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Determining the nutritional and metabolic
capabilities of a bacterial isolate is the
most common approach used for deter-
...
The methods available use a combination of tests
to establish the enzymatic capabilities of a given
bacterial isolate as w...
A.Establishing Enzymatic Capabilities
Enzyme based tests are designed to measure
the presence of a single enzyme as well a...
SINGLE ENZYME TESTS
Catalase test
Coagulase test
Pyrase test
Hippurate hydrolysis test
Oxidase test
Indole test
Dna...
ASSAYS FOR METABOLIC PATHWAYS
Carbohydrate oxidation and fermentation
oxidation fermentation tests
carbohydrate fermenta...
Amino acid degradation
 decarboxylase-dihydrolase reactions
 deamination reactions
 decarboxylation and deamination rea...
B. Establishing Inhibitor Profiles
 bacitracin susceptibility test
 bacitracin and sulfamethoxazole-trimethoprim
suscept...
C. Other more specific tests
 growth in various NaCl concentrations - Enterococci
and Vibrio species
 susceptibility to ...
PURPOSE
 To differentiate members of the family Microco-
coccaceae (including Staphylococcus) which are
catalase positive...
PRINCIPLE
 The enzyme catalase catalyzes the release of
water and oxygen from hydrogen peroxide.
catalase
2 H202 --------...
INTERPRETATION
 Positive – rapid and sustained appearance of
bubbles or effervescence
 Negative – lack of bubble formati...
A B
Catalase test
A.Positive – Staphylococcus aureus.
B.Negative – Streptococcus pyogenes
PURPOSE
 To determine the ability of the organism to produce
coagulase which clots plasma.
 To distinguish the pathogeni...
 Coagulase is an enzyme that converts soluble
fibrinogen into soluble fibrin.
Two forms of coagulase
 bound coagulase (c...
 free coagulase – detected in the coagulase tube test
reacts with a globulin plasma factor(coagulase
reacting factor-CRF)...
INTERPRETATION
Slide Coagulase test
 Positive – white fibrin clots in plasma
 Negative – smooth suspension
Tube Coagulas...
Slide coagulase test
A B
A. Negative – Staphylococcus epidermidis
B. Positive – Staphylococcus aureus
Tube coagulase test
A B
A. Positive – Staphylococcus aureus
B. Negative – Staphylococcus epidermidis
PURPOSE
 To determine the ability of the organism to hydrolyze
the substrate L-pyrrolidonyl-beta-napthylamide.
 To diffe...
PRINCIPLE
L-pyrrolidonyl-beta-napthylamide ------------
hydrolysis
pyrrolidonylarylamidase
Beta napthylamide + p-dimethyl...
INTERPRETATION
Positive – pink to cherry red color(after the addition
of color developer)
Negative – no color change in ...
PYRase(PYR) test
A B
A.Positive – Enterococcus
B.Negative – nonenterococcus
PURPOSE
 To determine the ability of the organism to produce
hippuricase which hydrolyzes the substrate hippurate.
 Usef...
PRINCIPLE
 The end products of hydrolysis of the substrate
hippurate by a constitutive enzyme hippuricase
include glycine...
INTERPRETATION
 Positive – deep purple color
 Negative – slightly yellow pink or colorless
Hippurate hydrolysis test
A B
A. Positive – Streptococcus
agalactiae
B. Negative- Enterococcus
PURPOSE
 To screen colonies suspected of being one
of the Enterobacteriaceae(all negative).
To identify colonies suspect...
PRINCIPLE
 The cytochrome oxidase test uses certain reagent
dyes, such as p-phenylenediamine dihydrochloride
that substit...
Tetramethyl-p-phenylene ----------- purple color
diamine hydrochloride
Dimethyl compound(1%) ----------- black color
P-p...
INTERPRETATION
Positive – blue/ dark purple/black color
Negative – no color development
A B
Oxidase test
A. Positive – Pseudomonas aeruginosa
B. Negative – Escherichia coli
PURPOSE
 To distinguish Enterobacteriaceae based on the
ability to produce indole from tryptophan.
 To identify lactose ...
PRINCIPLE
 Bacteria that possess the enzyme tryptophanase
are capable of hydrolyzing and deaminating
tryptophan with the ...
Tryptophan ------------------indol + pyruvic acid + NH3
tryptophanase
Indol + p-dimethylaminobenzaldehyde -----red compl...
Media used with tryptophan
 sulfide indol motility (SIM)
 motility indol ornithine(MIO)
 indole nitrate
 rapid spot te...
