1. Identification of Bacteria
B. Voc. in Medical Laboratory Technology
Semester – 3
MLT 7 – Bacteriology and Clinical Biochemistry - 2
Chapter – 16

2. Objectives
By the end of this chapter, you will be able to:
List the different methods of identifying bacteria.
Perform the following biochemical tests:
◦ Bile solubility test
◦ Catalase test
◦ Citrate utilization test
◦ Coagulate test
◦ Hydrogen sulphide production
◦ Indole test
◦ Nitrate reduction

3. Objectives (Contd.)
◦ Acetamide reaction Test
◦ Bacitracin Test
◦ Cetrimide Test
◦ Esculin hydrolysis Test
◦ Hippurate test
◦ Optocin test
◦ ONPG test
◦ Decarboxylase test

4. Objectives (Contd.)
◦ Oxidase test
◦ Oxidation Fermentation Test
◦ Urease Test
◦ Voges Proskauer (VP) Test
◦ Methyl Red Test
◦ Gelatin Liquefaction Test
◦ Biochemical Reactions on Triple Sugar Iron (TSI) Agar Slant
◦ Phenylalanine Deamination Test

5. Introduction
Definite characterisation
and identification of the
suspected organism is
done by studying the
bacterial growth.
Cultivation: Process of
growing micro organism
in a culture by using
specific media.
Identification
methods
Staining
reactions
Cultural
characteristics
Resistance
Metabolism
Biochemical
properties

6. The commonly used staining methods are:
 Gram Staining: Gram +ve and Gram – ve
 Ziehl – Neilsen stain: Acid fast and non acid fast type
 Fluorescent dyes: Brings out special characters
 Fluorescent antibody technique: Identify those characteristics
Staining Methods

7. Cultural characteristics
Solid Medium Streaked Medium Liquid Medium
• Size
• Shape
• Surface
• Elevation
• Margins
• Edge
• Colour
• Consistency
• Structure
• Degree of growth: scanty,
moderate or profuse
• Nature of colony:
discreet, confluent or
merging, spreading,
filliform or rhizoidal
• Other characteristics:
surface, edge, colour,
odour consistency etc.
• Degree of growth: absent,
scanty, moderate or abundant.
• Presence and nature of
turbidity
• Presence and nature of deposit
• Nature of growth on the
surface of the media.
• Ease of disintegrated and
odour

8.  Size : in mm
 Shape : circular, irregular, radiate/ filamentous
or rhizoid
 Surface : smooth, wavy, rough, granular,
papillate, glistening i.e. shining etc.
 Elevation : elevated, effuse, low convex,
concave, umbonate or umbllicate
 Margins : bevelled or otherwise
 Edge : entire, undulate or wavy, crenated,
fimbriate or curled
Cultural characteristics : Solid media
Colony Shape
Colony Edge
Colony Elevation
(American society of microbiology,2013)

9.  Colour: colourless, pink, black red etc.
 Consistency or texture: friable, vicid, butryous or membranous.
 Structure or opacity: transparent, translucent or opaque
Cultural characteristics : Solid media (Contd.)

10.  https://www.youtube.com/watch?v=SrfgQTzPU4w
Colony morphology video

11. Resistance and Metabolism
RESISTANCE
 The resistance of the organism is
tested for:
o Heat
o Low concentration of
disinfectant
o Antibiotics
o Chemotherapeutic agents
o Bacteriocins
METABOLISM
 Observing metabolic
characteristics like :
o Need of oxygen or carbon
dioxide
o Capacity to form pigments
o Haemolysis

12. Quiz 1 : Identify cultural characteristics
1 2

13. Quiz 1 : Identify cultural characteristics
3 4

14. Biochemical tests (23)
 Single enzyme test
o Catalase test
o Coagulation test
o Indole test
o Nitrate test
o Oxidase test
o Urease test
o Hippurate test
o ONPG test
 Carbohydrate Oxidation &
Fermentation
o Hydrogen sulphide production
o Oxidation fermentation
reaction
o VP test
o Methyl red
o TSI agar
 Inhibitor tests
o Bacitracin
o Cetrimide
o Optocin
 Amino acid degradation
o Phenylalanine Deamination
o Decarboxylase test
 Single substrate tests
o Citrate utilization test
o Acetamide reaction Test
 Other specific tests
o Bile solubility test
o Gel liquidation Test
o Esculin hydrolysis

15. Use: Differentiation between catalase producing Staphylococci and
non-catalase producing Streptococci.
Principle:
H2 O2 H2 O + O2 (bubbles)
Procedure:
Immerse colonies into test tube containing 2 - 3ml of H2 O2 with a
sterile stick and observe for immediate bubbling.
Catalase test
Catalase

16.  Observation :
o Appearance of bubbles: Catalase producing organism
o No bubbles: Non-catalase producing organism
 Precautions :
o Culture < 24 hours
o Use of blood free culture medium
Catalase test (Contd.)

