet and colonies around it
turning black/grey.
 A negative test is shown by the tablet remaining white and no
change in colour of the colonies.
30

31. • Bile Esculin hydrolysis
– Ability to grow in 40% bile
and hydrolyze Esculin are
features of streptococci
that possess Group D
antigen
Enterococci produce
a positive (left) bile
Esculin hydrolysis
test.

32. • Altematively, the test can be performed by making a dense suspension
of the test organism in 0.25 ml of physiological saline in a small tube,
adding a tablet, and incubating at 35-370C for 4 hours (or overnight).
• A positive reaction is shown by a black/grey colour in the medium.
6. Aesculin hydrolysis test (using saline tube)
32

33. 7. Oxidase test/Cytochrome oxidase test
• The oxidase test is used to detect bacteria that produce the
enzyme cytochrome oxidase which catalyze oxidation of
reduced cytochrome c by oxygen molecule
• It assist in the identification of Pseudomonas, Neisseria,
Vibrio, Brucella, and pasteurella species, which are oxidase
positive.
33

34. Oxidase test/Cytochrome oxidase test…Cont’d
Principle
• A piece of filer paper is soaked with a few drops of oxidase reagent. A
colony of the test organism is then smeared on the filter paper. When the
organism is oxidase-producing, the Phenylenediamine in the reagent will be
oxidized to a deep purple colour.
• Occasionally the test is performed by flooding the culture plate with
oxidase reagent but this technique is not recommended for routine use
because the reagent rapidly kills bacteria. It can however be useful when
attempting to isolate N.gonorrhoeae from mixed cultures in the absence
of a selective medium.
• The oxidase positive colonies must be removed and subcultured within 30
seconds of flooding the plate
34

35. 7. Oxidase test/Cytochrome oxidase test…Cont’d
Material Required
• Fresh Oxidase reagent (1% Tetramethyle-p-phenylenediamine
dihydrochloride)
• Filter paper or oxidase regent strip
Note:
• Fresh oxidase reagent is easily oxidized.
• When oxidized it appears blue and must not be used.
35

36. 7. Oxidase test/Cytochrome oxidase test…Cont’d
Method using filter paper
1. Place a piece of filter paper in a clean petridish
2. Add 2 or 3 drops of freshly prepared oxidase reagent,
3. Using a piece of stick or glass rod (not an oxidized wire loop),
remove a colony of the test organism and smear it on the filter
paper.
4. Look for the development of a blue-purple color within 10 sec
as shown in the figure.
36

37. 7. Oxidase test/Cytochrome oxidase test…Cont’d
Result
• Blue-purple color …..positive (Within 10 sec)
• No blue-purple color …Negative (Within 10 sec)
Note: Ignore any blue-purple color that develops after 10 sec.
37

38. 7. Oxidase test/Cytochrome oxidase test…Cont’d
Method using an oxidase regent strip
1. Moisten the strip with a drop of sterile water.
2. Using a piece of stick or glass rod (not an oxidized wire loop)
remove a colony of the test organism and rub it on the strip.
3. Look for a red-purple colour within 20 seconds.
NB: purple colour………….positive oxidase test.
Controls
• Positive control:--------Pseudomonas aeruginosa
• Negative control---------Escherichia coli
38

39. 39

40. 8. Urease test
• This test is used to detect the enzyme urease, which breaks
down urea into ammonia and carbon dioxide.
• Testing for urease enzyme activity is important in
differentiating Enterobacteriaceae
– Proteus, Morganella, and Providencia are strong
urease producers
– Klebsiella a weak urease producer,
– Y. enterocolitica also shows urease activity (weakly at
35-370C).
40

41. 8. Urease test
Principle
• The test organism is cultured in a medium which contains
urea and the indicator phenol red.
• When the strain is urease-producing, the enzyme break down
the urea to give ammonia and carbon dioxide.
• With the release of ammonia, the medium becomes alkaline
as shown by a change in colour of the indicator to pink-red .
41

42. 8. Urease test…Cont’d
Method
• Urea agar slant medium the sample is inoculated heavily over
the entire surface of the slant.
• Urease-positive cultures produce an alkaline reaction in the
medium, evidenced by a pinkish-red color.
• Urease-negative organisms do not change the color of the
medium, which is a pale yellowish
42