INTERPRETATION
 Positive – red ring at the interface of reagent and
broth (or reagent and xylene or chloroform)
 Negativ...
A B
Indole test
A.Positive – Escherichia coli
B.Negative – Klebsiella pneumoniae
Indole spot test
A B
A. Negative - Klebsiella pneumoniae
B. Positive - Escherichia coli
PURPOSE:
To detect Dnase activity in species of aerobic
bacteria.
To differentiate nonfermenting gram-negative
bacteria ...
Metachromatic dyes
 Toluidine blue is complexed with DNA. Hydrolysis
of DNA by the inoculated microorganism causes
change...
INTERPRETATION
Positive rose pink clear zone
Negative no change no clearing
Toluidine blue Methyl green
Deoxyribonuclease test
Positive – Staphylococcus aureus
Serratia marcescens
Negative – Staphylococcus epidermidis
Enteroba...
Deoxyribonuclase test
A. Positive – Staphylococcus aureus
B. Positive – Serratia marcescens
C.Negative –Staphylococcus epi...
PURPOSE
 To determine the presence of late or slow
fermenting strains.
 To detect the late lactose fermenting strains of...
PRINCIPLE
Two enzymes required for lactose fermentation
 lactose permease – actively transfers lactose into
the bacterial...
 ONPG(o-nitrophenyl-beta-D-galactopyranoside)
is useful in detecting late lactose fermenters, since
ONPG molecule is stru...
INTERPRETATION
Positive – yellow color within 20 minutes to 24 hours
Negative- no color change or colorless after 24 hou...
ONPG(O-nitrophenyl-beta-D-galactopyranoside) test
A B
A. Negative – Salmonella typhimurium
B. Positive – Escherichia coli(...
PURPOSE
To determine the ability of an organism to produce
the enzyme, urease, which hydrolyzes urea.
To identify the ra...
PRINCIPLE
 Urease splits the urea molecule into ammonia(NH3),
CO2 and water(H20).
 Ammonia reacts in solution to form an...
INTERPRETATION
Christensens agar
 Positive – rapid urease activity; red throughout
the medium
 Positive – slow urease ac...
A B C
A. Positive – Proteus spp.
B. Positive - Klebsiella spp.
C.Negative – Escherichia
coli
Urease test(Christensens agar)
Urease test
Stuart Urea broth
A B C
A. Uninoculated
B. Strong positive reaction-
Proteus spp.
C. Negative – Escherichia
co...
PURPOSE:
 To determine whether a substrate utilization is
an oxidative or fermentative process for the
identification of ...
COMPOSITION
 high concentration of carbohydrates (1%)
 small concentration of peptone(2%)
 Indicators
bromcresol purple...
Principle of glucose oxidative fermentation test
INTERPRETATION
 glucose fermenter – when acid production is
detected on both tubes since fermentation can
occur with or w...
Open tube Closed tube Metabolism
Acid(yellow) alkaline(green) oxidative
Acid(yellow) acid(yellow) fermentation
Alkaline(gr...
Oxidative Fermentative medium
(CDC method)
A. Fermenter – Escherichia coli
B. Oxidizer – Pseudomonas
aeruginosa
Oxidative fermentative medium
(CDC method)
Oxidative fermentative medium
(CDC method)
C. Nonutilizer- Alcaligenes
faecalis
1. As an initial step in the identification of
Enterobacteriaceae
PRINCIPLE:
1. The action of many species of microorganis...
TSIA – two reaction chamber
Aerobic slant portion
Anaerobic deep portion
 Protein sources – beef extract, peptone, yeast
extract, proteose peptone
 Sugars(lactose, sucrose, glucose)
 Indicator...
BIOCHEMICAL REACTIONS
 carbohydrate fermentation
acid production
yellow deep – glucose fermented
yellow slant – lactose a...
 A/@H2S(-)
Acid slant; acid butt; gas formation; no H2S
all sugars fermented; with gas formation;
no blackening of the bu...
 K/@H2S+
alkaline slant; acid butt; with gas formation
with H2S
glucose fermented; lactose and
or/sucrose not fermented; ...
K/A H2S( –)
alkaline slant; acid butt; no gas; no H2S
glucose is fermented; lactose
and/or sucrose not fermented;
no gas ...
 K/KH2S(-)
alkaline slant; alkaline butt; no gas;
no H2S
no sugars fermented; no gas;
no black precipitate in the butt
Ps...
A/@H2S+
acid slant;acid butt; with gas; with H2S
all sugars fermented; with gas formation;
with black precipitate in the ...