17. Use: Differentiation of Staphylococcus aeureus from S. Epidermidis and S.
Saprophyticus.
Principle:
Plasma (soluble fibrinogen) Insoluble fibrinogen (coagulate)
Procedure:
Two drops of saline at each end of slide. Add organisms to make thick suspension.
Add drop of plasma to one suspension and mix gently. Observe for coagulation in 10
seconds. Coagulase positive sample will have clumping of organisms.
Coagulate test
Coagulase

18.  Observation :
Coagulation (A)(10 sec): Staphylococcus aeureus
No coagulation (B) (10 sec): No coagulase
Coagulate test (Contd.)
(Dr Nabil,2014)

19. Use: Identification of species of Enterobacteria like E.coli, P. Vulgaris, P. Rettgeri etc.
which have the ability to produce indole.
Principle:
Tryptophan Other products + Indole
Indole Red coloured compound
Procedure:
Inoculate MUI media with test organisms and add 0.5 ml Kovac’s media or Kovac’s
strip at the neck and incubate at 37°C. Observe for red test strip or red reaction
mixture.
Indole test
breakdown
Kovac’s /Ehlrich reagent

20.  Observation :
Indole test (Contd.)
L: Negative R: Positive

21. Use: Differentiate bacteria which produce enzyme nitrate reductase and
differentiating mycobacterium species.
Principle:
NO3
-
NO2
-
NO2
- Pink red coloured compound
Procedure:
Incubate sterile nitrate broth at 37°C for 4 hours. Add one drop of sulfanilic acid and α
– naphthylamine reagent. Mix well and observe for red colour.
Nitrate reduction
nitrate reductase
Sulfanilic acid
α – napthylamine reagent

22.  Observation :
Red colour (B) : Positive
No red colour (A) : Negative (no nitrate
reduction)
After addition of Zinc:
Red colour (D) : Negative
No red colour (C) : Positive (nitrate
reduced to ammonia or nitrogen gas)
Nitrate reduction (Contd.)
A B C D
Dr. T V Rao, 2010

23. Use: Identify organism which produce enzyme oxidase. E.g. Pseudomonas, Neisseria,
Vibrio, Pasteurella
Principle:
Phenylenediamine (oxidase) reagent Deep purple colour
Procedure:
Place filter paper in petri dish and add 2 to 3 drops of fresh oxidase reagent. Colony is
smeared on the filter paper and reaction is observed.
Oxidase test
Oxidase

24.  Observation :
Blue purple (R) : Positive
No blue purple (L) : Negative
Oxidase test (Contd.)
L: Negative R: Positive
Dr. T V Rao, 2010

25. Use: To identify Proteus strain in Enterobacteria, which are strong urease producers.
Principle:
MUI (Urea ) Ammonium carbonate + NH 4
Ammonia being alkaline changes the colour of media to red pink
Procedure:
Inoculate MIU media and incubated at 35°C overnight. Observe for red pink colour.
Urease Test
Urease

26.  Observation :
Urease Test (Contd.)
Red Pink Medium: Positive
No Red Pink Medium: Negative
Dr. T V Rao, 2010

27. Use: To identify organism producing enzyme hippuricase.
Principle: Organisms like Streptococcus agalactiae which acts on hippuric acid in the
disk to produce glycine and benzoic acid. Ninhydrin when added deaminates glycine
and itself undergoes oxidation which results in formation of blue colour.
Procedure:
Make heavy suspension of the organism in a test tube with 0.1 ml sterile water. Place
hippurate disk in it aseptically with forceps. Incubate at 35°C for 2 hours. Add 0.2 ml of
ninhydrin and re incubate at 35°C for 30 minutes. Observe for change of suspension
colour to blue.
Hippurate Test

28.  Observation :
Formation of blue colour:
Organism like Streptococcus
agalactiae
No blackening of slant: Negative
test
Hippurate Test (Contd.)