43. 8. Urease test
43

44. 9. Indole test
• The test detect the ability of an organism to produce indole
from Tryptophan.
• Testing for indole production is important in the identification of
Enterobacteriaceae
• Production of indole from tryptophan is an important
biochemical property of most strains of
» E. coli,
» Edwardsiella tarda,
» Klebsiella oxytoca,
» Providencia species and
» Proteus vulgaris 44

45. 9. Indole test …Cont’d
Principle
• The test organism is cultured in a medium which contains
tryptophan.
• Tryptophan is degraded by tryptophanase into indole,
ammonia, pyruvic acid.
• Indole production is detected by Kovac’s or Ehrlich’s reagent
which contains 4(p)-dimethylamino-benzaldehyde.
• This reacts with the indole to produce a red coloured
compound.
45

46. 9. Indole test …Cont’d
Material required
• Kovac’s or Ehrlich’s reagent
• Bijou bottle/test tube
Method
Indole test using tryptone water and kovac’s reagent.
1. Inoculate the test organism in a bijou bottle containing 3 ml of
sterile tryptone water.
2. Incubate at 35 – 37oC for up to 48 hr
3. Test for indole by adding 0.5ml of Kovac’s reagent and shake
gently.
4. Examine for a red color in the surface layer within 10 min.
46

47. 9. Indole test …Cont’d
Results
• Red surface layer…………………Positive indole test
• No red surface layer………………. Negative indole test
Control
• Positive control
….. E.coli
• Negative control
…. K. pneumoniae
Fig. Indole test: The tube on the left with the red ring is positive for indole
production while the tube on the right shows a negative result.
47

48. 10. Citrate utilization test
• The test detect the ability of an organism to use citrate as its
only source of carbon.
• Citrate utilization is a key biochemical property of
» Salmonella,
» Citrobacter,
» Klebsiella,
» Enterobacter, and
» Serratia.
48

49. 10. Citrate utilization test
Principle
• The utilization of citrate by a test bacterium is detected in citrate
medium by the production of alkaline by-products.
• The medium includes sodium citrate as the sole source of
carbon and ammonium phosphate as the sole source of
nitrogen.
• Bacteria that can use citrate can also extract nitrogen from the
ammonium salt, with the production of ammonia (NH+), leading
to alkalization of the medium.
• In the presence of the indicator bromothymol blue the medium
will be converted from green (at pH 6.0) to blue (at a pH above
7.6).
49

50. Ways of performing a citrate test
– Using Rosco citrate identification tablets
– Using simmon’s citrate agar
Citrate utilization using a Rosco citrate tablet
– Prepare a dense bacterial suspension of the test organism
in 0.25 ml sterile physiological saline in small tube.
– Add a citrate tablet and stopper the tube
– Incubate overnight at 35 – 37oC
Result
– Red color - ------------------ Positive citrate test
– Yellow orange color ------- Negative citrate test
10. Citrate utilization test
50

51. Citrate utilization using Simmon’s citrate agar
Material required
• Simmon’s citrate medium/agar
• Inoculating loop
Method
1.Prepare slopes of the medium in bijou bottles as
recommended by the manufacturer (store at 2-8 C)
2.Using a sterile straight wire, first streak the slope with a
saline suspension of the test organism and then stab the
butt.
3.Incubate at 35 0C for 48 hours
4.Look for a bright blue colour in the medium
51

52. Fig. Citrate utilization test: Left tube is a negative result. Right tube is a positive result.
10. Citrate utilization test
Results
• Bright blue-----------------------------------------Positive citrate test
• No change in colour of medium ------------Negative citrate test
Controls
• Positive control ---------------------------- k.pneumoniae
• Negative control---------------------------E. coli
52

53. 11. MRVP (methyl red-Vogues Proskauer) test
• This test is used to determine two things.
– The MR portion is used to determine if glucose can be converted to
acidic products like lactate, acetate, and formate.
– The VP portion is used to determine if glucose can be converted to
acetoin.
• These tests are performed by inoculating a single tube of MRVP media with
a transfer loop and then allowing the culture to grow for 3-5 days.
• After the culture is grown, about half of the culture is transferred to a clean
tube.
• One tube of culture will be used to conduct the MR test, the second tube
serves as the VP test.
53