PURPOSE:
 To identify the lactose fermenting Enterobacte-
riaceae such as Escherichia coli (MR positive
and VP negative) ...
Metabolism of glucose using MR and VP pathways
Glucose
Acetoin Pyruvic acid Mixed acid fermentation
KOH + air pH less than...
 In the first pathway, mixed acid products (lactic,
acetic, formic and succinic) result, leading to
a decrease in the pH ...
 In the second pathway, acetylmethyl carbinol
acetoin is an intermediate product to butylene
glycol.
 It is the neutral ...
INTERPRETATION
Methyl red test
 Positive – distinct red color at surface of the medium
 Negative – yellow color at the s...
A B
Methyl Red test
A. Positive – Escherichia coli
B. Negative – Klebsiella pneumoniae
A B
Voges Proskauer test
A. Positive – Klebsiella pneumoniae
B. Negative – Escherichia coli
PURPOSE:
 To determine the production of decarboxylase
by bacteria(Enterobacteriaceae).
Composition – Moeller decarboxylase medium
1. Glucose
2. Amino acid substrate(1% lysine, 1% arginine
1% ornithine)
3. pH i...
PRINCIPLE
 Decarboxylase enzyme - removes carboxyl groups
from the amino acids lysine and ornithine.
 Dihydrolase enzyme...
Lysine ----------------- cadaverine
Ornithine-------------- putrescine
Arginine--------------- citrulline----------- o...
 Early incubation – both tubes yellow due to
acidification of the indicator (bromcresol purple)
by the acid end products ...
INTERPRETATION
 Control tube – yellow- glucose fermentation;
viable organisms; pH of the medium has been
lowered sufficie...
A B
A. Positive – purple; decarboxylation
B. Negative – yellow; no decarboxylation;
only glucose fermentation
Moeller deca...
A B C D
Decarboxylase-dihydrolase reactions – Enterobacter cloacae)
A. Control – without amino acid C. lysine-negative
B. ...
Enterobacter cloacae Klebsiella pneumoniae
Arginine +(purple)alkaline -(yellow) acid
Lysine -(yellow)acid +(purple)alkalin...
PURPOSE
To determine the deaminase activity using the
amino acids phenylalanine or tryptophan.
Only Proteus, Providencia...
PRINCIPLE
 Deamination of the amino acid results in a colored
compound with the addition of 10% ferric chloride
Phenylala...
INTERPRETATION
 Positive deamination for phenylalanine – intense
green color
 Positive deamination for tryptophan – brow...
A. Negative – Escherichia coli
B. Positive – Proteus vulgaris
A B
Phenylalanine deamination test
PURPOSE:
To determine the ability of the organism to deaminate
lysine, decarboxylate lysine and produce H2S.
To identify...
COMPOSITION
1. Proteins
2. Sugar- Glucose
3. Amino acid - Lysine
4. Sulfur
5. indicators
a. ferric ammonium citrate –
H2S ...
PRINCIPLE:
As glucose fermentation occurs, deep of the tube
turns yellow.
Lysine decarboxylation produces alkaline cadav...
INTERPRETATION
Lysine decarboxylation - butt
 Positive – purple
 Negative – yellow
Lysine deamination - slant
 Positive...
K/K alkaline slant/alkaline butt H2S(-)
purple/ purple
Negative deamination
Positive decarboxylation
No blackening of the ...
K/K alkaline slant/alkaline butt H2S +
purple/purple
Negative deamination
Positive decarboxylation
With black precipitate ...
K/A alkaline slant/acid butt H2S(-)
(purple/yellow)
Negative deamination
Negative decarboxylation
No black precipitate in ...
R/A red slant/acid butt H2S(-)
red/yellow
Positive deamination
Negative decarboxylation
No black precipitate in the butt
P...
PURPOSE:
To determine if a member of the Enterobacteriaceae
is capable of utilizing citrate as the sole source
of carbon....
PRINCIPLE
Sodium citrate is the only carbon source in
Simmons citrate agar.
If the organism can utilize citrate, the sod...
INTERPRETATION
 Positive – growth with an intense blue color on the
slant or solely the presence of growth
 Negative – a...
Citrate Utilization test
A. Positive - Klebsiella pneumoniae
B. Negative - Escherichia coli
A B
PURPOSE
 To determine the ability of an organism to use
acetate as the sole source of carbon.
PRINCIPLE
 Breakdown of th...
INTERPRETATION
Positive – Medium becomes alkalinized(blue)
because of the growth of the organism
Negative – no growth or...