29. Use: To detect organism which hydrolyses ONPG.
Principle:
ONPG Orthonitrophenol (yellow)
Procedure:
Make a suspension of loop, full of organism in a normal saline. Place ONPG disk in the
tube and incubate it at 37°C for 4 hours. Observe the tube for colour change.
O – Nitrophenyl –β –D – Galactopyranoside (ONPG) Test
Β –galactosidase enzyme
Hydrolyses

30.  Result :
Yellow coloured disk: Organism
like E. coli
No yellow coloured disk:
Negative test
ONPG Test (Contd.)

31. Use : Assist in identification of Enterobacteria.
Principle :
Enterobacteria + Amino acid (-SH) H2 S
H2 S + (TSI) Fe
+
Fe S (black colour)
Procedure :
Stab the butt and streak the TSI slant. Incubate at 37°C and observe daily for 7 days
for blackening.
Hydrogen Sulphide Production
decomposed

32.  Observation :
Blackening : Positive
No blackening: Negative (no H2 S produced)
Hydrogen Sulphide Production (Contd.)
L: Negative R: Positive

33. Use : Differentiate organism which oxidise carbohydrate (aerobic) and which
ferments carbohydrate (anaerobic)
Principle :
Oxidative organisms : Carbohydrate Acid (yellow)
Fermentative organisms: Carbohydrate Acid (yellow)
Procedure :
Inoculate of media with heavy inoculation at the bottom of the tubes. Cover one tube
with 1cm layer of paraffin oil. Incubate at 35°C for 14 days. Observe them daily.
Oxidation Fermentation Test
O2
Bromothymol
blue indicator
Bromothymol
blue indicator
Absence of O2

34.  Result :
Oxidation Fermentation Test (Contd.)
Open tube Paraffin tube Interpretation
Yellow Green Oxidative organisms
Yellow Yellow Fermentative
organism
Green or blue Green No carbohydrate
utilization

35. Use : To identify Enterobacteria strain which can ferment sugar with the formation of
acetoin. E.g. Vibrio cholerea, K. pneumonia
Principle :
Glucose phosphate peptone water Acetoin Diacetyl
+ Creatine Powder Pink compound
Procedure :
Inoculate 2 ml of Glucose phosphate peptone water and incubate at 35°C for 48
hours. Add small amount of creatine powder and 3 ml of sodium hydroxide reagent
an mix. Keep at 25°C to ± 5° C for one hour. Observe colour.
Voges Proskauer (VP) Test
48 hours
35° C
NaOH

36.  Observation :
Voges Proskauer (VP) Test (Contd.)

37. Use : To identify Enterobacteria strain which can ferment sugar with the formation of
acid.
Principle :
Glucose phosphate acetone water Acid + Methyl red
(indicator) Red colour
Procedure :
Inoculate 2ml of Glucose phosphate acetone water and incubate at 35° to 37°C
overnight . Add methyl red indicator. Observe colour.
Methyl Red Test
overnight
35° to 37°C

38.  Observation :
Methyl Red Test (Contd.)
L: Positive R: Negative
Dr. T V Rao, 2010

39. Use: To identify enteric bacteria including Shigella and Salmonella
Principle:
TSI Agar (Phenol red+lactose+sucrose+glucose+sodium thiosulphate+ferrous sulphate)
------- Fermentation by
inoculated bacteria---acids (by product)---- change of colour from red to yellow
Procedure:
After streaking the TSI agar with loop, pierce it with needle. Then incubate it for 18-24
hours at 37 degree Celsius. Then observe the reactions produced with change of
colour.
Triple Sugar Iron (TSI) Agar Slant

40. Triple Sugar Iron (TSI) Agar Slant (Contd.)
Tube A – Acid slant , acid butt and gas
production.
Tube B – Alkaline slant and alkaline
butt
Tube C – Alkaline slant and acid butt
Observation :

41. a) The test is used to differentiate between Stephyloccoci’s and Streptococci’ s.
The positive reaction to the test is production of oxygen bubbles.
b) It is used to identify certain species of Enterobacteria. In this tryptophan in
the medium is broken down to give an end product which is indicated by the
formation of a red coloured complex.
c)
Q. 2 : Identify the test
Dr. T V Rao, 2010

42. a) Coagulate test
b) Methyl Red
c) Oxidation fermentation reaction
Q. 3: Give the principle for

