1. Determining an isolate's nutritional and metabolic capabilities through a combination of enzyme and inhibitor tests is commonly used to identify its genus and species. 2. Tests establish enzymatic capabilities and ability to grow under stressors like salts, surfactants, toxins and antibiotics. Enzyme tests measure single enzymes or full pathways, while inhibitor tests examine susceptibility. 3. Common tests include catalase, oxidase, urease, and carbohydrate fermentation, as well as assays for metabolic pathways and tolerance of stressors like antibiotics. Together these provide a metabolic and inhibitory profile for identification.

2. Determining the nutritional and metabolic
capabilities of a bacterial isolate is the
most common approach used for deter-
mining the genus and species of an
organism.

3. The methods available use a combination of tests
to establish the enzymatic capabilities of a given
bacterial isolate as well as the isolates ability to
grow or survive the presence of certain inhibitors
(e.g. salts, surfactants, toxins and antibiotics)

4. A.Establishing Enzymatic Capabilities
Enzyme based tests are designed to measure
the presence of a single enzyme as well as a
complete metabolic pathway.

5. SINGLE ENZYME TESTS
Catalase test
Coagulase test
Pyrase test
Hippurate hydrolysis test
Oxidase test
Indole test
Dnase test
ONPG(B-galactosidase)test
Urease test

6. ASSAYS FOR METABOLIC PATHWAYS
Carbohydrate oxidation and fermentation
oxidation fermentation tests
carbohydrate fermentation in TSIA
methyl red test
Voges Proskauer test

7. Amino acid degradation
 decarboxylase-dihydrolase reactions
 deamination reactions
 decarboxylation and deamination reactions in LIA
Single substrate utilization
 citrate utilization test
 acetate utilization test
 acetamide utilization test

8. B. Establishing Inhibitor Profiles
 bacitracin susceptibility test
 bacitracin and sulfamethoxazole-trimethoprim
susceptibility test
 novobiocin susceptibility test
 vancomycin susceptibility test
 antibiotic disks for presumptive identification
of anaerobes

9. C. Other more specific tests
 growth in various NaCl concentrations - Enterococci
and Vibrio species
 susceptibility to optochin and solubility in bile –
Streptococcus pneumoniae
 ability to hydrolyze esculin in the presence of bile –
Enterococcus spp.and Group D streptococcus
 CAMP – Streptococcus agalactiae

13. PURPOSE
 To differentiate members of the family Microco-
coccaceae (including Staphylococcus) which are
catalase positive from Streptococcus species
which are catalase negative.
 To differentiate Listeria monocytogenes and
corynebacteria(catalase positive) from other gram
positive, non-sporeforming bacilli.

14. PRINCIPLE
 The enzyme catalase catalyzes the release of
water and oxygen from hydrogen peroxide.
catalase
2 H202 -------------- 2 H20 + O2
bubbles or
effervescence

15. INTERPRETATION
 Positive – rapid and sustained appearance of
bubbles or effervescence
 Negative – lack of bubble formation 30 seconds
later

16. A B
Catalase test
A.Positive – Staphylococcus aureus.
B.Negative – Streptococcus pyogenes

18. PURPOSE
 To determine the ability of the organism to produce
coagulase which clots plasma.
 To distinguish the pathogenic coagulase positive
staphylococcus from the nonpathogenic coagulase
negative staphylococcus.