A B
Acetate utilization test
A. Positive - Klebsiella pneumoniae
B. Negative – Escherichia coli
PURPOSE
 To determine the ability of an organism to use
acetamide as the sole source of carbon.
PRINCIPLE
 Bacteria that...
INTERPRETATION
Positive – deamination of the acetamide resulting
in a blue color
Negative – no color change
Acetamide utilization test
A. Positive – Klebsiella pneumoniae
B. Negative – Escherichia coli
PURPOSE:
 To differentiate Micrococccus and Stomatococcus
from Staphylococcus when combined with other
procedures such as...
 Bacitracin(0.04 units) inhibits the growth of
Micrococcus and Stomatococcus and Group A
streptococcus while having no ef...
INTERPRETATION
 susceptible – zones of inhibition greater than or
equal to 10 mm
 resistant – zones of inhibition less t...
A. Susceptible – Micrococcus and Stomatococcus
B. Resistant – Staphylococcus epidermidis
A B
Bacitracin susceptibility test
PURPOSE:
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 resis...
INTERPRETATION
Susceptible: any zone of inhibition around either
disk
Resistant: growth up to the disk(no zone of
inhibi...
Organism Bacitracin SXT
Group A susceptible resistant
Group B resistant resistant
Group C,F,G resistant susceptible
PURPOSE
 To differentiate the different species of coagulase
negative staphylococci.
PRINCIPLE
 After incubation with 5 ...
INTERPRETATION:
 susceptible – zone greater than 16 mm
 resistant – zone diameter less than or equal to
16 mm
A B
Novobiocin susceptibility test
A. Susceptible - Staphylococcus epidermidis
B. Resistant - Staphylococcus saprophyticus
PURPOSE
 To differentiate Pediococcus from other alpha
hemolytic streptococcus.
PRINCIPLE
 After incubation with 5 ug of...
INTERPRETATION
 Susceptible – zone of inhibition
 Resistant – no zone of inhibition
Vancomycin susceptibility test
A. Susceptible - Viridans streptococcus
B. Resistant - Pediococcus
A B
PURPOSE
 To determine an anaerobe’s inhibition that can
be used for presumptive identification based on
its characteristi...
PRINCIPLE
 Most anaerobes have a characteristic susceptibility
pattern to colistin(10 ug), vancomycin( 5 ug), and
kanamyc...
K Va
Co
INTERPRETATION
Susceptible – zone greater than 10 mm
Resistant – zone of 10 mm. or less
Antibiotic Disks for the P...
PURPOSE
To classify bacteria based on their ability to grow
in the presence of 6.5% NaCl, a characteristic
of certain spe...
PRINCIPLE
Nutrient broth or 6.5%NaCl
Trypticase broth-salt
free medium
Positive equal equal
Negative good very weak
INTERPRETATION
Positive – if growth is equivalent to both media –
tolerant of salt
Negative- growth on the salt containi...
SALT TOLERANCE TEST
A. Positive - Enterococcus faecalis ( salt tolerant)
B. Negative - Streptococcus bovis(salt intolerant)
 To distinguish Group D streptococci and Enterococcus
species from other Lancefield group of streptococci
 based on the ...
INTERPRETATION
Positive
growth indicates tolerance to 40% bile(40% oxygall)
blackening indicates hydrolysis of esculin
N...
A. Positive - Enterococcus faecalis
B. Negative - Streptococcus viridans
Bile esculin agar
A B
PURPOSE
 To differentiate Streptococcus pneumoniae from
other alpha hemolytic streptococci
PRINCIPLE
 In the presence of...
 Positive – zone of inhibition at least 14 mm. in
diameter using a 10 ug P disk and at least 10 mm.
using a 6 mg P disk
...
Optochin susceptibility test
A. Positive – Streptococcus pneumoniae
B. Negative – Viridans streptococci
A B
PURPOSE
 To differentiate Streptococcus pneumoniae(positive)
from other alpha hemolytic streptococci.
PRINCIPLE
 Pneumococcal colonies are rapidly lysed by bile
or a solution of a bile salt such as sodium
deoxycholate.
 Ly...
INTERPRETATION
 Positive – colony disintegrates; an imprint of the
lysed colony may remain within the zone
 Negative – i...
B
A
Bile solubility test
A. Positive – Streptococcus pneumoniae
B. Negative – Viridans Streptococci
PURPOSE
 to demonstrate the phenomena of synergistic
hemolysis between group B streptococcus and
beta hemolytic Staphyloc...