43. Use : To identify Proteus species which carry out deamination of phenylalanine.
Principle :
Phenylalanine Phenyl pyruvic acid + Ferric chloride ions
Green colour on top of culture
Procedure :
Inoculate slope of phenylalanine agar and incubate at 35° to 37°C overnight. Add 3 –
4 drops of ferric chloride reagent . Observe colour.
Phenylalanine Deamination Test
Deamination

44.  Observation :
Phenylalanine Deamination Test (Contd.)
Green colour: Positive test
No green colour: Negative
test
L: Negative R: Positive

45. Use : To detect an organism’s enzymatic ability to decarboxylase/ hydrolase an amino
acid to amine.
Principle :
The organisms which can produce decarboxylase or hydrolase enzymes an amino
acid to amine. This results in an alkaline pH which changes the solution to purple
colour.
Requirement : Glucose fermenting & glucose non fermenting organism,
decarboxylase broth containing lysine, arginine or ornithine, sterile mineral oil,
suspensions of suspected organism grown on 5% sheep blood agar for 18 to 24 hours
in brain heart infusion broth.
Decarboxylase Test (Moeller’s Reagent)

46.  Procedure:
Glucose non fermenting organism: Inoculate each of the decarboxylase broth and
one control broth (without amino acid). Add 4 ml of mineral oil and incubate at
37°C up to 7 days. Observe colour changes.
Glucose fermenting organism: Inoculate each of the decarboxylase broth with
one drop of the broth. Add 4 ml of mineral oil and incubate at 37°C up to 7 days.
Observe colour changes.
Decarboxylase Test (Contd.)

47.  Observation : On comparison with
control tube:
Positive: Purple colour
Lysine: Klebsiella pneumonia
Arginine: Enterobacter cloacae
Ornithine: Enterobacter cloacae
Negative: No colour change or yellow
colour due to fermentation of glucose
in BHIB.
Decarboxylase Test (Contd.)
Bianca Isaguirre (2013)

48. Decarboxylase Test (Contd.)
Bianca Isaguirre (2013)

49. Use : Differentiate between Streptococcus pyogenes and other β - hemolytic
organisms.
Principle :
Growth of Streptococcus pyogenes is inhibited by small amount of Bacitracin.
Procedure :
Streak 2 to 3 of the suspected colonies on to a blood agar plate. Bacitracin disks are
placed in the first quadrant of the plate and incubate it at 35°C for 18 to 24 hours.
Observe for clearing around the bacitracin disk.
Bacitracin Test

50.  Result:
Zone of inhibition Group A :
Streptococcus pyogenes
No zone of inhibition Group C:
Negative test
Bacitra cin Test (Contd.)

51. Use : To confirm the presence of organism which grow in the presence of the toxic
substance, Cetrimide .
Principle:
Organisms like Pseudomonas aeruginosa grow in the presence of toxic substance
Cetrimide which inhibits the growth of many bacteria.
Procedure:
Inoculate in Cetrimide agar slate a drop of 18 to 24 hour culture in brain heart infusion
broth. Incubate the slant at 35°C for 7 days. Observe for growth.
Cetrimide Test

52.  Observation:
Growth: Pseudomonas aeruginosa
No growth: Negative test
Cetrimide Test (Contd.)

53. Use: To identify pneumococci.
Principle:
Optocin lyses pneumococci while α streptococci are resistant to optocin.
Procedure:
Streak 2 to 3 of the suspected colonies of a pure culture on to half of 5% sheep blood
agar. 6mm optocin disks are placed aseptically on the upper third of the streaked
area. Incubate the plate for 18 to 24 hours. Observe and measure the zone of
inhibition and the diameter of the disk.
.
Optocin Test

54.  Result:
Positive (image): Zone of
inhibition > 14 mm for 6mm
disk
No zone of inhibition: Negative
test
Equivocal : Zone of inhibition <
14 mm
Optocin Test (Contd.)