19.  Coagulase is an enzyme that converts soluble
fibrinogen into soluble fibrin.
Two forms of coagulase
 bound coagulase (clumping factor) – detected in the
coagulase slide test
can directly convert fibrinogen to insoluble fibrin
and causes the staphylococci to clump together
PRINCIPLE

20.  free coagulase – detected in the coagulase tube test
reacts with a globulin plasma factor(coagulase
reacting factor-CRF) to form a thrombinlike factor,
staphylothrombin--- catalyzes the conversion
of fibrinogen to insoluble fibrin

21. INTERPRETATION
Slide Coagulase test
 Positive – white fibrin clots in plasma
 Negative – smooth suspension
Tube Coagulase test
 Positive – formation of fibrin clot
 Negative – no clot is formed

22. Slide coagulase test
A B
A. Negative – Staphylococcus epidermidis
B. Positive – Staphylococcus aureus

23. Tube coagulase test
A B
A. Positive – Staphylococcus aureus
B. Negative – Staphylococcus epidermidis

25. PURPOSE
 To determine the ability of the organism to hydrolyze
the substrate L-pyrrolidonyl-beta-napthylamide.
 To differentiate the Enterococcus species from the
nonenterococcus species.
 Useful for presumptive identification of Group A
beta hemolytic streptococcus(Streptococcus
pyogenes)

26. PRINCIPLE
L-pyrrolidonyl-beta-napthylamide ------------
hydrolysis
pyrrolidonylarylamidase
Beta napthylamide + p-dimethylaminocinnamaldehyde
Pink to cherry red color
(color developer)

27. INTERPRETATION
Positive – pink to cherry red color(after the addition
of color developer)
Negative – no color change in inoculated portion of
the disk

28. PYRase(PYR) test
A B
A.Positive – Enterococcus
B.Negative – nonenterococcus

30. PURPOSE
 To determine the ability of the organism to produce
hippuricase which hydrolyzes the substrate hippurate.
 Useful in the identification of Streptococcus agalactiae,
Camphylobacter jejuni and Listeria monocytogenes.

31. PRINCIPLE
 The end products of hydrolysis of the substrate
hippurate by a constitutive enzyme hippuricase
include glycine and benzoic acid.
 Glycine is deaminated by the oxidizing agent,
ninhydrin, which is reduced during the process.
 The end products of ninhydrin oxidation react to
form a purple colored product.

32. INTERPRETATION
 Positive – deep purple color
 Negative – slightly yellow pink or colorless

33. Hippurate hydrolysis test
A B
A. Positive – Streptococcus
agalactiae
B. Negative- Enterococcus

35. PURPOSE
 To screen colonies suspected of being one
of the Enterobacteriaceae(all negative).
To identify colonies suspected of belonging to
other genera such as Aeromonas, Pseudomonas,
Neisseria, Camphylobacter and Pasteurella.

36. PRINCIPLE
 The cytochrome oxidase test uses certain reagent
dyes, such as p-phenylenediamine dihydrochloride
that substitute for oxygen as artificial electron
acceptors
 It is colorless in the reduced state.
 In the presence of cytochrome oxidase and
atmospheric oxygen, p-phenylenediamine is
oxidized forming indophenol blue.

37. Tetramethyl-p-phenylene ----------- purple color
diamine hydrochloride
Dimethyl compound(1%) ----------- black color
P-phenylenediamine -----------------
dihydrochloride
cytochrome oxidase
+
atmospheric air
Indophenol blue
oxidation

38. INTERPRETATION
Positive – blue/ dark purple/black color
Negative – no color development

39. A B
Oxidase test
A. Positive – Pseudomonas aeruginosa
B. Negative – Escherichia coli

41. PURPOSE
 To distinguish Enterobacteriaceae based on the
ability to produce indole from tryptophan.
 To identify lactose fermenting members of
Enterobacteriaceae, Escherichia coli(indol positive)
from Klebsiella pneumoniae(indol negative).
 To speciate Proteus:
Proteus mirabilis – indole negative
Proteus vulgaris – indole positive

42. PRINCIPLE
 Bacteria that possess the enzyme tryptophanase
are capable of hydrolyzing and deaminating
tryptophan with the production of indole, pyruvic
acid and ammonia.
 A red complex is formed when indole reacts
with the aldehyde group of p-dimethylaminobenzal-
dehyde, the active chemical in Kovac’s and Ehrlich’s
reagent.