54. MRVP Cont’d
• VP (Vogues Proskauer) test positive bacteria include
– Klebsilla spp, Enterobacter spp and Serratia spp, Vibrio,
Staphylococci
• Methyl Red positive bacteria include
– Escherchia spp., Citobacter spp., Salmonella spp,
Proteus spp., Yersinia spp., Staphylococci etc..
• MR-VP test positive bacteria include
– Staphylococci
54

55. 11. MRVP test…Cont’d
A. Methyl red (MR) test:
• Methyl red is added to the MR tube.
 A red color indicates a positive result
– glucose can be converted into acidic end products
such as lactate, acetate, and formate.
 A yellow color indicates a negative result,
– glucose is converted into neutral end products.
55

56. Figure: methyl red test
56

57. 11. MRVP test…Cont’d
B. VP (Vogues Proskauer) test:
• First alpha-napthol (also called Barritt’s reagent A) and then
KOH (also called Barritt’s reagent B) are added to the VP tube.
• The culture should be allowed to sit for about 15 minutes for
color development to occur.
• If acetoin is produced then the culture turns to red color
(positive result);
• if acetoin is not produced then the culture appears yellowish
in color (a negative result).
57

58. Figure: VP(Vogues Proskauer) test MRVP 58

59. 12. Triple sugar Iron (TSI)
• TSI is a composite medium containing glucose, lactose, sucrose,
phenol red and ferric citrate.
• Looks at fermentation of glucose, lactose, and sucrose and
checks if hydrogen sulfide and gas is produced in the process.
• The color change that occurs in the tube will indicate what sugar
or sugars were fermented.
• The presence of a black color indicates that H2S was produced.
• In this media, H2S reacts with the ferrous sulfate in the media to
make ferrous sulfide, which is black in colour.
• To inoculate, use a needle to stab agar and then use a loop to
streak the top slanted region.
59

60. 12. TSI…Cont’d
60

61. 61

62. 62
TSI

63. 13. Kligler Iron Agar (KIA)
• KIA is a composite medium containing glucose, lactose,
phenol red and ferric citrate.
• A yellow base indicates glucose fermentation
• A yellow base and slope indicates both glucose and lactose
fermentation.
• Bubble in the medium indicate gas production from glucose
• Blackening of the medium indicate H2S production
63

64. 64
KIA
KIA

65. 14. Nitrate reduction test
• Nitrate broth is used for the test
Principle
• The test detects the ability of the organism to produce the
enzyme nitrate reductase which reduces nitrate to nitrite
Method
• Growing the bacteria for 5 days at 370C in a broth containing 1%
KNO3
• Add 0.1 ml of the test reagent to the culture
• The test reagent consists of a mixture of equal volume of
solutions of Sulphonilic acid and -naphthylamine in 5 N acetic
acid. Mixed just before use
65

66. 14. Nitrate reduction test…Cont’d
Result
• Positive ---------Red colour developing within a few minutes
• Negative--------No colour change
66
Nitrate

67. 15. Additional non biochemical test
Motility test
• The motility test is not a biochemical test since we are not
looking at metabolic properties of the bacteria.
• Rather, this test can be used to check for the ability of bacteria
to migrate away from a line of inoculation.
• To perform this test, the bacterial sample is inoculated into
motility media using inoculating straight wire.
• Simply stab the media in as straight a line as possible and
withdraw the needle very carefully to avoid destroying the
straight line.
67

68. • After incubating the sample for 24-48 hours, observations
can be made.
• Check to see if the bacteria have migrated away from the
original line of inoculation.
• If migration away from the line of inoculation is evident then
you can conclude that the test organism is motile (positive
test).
• Lack of migration away from the line of inoculation indicates
a lack of motility (negative test result).
Motility test…
68

69. Fig. Motility test: Left tube is the
result for a non-motile bacterium.
Right tube is the result for a motile
organism.
Figure: Motility agar is a differential
medium .The organisms in the two tubes
pictured on the right are motile.
Motility test…
69