55. Use : Identification of Enterobacteria.
Principle :
Organism uses citrate (C – source) and ammonium salt (N – source) in the Simmon’s
citrate medium causing an alkaline reaction turning light green solution to blue.
Procedure :
Simmon’s citrate medium + test organism Blue turbid medium
(light green)
Citrate utilization test
Incubate 35°C
for 4 days
Bromothymol
indicator

56.  Observation :
Blue colour and turbidity: Positive
No growth (no turbidity and no change in colour):
Negative
Citrate utilization test (Contd.)
L: Negative R: Positive

57. Use : To identify non fermentative Gram negative bacteria like pseudomonas
aeruginosa, which utilize acetamide.
Principle :
Acetamide (green) NH 4 Royal blue coloured complex
Procedure :
Inoculate acetamide slant with a needle using growth of a 18 to 24 hour culture.
Incubate at 35°C up to 7 days. Observe for blue colour of media
Acetamide reaction Test
Deamination indicator

58.  Observation :
Colour of medium blue (A):
Positive
Colour of medium green (B):
Negative
Acetamide reaction Test (Contd.)
Bianca Isaguirre, 2013

59. Use : Differentiation between S. Pneumoniae (bile soluble) and Viridans
Streptococci (bile insoluble)
Principle :
S. Pneumoniae + Bile salts Clearance of turbidity
Viridans Streptococci + Bile salts Persistence of turbidity
Bile solubility test
Soluble
Insoluble

60. Bile solubility test (Contd.)
2 ml normal saline Several colonies
Test Control
2 drops sodium
deoxycholate 2 ml sterile distilled water
Observation :
Clearing of turbidity (B): Probably S. Pneumoniae
No clearance of turbidity (A): Probably not S.
Pneumoniae
Procedure:
Dr Nabil (2014)

61. Use: To identify organisms producing gelatinase enzyme which liquefy gelatin. E.g.
Pseudomnos and Vibrio cholerea
Principle:
Gelatin Liquefied gelatin
Procedure:
Inoculate medium and incubate at 35° to 37°C for 72 hours. Chill tubes at 4° C for 30
minutes. Observe for positive gelatin liquefaction.
Gelatin Liquefaction Test
4° C for 30 min
Gelatinase

62.  Observation :
Gelatin Liquefaction Test
Liquid media: Positive test
Solid media: Negative test

63. Use : To identify bacteria which can hydrolyze Esculin.
Principle :
Organisms like Klebsiella pneumonia can hydrolyze Esculin a glycoside.
Procedure :
Inoculate of Esculin slant with one drop of 24 hour broth culture. Incubate the slant at
37°C for 7 days. Examine the slant for blackening and confirmation with loss of
fluorescence with Wood’s lamp.
Esculin Hydrolysis Test

64.  Observation :
Blackening of slant: Klebsiella
pneumonia
No blackening of slant: Negative
test
Esculin Hydrolysis Test (Contd.)

65. a) Citrate utilization test
b) Bile solubility test
c) Esculin hydrolysis test
Q. 5: Give the principle for:

66. Q. 6: Explain this result for decarboxylase test:

67. Summary
Now, you will be able to:
List the different methods of identifying bacteria.
Perform the following biochemical tests:
◦ Bile solubility test
◦ Catalase test
◦ Citrate utilization test
◦ Coagulate test
◦ Hydrogen sulphide production
◦ Indole test
◦ Nitrate reduction

68. Summary (Contd.)
◦ Oxidase test
◦ Oxidation Fermentation Test
◦ Urease Test
◦ Voges Proskauer (VP) Test
◦ Methyl Red Test
◦ Gelatin Liquefaction Test
◦ Biochemical Reactions on Triple Sugar Iron (TSI) Agar Slant
◦ Phenylalanine Deamination Test

69. Summary (Contd.)
◦ Acetamide reaction Test
◦ Bacitracin Test
◦ Cetrimide Test
◦ Esculin hydrolysis Test
◦ Hippurate test
◦ Optocin test
◦ ONPG test
◦ Decarboxylase test

70.  American society of microbiology (2013) Colony Morphology
protocol [Online] Available at:
http://www.microbelibrary.org/component/resource/laboratory-test/3136-
colony-morphology-protocol
[Accessed: 22nd September 2015]
 Bianca Isaguirre (2013) Biochemical identification of bacteria
[Online] Available at:
http://www.slideshare.net/anwarsh148/slides-exam
[Accessed: 22nd September 2015]
References

71. References (Contd.)
Dr T. V. Rao (2010) Biochemical reaction Bacteriology [Online]
Available at:
http://www.slideshare.net/doctorrao/cdocuments-and-
settingsadministratordesktopbiochemical-reaction-in-bacteriology?related=3
[Accessed: 22nd September 2015]
 Godkar P (2008) Textbook Of Medical Laboratory Technology , 2nd
edition Bhalani Publishing House, Mumbai
 Good source (2015) Biochemical reaction Bacteriology [Online]
Available at:
http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/colony_m
orph.pdf
[Accessed: 22nd September 2015]