43. Tryptophan ------------------indol + pyruvic acid + NH3
tryptophanase
Indol + p-dimethylaminobenzaldehyde -----red complex
Reagents used to detect indole
 Ehrlich’s – to detect indol in anaerobic and
nonfermentative bacteria
 Kovac’s – to identify members of Enterobacteriaceae

44. Media used with tryptophan
 sulfide indol motility (SIM)
 motility indol ornithine(MIO)
 indole nitrate
 rapid spot tests – filter paper strips impregnated
with p-diaminocinnamaldehyde reagent – useful
in screening bacteria that are prompt indole
producers

45. INTERPRETATION
 Positive – red ring at the interface of reagent and
broth (or reagent and xylene or chloroform)
 Negative – no color development
 Variable results – orange color, indicates products
of skatole, a methylated intermediate that maybe
a precursor to indole production
Rapid spot test
paradimethylaminocinnamaldehyde – blue green
paradimethylaminobenzaldehyde – bright pink color

46. A B
Indole test
A.Positive – Escherichia coli
B.Negative – Klebsiella pneumoniae

47. Indole spot test
A B
A. Negative - Klebsiella pneumoniae
B. Positive - Escherichia coli

49. PURPOSE:
To detect Dnase activity in species of aerobic
bacteria.
To differentiate nonfermenting gram-negative
bacteria as well as Staphylococcus aureus and
Serratia marcescens.

50. Metachromatic dyes
 Toluidine blue is complexed with DNA. Hydrolysis
of DNA by the inoculated microorganism causes
changes of structure of the dye to yield a pink color.
 Methyl green is also complexed with DNA. If the
organism growing on the medium hydrolyzes DNA,
the green color fades and the colony is surrounded
by a colorless zone.
PRINCIPLE

51. INTERPRETATION
Positive rose pink clear zone
Negative no change no clearing
Toluidine blue Methyl green

52. Deoxyribonuclease test
Positive – Staphylococcus aureus
Serratia marcescens
Negative – Staphylococcus epidermidis
Enterobacter cloacae

53. Deoxyribonuclase test
A. Positive – Staphylococcus aureus
B. Positive – Serratia marcescens
C.Negative –Staphylococcus epidermidis
A
BC

55. PURPOSE
 To determine the presence of late or slow
fermenting strains.
 To detect the late lactose fermenting strains of
Escherichia coli
 To distinguish some Citrobacter species and
arizonae subspecies(ONPG positive) from similar
Salmonella subspecies(ONPG negative)
 To speciate Shigella, since Shigella sonnei is the
only ONPG-positive Shigella species.

56. PRINCIPLE
Two enzymes required for lactose fermentation
 lactose permease – actively transfers lactose into
the bacterial cell
 beta galactosidase- degrades lactose into glucose
and galactose
Lactose fermenters – possess both enzymes
Slow or late lactose fermenters – no permease ; only
beta galactosidase
Non lactose fermenters – lack both enzymes

57.  ONPG(o-nitrophenyl-beta-D-galactopyranoside)
is useful in detecting late lactose fermenters, since
ONPG molecule is structurally similar to lactose.
 It can enter the bacterial cell without a permease.
 In the presence of galactosidase, ONPG(colorless)
is converted into galactose and o-nitrophenyl,
which is a yellow chromogen and the alkaline end
product.