INTERPRETATION
 Positive – a zone of enhanced hemolysis given by
an arrowhead appearance at the junction of the
Staphyloc...
CAMP REACTION
A. Positive - Streptococcus agalactiae
B. Negative - Streptococcus bovis
A B
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
Biochemical identification of bacteria
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Biochemical identification of bacteria Slide 1 Biochemical identification of bacteria Slide 2 Biochemical identification of bacteria Slide 3 Biochemical identification of bacteria Slide 4 Biochemical identification of bacteria Slide 5 Biochemical identification of bacteria Slide 6 Biochemical identification of bacteria Slide 7 Biochemical identification of bacteria Slide 8 Biochemical identification of bacteria Slide 9 Biochemical identification of bacteria Slide 10 Biochemical identification of bacteria Slide 11 Biochemical identification of bacteria Slide 12 Biochemical identification of bacteria Slide 13 Biochemical identification of bacteria Slide 14 Biochemical identification of bacteria Slide 15 Biochemical identification of bacteria Slide 16 Biochemical identification of bacteria Slide 17 Biochemical identification of bacteria Slide 18 Biochemical identification of bacteria Slide 19 Biochemical identification of bacteria Slide 20 Biochemical identification of bacteria Slide 21 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Biochemical identification of bacteria

  1. 1. 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.
  2. 2. 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)
  3. 3. A.Establishing Enzymatic Capabilities Enzyme based tests are designed to measure the presence of a single enzyme as well as a complete metabolic pathway.
  4. 4. SINGLE ENZYME TESTS Catalase test Coagulase test Pyrase test Hippurate hydrolysis test Oxidase test Indole test Dnase test ONPG(B-galactosidase)test Urease test
  5. 5. ASSAYS FOR METABOLIC PATHWAYS Carbohydrate oxidation and fermentation oxidation fermentation tests carbohydrate fermentation in TSIA methyl red test Voges Proskauer test
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. 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.
  10. 10. PRINCIPLE  The enzyme catalase catalyzes the release of water and oxygen from hydrogen peroxide. catalase 2 H202 -------------- 2 H20 + O2 bubbles or effervescence
  11. 11. INTERPRETATION  Positive – rapid and sustained appearance of bubbles or effervescence  Negative – lack of bubble formation 30 seconds later
  12. 12. A B Catalase test A.Positive – Staphylococcus aureus. B.Negative – Streptococcus pyogenes
  13. 13. 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.
  14. 14.  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
  15. 15.  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
  16. 16. 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
  17. 17. Slide coagulase test A B A. Negative – Staphylococcus epidermidis B. Positive – Staphylococcus aureus
  18. 18. Tube coagulase test A B A. Positive – Staphylococcus aureus B. Negative – Staphylococcus epidermidis
  19. 19. 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)
  20. 20. PRINCIPLE L-pyrrolidonyl-beta-napthylamide ------------ hydrolysis pyrrolidonylarylamidase Beta napthylamide + p-dimethylaminocinnamaldehyde Pink to cherry red color (color developer)
  21. 21. INTERPRETATION Positive – pink to cherry red color(after the addition of color developer) Negative – no color change in inoculated portion of the disk
  22. 22. PYRase(PYR) test A B A.Positive – Enterococcus B.Negative – nonenterococcus
  23. 23. 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.
  24. 24. 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.
  25. 25. INTERPRETATION  Positive – deep purple color  Negative – slightly yellow pink or colorless
  26. 26. Hippurate hydrolysis test A B A. Positive – Streptococcus agalactiae B. Negative- Enterococcus
  27. 27. 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.
  28. 28. 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.
  29. 29. Tetramethyl-p-phenylene ----------- purple color diamine hydrochloride Dimethyl compound(1%) ----------- black color P-phenylenediamine ----------------- dihydrochloride cytochrome oxidase + atmospheric air Indophenol blue oxidation
  30. 30. INTERPRETATION Positive – blue/ dark purple/black color Negative – no color development
  31. 31. A B Oxidase test A. Positive – Pseudomonas aeruginosa B. Negative – Escherichia coli
  32. 32. 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
  33. 33. 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.
  34. 34. 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
  35. 35. 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
  36. 36. 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
  37. 37. A B Indole test A.Positive – Escherichia coli B.Negative – Klebsiella pneumoniae
  38. 38. Indole spot test A B A. Negative - Klebsiella pneumoniae B. Positive - Escherichia coli
  39. 39. PURPOSE: To detect Dnase activity in species of aerobic bacteria. To differentiate nonfermenting gram-negative bacteria as well as Staphylococcus aureus and Serratia marcescens.