70. 16. Motility-Indole-Urea (MIU)
• MIU is a composite medium containing tryptophan, phenol
red, urea and a paper strip moistened in kovac’s reagent.
• It is inoculated by straight wire through the center of the
medium
• Non-motile organism grow only in the line of the inoculum,
but motile organism grow through out the medium which
become turbid
• Urease positive organism turn the medium red
• Indol positive organism turn the Kovac’s strips red
70

71. 71
MIU

72. SIM (Sulfur, Indole, Motility) Medium
• SIM Medium is used for the differentiation of microorganisms on the
basis of hydrogen sulfide production, indole production, and motility in
a laboratory setting. SIM Medium is not intended for use in the
diagnosis of disease or other conditions in humans.
72

73. • Carbohydrates are complex chemical substrates that serve as energy
sources when broken down by bacteria and other cells.
• Facultative anaerobic and anaerobic bacteria are capable of fermentation,
an anaerobic process during which carbohydrates are broken down for
energy production.
• We can detect whether a specific carbohydrate has been fermented by
looking for common end products of fermentation.
• When carbohydrates are fermented as a result of bacterial enzymes, the
following fermentation end products may be produced:
1. acid end products, or
2. acid and gas end products.
17. Fermentation of carbohydrates
73

74. • In order to test for these fermentation products, inoculate
and incubate tubes of media containing a single carbohydrate
(such as lactose or maltose), a pH indicator (such as phenol
red) and a Durham tube (a small inverted tube to detect gas
production).
• If the particular carbohydrate is fermented by the bacterium,
acid end products will be produced which lowers the pH,
causing the pH indicator to change color (phenol red turns
yellow).
17. Fermentation …
74

75. If the carbohydrate is not fermented, no acid or gas will be produced and the
phenol red will remain red.
17. FERMENTATION …
Fig; If gas is produced along with the acid, it collects in the Durham tube as a
gas bubble 75

76. Antimicrobial Susceptibility Typing
• This typing technique involves comparison of different
isolates to a set of antibiotics.
• Isolates differing in their susceptibilities are considered as
different strains
76

77. Bacitracin

78. Optochin susceptibility test
• The optochin susceptibility test is performed with a 6-mm, 5-
μg optochin disk and is used to differentiate between S.
pneumoniae and viridans streptococci.
• Optochin-susceptible strains can be identified as S.
pneumoniae
Optochin

79. Christie-Atkins, Munch-Petersen (CAMP) test
This test detects a diffusible, heat-stable, extracellular protein produced
by Group B Streptococcus that enhances the hemolysis by S. aureus.
Group B streptococci showing the classical “arrow-shaped hemolysis near the staphylococcus
streak 79
CAMP

80. Biochemical test reading chart for G-ve rods
80
Lactose Indol Urea Manitol H2s Gas in
glucose
Citrate Motility LDC Organisms
Fermen
ter
_
+
+ + + + + - Citrobacter
- - +
+ + - Entrobacter cloacae
- + Klepsella pneumoniia
-/+ - -/+ Klepsella ozenae
- + -
- - - - Klepsella rhinose / Shigella / salmonella
- - + Serratia
+ + + + Entrobacter aerogens / haznia
- Entrobacter cloacae / citrobacter
+
+ + + - citrobacter
+ Arizona
+
+ + - + + - - Klepsella pneumonia / oxitonia
- + - +
+ + - Citrobacter diveras
- + + E.coli
- - E.coli
- - - - E.coli (A-D)
- - - - - - Shigella dysentery/ E.coli

81. Lactose Indol Urea Manitol H2s Gas Citrate Motility LDC Organisms
Non
lactose
ferment
er
+ +
+ /- - + + + - Providential reffigeri
- - + - + - Morganella morgani
+ + +/- + - Proteus vulgaris
- -
-
+ + + - Providential / colizonoves
-
+ + - Providential stuarti
- - - Shigella dysentery
+ + - + + Edwidisella
+ - -
- - + E.colli (A-D)
- Shigella specious
+ + - Providential stuarti (C-D)
- -
- - - - - - S. dysentery
+ -
- - - - Shigella specious
+ - + - Salmonella group A
+
+ + + + S.arizana
- - + + S. typhi
+ - + + + /- + - Proteus mirabilis
81
Biochemical test reading chart for G-ve rods

82. 82