58. INTERPRETATION
Positive – yellow color within 20 minutes to 24 hours
Negative- no color change or colorless after 24 hours

59. ONPG(O-nitrophenyl-beta-D-galactopyranoside) test
A B
A. Negative – Salmonella typhimurium
B. Positive – Escherichia coli(EHEC)

61. PURPOSE
To determine the ability of an organism to produce
the enzyme, urease, which hydrolyzes urea.
To identify the rapid urease producers(Proteus and
Morganella) and weak urease producers(Klebsiella
pneumoniae and species of Enterobacter)

62. PRINCIPLE
 Urease splits the urea molecule into ammonia(NH3),
CO2 and water(H20).
 Ammonia reacts in solution to form an alkaline
compound, ammonium carbonate, which results in
an increased pH of the medium and a color change
in the indicator to pink red.
Urea + 2H2O --------------- CO2 + H2O +2NH3
urease
(NH4)2CO3

63. INTERPRETATION
Christensens agar
 Positive – rapid urease activity; red throughout
the medium
 Positive – slow urease activity: red in slant initially
gradually converting the entire tube
 Negative – no urease activity; medium remains yellow
Stuart (urea) broth
Positive - red color in the medium
Negative – no color change(buff to pale yellow)

64. A B C
A. Positive – Proteus spp.
B. Positive - Klebsiella spp.
C.Negative – Escherichia
coli
Urease test(Christensens agar)

65. Urease test
Stuart Urea broth
A B C
A. Uninoculated
B. Strong positive reaction-
Proteus spp.
C. Negative – Escherichia
coli

69. PURPOSE:
 To determine whether a substrate utilization is
an oxidative or fermentative process for the
identification of several different bacteria
 To separate organisms into two major groups:
Enterobacteriaceae – fermentative
Pseudomonas – oxidative

70. COMPOSITION
 high concentration of carbohydrates (1%)
 small concentration of peptone(2%)
 Indicators
bromcresol purple – purple to yellow
Andrade’s acid fuchsin – pale yellow to pink
phenol red – red to yellow
bromthymol blue – green to yellow

71. Principle of glucose oxidative fermentation test

72. 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

73. Open tube Closed tube Metabolism
Acid(yellow) alkaline(green) oxidative
Acid(yellow) acid(yellow) fermentation
Alkaline(green) alkaline(green) nonsaccharolytic
(nonutilizer)
Oxidative-Fermentation Medium of Hugh and Leifson

74. Oxidative Fermentative medium
(CDC method)
A. Fermenter – Escherichia coli

75. B. Oxidizer – Pseudomonas
aeruginosa
Oxidative fermentative medium
(CDC method)

76. Oxidative fermentative medium
(CDC method)
C. Nonutilizer- Alcaligenes
faecalis

78. 1. As an initial step in the identification of
Enterobacteriaceae
PRINCIPLE:
1. The action of many species of microorganisms on a
carbohydrate substrate results in the acidification of
the medium with or without gas formation.
2. Iron salts(ferrous sulfate and ferric ammonium
citrate) reacts with H2S to produce an insoluble black
precipitate(ferrous sulfide).
PURPOSE

79. TSIA – two reaction chamber
Aerobic slant portion
Anaerobic deep portion

80.  Protein sources – beef extract, peptone, yeast
extract, proteose peptone
 Sugars(lactose, sucrose, glucose)
 Indicators
a. phenol red – carbohydrate fermentation
b. ferrous sulfate – hydrogen sulfide production
 Sodium thiosulfate – source of sulfur atoms
 Sodium chloride – osmotic stabilizer
COMPOSITION

81. BIOCHEMICAL REACTIONS
 carbohydrate fermentation
acid production
yellow deep – glucose fermented
yellow slant – lactose and/ or sucrose fermented
gas formation
bubble formation
cracking or splitting of the agar
upward displacement of the agar
pulling away of the medium from the walls of test tube
 H2S production
blackening of the butt(FeS – black precipitate)

82.  A/@H2S(-)
Acid slant; acid butt; gas formation; no H2S
all sugars fermented; with gas formation;
no blackening of the butt
Escherichia
Klebsiella
Enterobacter

83.  K/@H2S+
alkaline slant; acid butt; with gas formation
with H2S
glucose fermented; lactose and
or/sucrose not fermented; with gas
formation and black precipitate
Salmonella
Proteus
Citrobacter