  40. 40. 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
  41. 41. INTERPRETATION Positive rose pink clear zone Negative no change no clearing Toluidine blue Methyl green
  42. 42. Deoxyribonuclease test Positive – Staphylococcus aureus Serratia marcescens Negative – Staphylococcus epidermidis Enterobacter cloacae
  43. 43. Deoxyribonuclase test A. Positive – Staphylococcus aureus B. Positive – Serratia marcescens C.Negative –Staphylococcus epidermidis A BC
  44. 44. 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.
  45. 45. 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
  46. 46.  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.
  47. 47. INTERPRETATION Positive – yellow color within 20 minutes to 24 hours Negative- no color change or colorless after 24 hours
  48. 48. ONPG(O-nitrophenyl-beta-D-galactopyranoside) test A B A. Negative – Salmonella typhimurium B. Positive – Escherichia coli(EHEC)
  49. 49. 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)
  50. 50. 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
  51. 51. 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)
  52. 52. A B C A. Positive – Proteus spp. B. Positive - Klebsiella spp. C.Negative – Escherichia coli Urease test(Christensens agar)
  53. 53. Urease test Stuart Urea broth A B C A. Uninoculated B. Strong positive reaction- Proteus spp. C. Negative – Escherichia coli
  54. 54. 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
  55. 55. 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
  56. 56. Principle of glucose oxidative fermentation test
  57. 57. 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
  58. 58. 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
  59. 59. Oxidative Fermentative medium (CDC method) A. Fermenter – Escherichia coli
  60. 60. B. Oxidizer – Pseudomonas aeruginosa Oxidative fermentative medium (CDC method)
  61. 61. Oxidative fermentative medium (CDC method) C. Nonutilizer- Alcaligenes faecalis
  62. 62. 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
  63. 63. TSIA – two reaction chamber Aerobic slant portion Anaerobic deep portion
  64. 64.  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
  65. 65. 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)
  66. 66.  A/@H2S(-) Acid slant; acid butt; gas formation; no H2S all sugars fermented; with gas formation; no blackening of the butt Escherichia Klebsiella Enterobacter
  67. 67.  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
  68. 68. 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
  69. 69.  K/KH2S(-) alkaline slant; alkaline butt; no gas; no H2S no sugars fermented; no gas; no black precipitate in the butt Pseudomonas Alcaligenes
  70. 70. A/@H2S+ acid slant;acid butt; with gas; with H2S all sugars fermented; with gas formation; with black precipitate in the butt Citrobacter freundii
  71. 71. 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.
  72. 72. 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
  73. 73.  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
  74. 74.  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
  75. 75. 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
  76. 76. A B Methyl Red test A. Positive – Escherichia coli B. Negative – Klebsiella pneumoniae
  77. 77. A B Voges Proskauer test A. Positive – Klebsiella pneumoniae B. Negative – Escherichia coli
  78. 78. PURPOSE:  To determine the production of decarboxylase by bacteria(Enterobacteriaceae).
  79. 79. 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
  80. 80. 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.
  81. 81. Lysine ----------------- cadaverine Ornithine-------------- putrescine Arginine--------------- citrulline----------- ornithine dihydrolase reaction decarboxylation putrescine Specific amine products
  82. 82.  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.
  83. 83. 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
  84. 84. A B A. Positive – purple; decarboxylation B. Negative – yellow; no decarboxylation; only glucose fermentation Moeller decarboxylase medium
  85. 85. A B C D Decarboxylase-dihydrolase reactions – Enterobacter cloacae) A. Control – without amino acid C. lysine-negative B. Arginine – positive D. ornithine-positive
  86. 86. Enterobacter cloacae Klebsiella pneumoniae Arginine +(purple)alkaline -(yellow) acid Lysine -(yellow)acid +(purple)alkaline Ornithine +(purple)alkaline -(yellow)acid
  87. 87. PURPOSE To determine the deaminase activity using the amino acids phenylalanine or tryptophan. Only Proteus, Providencia and Morganella species possess the deaminase enzyme.