84. K/A H2S( –)
alkaline slant; acid butt; no gas; no H2S
glucose is fermented; lactose
and/or sucrose not fermented;
no gas formation; no black
precipitate
Shigella
Providencia
Serratia
anaerogenic Escherichia coli

85.  K/KH2S(-)
alkaline slant; alkaline butt; no gas;
no H2S
no sugars fermented; no gas;
no black precipitate in the butt
Pseudomonas
Alcaligenes

86. A/@H2S+
acid slant;acid butt; with gas; with H2S
all sugars fermented; with gas formation;
with black precipitate in the butt
Citrobacter freundii

88. PURPOSE:
 To identify the lactose fermenting Enterobacte-
riaceae such as Escherichia coli (MR positive
and VP negative) whereas most members of
the Klebsiella-Enterobacter-Serratia-Hafnia
group are VP positive.

89. Metabolism of glucose using MR and VP pathways
Glucose
Acetoin Pyruvic acid Mixed acid fermentation
KOH + air pH less than 4.4(red)
Diacetyl
Napthol + creatine
pink red complex
Positive VP

90.  In the first pathway, mixed acid products (lactic,
acetic, formic and succinic) result, leading to
a decrease in the pH of the medium and a
positive MR test.
 The pH must drop to 4.4 or less for the MR indicator
to take on its acidic red color.
PRINCIPLE – METHYL RED TEST

91.  In the second pathway, acetylmethyl carbinol
acetoin is an intermediate product to butylene
glycol.
 It is the neutral product detected in the
VP reaction.
 In the presence of oxygen and 40% potassium
hydroxide, acetoin is converted to the diacetyl form,
which results in a red color in the presence of
alpha-napthol.
PRINCIPLE – VOGES PROSKAUER TEST

92. INTERPRETATION
Methyl red test
 Positive – distinct red color at surface of the medium
 Negative – yellow color at the surface of the medium
Voges Proskauer test
 Positive – pink red color at surface of the medium
 Negative – yellow color at surface of the medium

93. A B
Methyl Red test
A. Positive – Escherichia coli
B. Negative – Klebsiella pneumoniae

94. A B
Voges Proskauer test
A. Positive – Klebsiella pneumoniae
B. Negative – Escherichia coli

97. PURPOSE:
 To determine the production of decarboxylase
by bacteria(Enterobacteriaceae).

98. Composition – Moeller decarboxylase medium
1. Glucose
2. Amino acid substrate(1% lysine, 1% arginine
1% ornithine)
3. pH indicator
a. bromcresol purple
1. alkaline pH- purple
2. acid ph-yellow
b. phenol red
1. alkaline pH– red
2. acid pH-yellow

99. PRINCIPLE
 Decarboxylase enzyme - removes carboxyl groups
from the amino acids lysine and ornithine.
 Dihydrolase enzyme - removes a carboxyl group
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 to exclude air.

100. Lysine ----------------- cadaverine
Ornithine-------------- putrescine
Arginine--------------- citrulline----------- ornithine
dihydrolase
reaction decarboxylation
putrescine
Specific amine products

101.  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.

102. INTERPRETATION
 Control tube – yellow- glucose fermentation;
viable organisms; pH of the medium has been
lowered sufficient to activate the decarboxylase
enzyme
 Positive test – purple – decarboxylation;
formation of the amino acids from the
decarboxylation

103. A B
A. Positive – purple; decarboxylation
B. Negative – yellow; no decarboxylation;
only glucose fermentation
Moeller decarboxylase medium

104. A B C D
Decarboxylase-dihydrolase reactions – Enterobacter cloacae)
A. Control – without amino acid C. lysine-negative
B. Arginine – positive D. ornithine-positive

105. Enterobacter cloacae Klebsiella pneumoniae
Arginine +(purple)alkaline -(yellow) acid
Lysine -(yellow)acid +(purple)alkaline
Ornithine +(purple)alkaline -(yellow)acid