  88. 88. PRINCIPLE  Deamination of the amino acid results in a colored compound with the addition of 10% ferric chloride Phenylalanine ----------------PPA + 10% FeCl3 Phenylalanine deaminase green Tryptophan-------------indole-pyruvic acid +10% FeCl3 Tryptophan deaminase brown
  89. 89. INTERPRETATION  Positive deamination for phenylalanine – intense green color  Positive deamination for tryptophan – brown color  Negative – slant retains its original color after the addition of ferric chloride
  90. 90. A. Negative – Escherichia coli B. Positive – Proteus vulgaris A B Phenylalanine deamination test
  91. 91. PURPOSE: To determine the ability of the organism to deaminate lysine, decarboxylate lysine and produce H2S. To identify Salmonella, Proteus, Providencia and Morganella.
  92. 92. COMPOSITION 1. Proteins 2. Sugar- Glucose 3. Amino acid - Lysine 4. Sulfur 5. indicators a. ferric ammonium citrate – H2S production b. bromcresol purple – carbohydrate fermentation
  93. 93. PRINCIPLE: As glucose fermentation occurs, deep of the tube turns yellow. Lysine decarboxylation produces alkaline cadaverine and leads to reversion of the deep from yellow to purple. Lysine deaminatiion occurs in the presence of oxygen (on the slant) and results in the production of a red color. H2S production is noted by a black precipitate in the deep as H2S reacts with ferric ammonium citrate.
  94. 94. INTERPRETATION Lysine decarboxylation - butt  Positive – purple  Negative – yellow Lysine deamination - slant  Positive – red  Negative –purple
  95. 95. K/K alkaline slant/alkaline butt H2S(-) purple/ purple Negative deamination Positive decarboxylation No blackening of the butt Escherichia coli Lysine iron agar
  96. 96. K/K alkaline slant/alkaline butt H2S + purple/purple Negative deamination Positive decarboxylation With black precipitate in the butt Salmonella typhimurium Lysine iron agar
  97. 97. K/A alkaline slant/acid butt H2S(-) (purple/yellow) Negative deamination Negative decarboxylation No black precipitate in the butt Shigella flexneri Lysine iron agar
  98. 98. R/A red slant/acid butt H2S(-) red/yellow Positive deamination Negative decarboxylation No black precipitate in the butt Proteus vulgaris Lysine iron agar
  99. 99. PURPOSE: 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
  100. 100. PRINCIPLE Sodium citrate is the only carbon source in Simmons citrate agar. If the organism can utilize citrate, the sodium citrate is converted to ammonia, which is then converted to ammonium hydroxide. The alkalinity of the compound formed raises the pH of the medium, and the bromthymol blue indicator takes on its alkaline color which is blue.
  101. 101. INTERPRETATION  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)
  102. 102. Citrate Utilization test A. Positive - Klebsiella pneumoniae B. Negative - Escherichia coli A B
  103. 103. PURPOSE  To determine the ability of an organism to use acetate as the sole source of carbon. PRINCIPLE  Breakdown of the sodium acetate causes the pH of the medium to shift toward the alkaline range, turning the indicator from green to blue.
  104. 104. INTERPRETATION Positive – Medium becomes alkalinized(blue) because of the growth of the organism Negative – no growth or growth with no indicator change to blue
  105. 105. A B Acetate utilization test A. Positive - Klebsiella pneumoniae B. Negative – Escherichia coli
  106. 106. PURPOSE  To determine the ability of an organism to use acetamide as the sole source of carbon. PRINCIPLE  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.
  107. 107. INTERPRETATION Positive – deamination of the acetamide resulting in a blue color Negative – no color change
  108. 108. Acetamide utilization test A. Positive – Klebsiella pneumoniae B. Negative – Escherichia coli
  109. 109. PURPOSE:  To differentiate Micrococccus and Stomatococcus from Staphylococcus when combined with other procedures such as the modified oxidase test.  For presumptive identification of Group A streptococcus
  110. 110.  Bacitracin(0.04 units) inhibits the growth of Micrococcus and Stomatococcus and Group A streptococcus while having no effect on Staphylo- coccus which is resistant. PRINCIPLE:
  111. 111. INTERPRETATION  susceptible – zones of inhibition greater than or equal to 10 mm  resistant – zones of inhibition less than or equal to 9 mm.
  112. 112. A. Susceptible – Micrococcus and Stomatococcus B. Resistant – Staphylococcus epidermidis A B Bacitracin susceptibility test
  113. 113. PURPOSE: To identify the different species of Streptococcus especially Group A and Group B beta hemolytic streptococci.
  114. 114. 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 – resistant to both bacitracin and SXT
  115. 115. INTERPRETATION Susceptible: any zone of inhibition around either disk Resistant: growth up to the disk(no zone of inhibition
  116. 116. Organism Bacitracin SXT Group A susceptible resistant Group B resistant resistant Group C,F,G resistant susceptible
  117. 117. PURPOSE  To differentiate the different species of coagulase negative staphylococci. PRINCIPLE  After incubation with 5 ug of novobiocin, Staphylococcus saprophyticus is not inhibited by the antibiotic whereas Staphylococcus epidermidis are susceptible to novobiocin.
  118. 118. INTERPRETATION:  susceptible – zone greater than 16 mm  resistant – zone diameter less than or equal to 16 mm
  119. 119. A B Novobiocin susceptibility test A. Susceptible - Staphylococcus epidermidis B. Resistant - Staphylococcus saprophyticus
  120. 120. PURPOSE  To differentiate Pediococcus from other alpha hemolytic streptococcus. PRINCIPLE  After incubation with 5 ug of vancomycin, Pediococcus is not inhibited by the antibiotic whereas Viridans streptococcus is susceptible to vancomycin.
  121. 121. INTERPRETATION  Susceptible – zone of inhibition  Resistant – no zone of inhibition
  122. 122. Vancomycin susceptibility test A. Susceptible - Viridans streptococcus B. Resistant - Pediococcus A B
  123. 123. PURPOSE  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).
  124. 124. PRINCIPLE  Most anaerobes have a characteristic susceptibility pattern to colistin(10 ug), vancomycin( 5 ug), and kanamycin(1 mg) disks. kanamycin – inhibits facultative gram-negative bacilli vancomycin- inhibits facultative and obligate gram-positive bacteria colistin- inhibits facultative gram-negative bacilli
  125. 125. K Va Co INTERPRETATION Susceptible – zone greater than 10 mm Resistant – zone of 10 mm. or less Antibiotic Disks for the Presumptive Identification of Anaerobes
  126. 126. PURPOSE 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).
  127. 127. PRINCIPLE Nutrient broth or 6.5%NaCl Trypticase broth-salt free medium Positive equal equal Negative good very weak
  128. 128. INTERPRETATION Positive – if growth is equivalent to both media – tolerant of salt Negative- growth on the salt containing medium is very weak or absent growth in the salt free medium is good - intolerant of salt Indicator: bromcresol purple Positive: medium turns yellow from purple or the appearance of growth
  129. 129. SALT TOLERANCE TEST A. Positive - Enterococcus faecalis ( salt tolerant) B. Negative - Streptococcus bovis(salt intolerant)
  130. 130.  To distinguish Group D streptococci and Enterococcus species from other Lancefield group of streptococci  based on the organisms ability to grow in 40% bile and to hydrolyze esculin to produce esculitin  Esculin reacts with ferric citrate to form a brown black precipitate. PURPOSE PRINCIPLE
  131. 131. INTERPRETATION Positive growth indicates tolerance to 40% bile(40% oxygall) blackening indicates hydrolysis of esculin Negative lack of growth indicates inability to grow in 40% bile lack of color change indicates inability to hydrolyze esculin
  132. 132. A. Positive - Enterococcus faecalis B. Negative - Streptococcus viridans Bile esculin agar A B
  133. 133. PURPOSE  To differentiate Streptococcus pneumoniae from other alpha hemolytic streptococci PRINCIPLE  In the presence of optochin, colonies of Strepto- coccus pneumoniae are selectively lysed indicated by a zone of inhibition after incubation under increased CO2.  Other alpha hemolytic streptococci are resistant to optochin.
  134. 134.  Positive – zone of inhibition at least 14 mm. in diameter using a 10 ug P disk and at least 10 mm. using a 6 mg P disk  Negative – growth up to the disk or a zone of inhibition less than 14 mm with a 10 ug P disk or less than 10 mm. with a 6 ug P disk INTERPRETATION
  135. 135. Optochin susceptibility test A. Positive – Streptococcus pneumoniae B. Negative – Viridans streptococci A B
  136. 136. PURPOSE  To differentiate Streptococcus pneumoniae(positive) from other alpha hemolytic streptococci.
  137. 137. 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.
  138. 138. INTERPRETATION  Positive – colony disintegrates; an imprint of the lysed colony may remain within the zone  Negative – intact colonies
  139. 139. B A Bile solubility test A. Positive – Streptococcus pneumoniae B. Negative – Viridans Streptococci
  140. 140. PURPOSE  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 perpen- dicular to beta hemolytic Staphylococcus aureus
  141. 141. 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 streptococcus
  142. 142. CAMP REACTION A. Positive - Streptococcus agalactiae B. Negative - Streptococcus bovis A B
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