Laboratory # 3

Biochemical Differentiation of some Medically

Important Gram Positive Cocci

Streptococci: Gram positive Staphylococci: Gram positive

cocci in chains cocci in clusters
 Catalase Test
Principle: to determine the ability of an organism to produce the enzyme catalase
which breaks down H2O2 into water & O2 gas.
The test is helpful in differentiating Staphylococcus spp (+) from Streptococcus spp
(-)

- +
Flow chart for identifying staphylococci
Staphylococcus aureus
Positive
Staphylococcus Coagulase
species Test

Coagulase
negative
Negative staphylococci

Mannitol
fermentation Negative

Staphylococcus aureus

Positive
Coagulase test
• Principle: to determine the ability of an
organism to produce the enzyme coagulase
which converts fibrinogen into fibrin resulting
in the formation of a visible clot

This test differentiates S. aureus (+) from other

Staphylococcus

• Procedure: mix 0.5 ml of rabbit plasma with 2-

3 colonies or 0.5 ml 18-24 hrs broth in a sterile
tube, incubate @ 37°C & examine for clot
formation after 4 hrs

• NB: do not leave the test for more than 8 hrs

without examination since coagulation may be
followed by liquefaction due to the
production of enzyme (Staphylokinase)
 Mannitol Fermentation Test

+ - (same as uninoculated media)

Phenol red indicator turns
yellow at acid pH

Principle: to determine the ability of an organism to ferment mannitol with the production of
acids
Broth with 1% mannitol & phenol red indicator
Pastorex Staph Plus is a rapid
agglutination test for the
simultaneous detection of the
fibrinogen affinity antigen
(clumping factor), protein A, and
the capsular polysaccharides of
Staphylococcus aureus.
 Beta-hemolytic Streptococci: species
serological groups (lancefield grouping)
Species/ Group Serological groups
S. pyogenes A
S. agalactiae B
S. equi C
S. equisimillis C
S. zooepidemics C
Lancefield group G G
S. anguinosus A,C,F,G OR none
Flow chart for
identification of Beta hemolytic
beta hemolytic
Streptococci

Growth in presence of bacitracin

Sensitive Resistnant CAMP test

Streptococcus pyogenes Other beta-hemolytic
Streptococci
+
Streptococcus agalactiae
-
Other beta-hemolytic
Streptococci
Bacitracin susceptibility
• Principle: to determine whether the growth of
an organism is inhibited by bacitracin disk
(0.04 units) or not.
The test is helpful in differentiating S. pyogenes
(+) from other β- hemolytic Streptococcus (-)

• Procedure:
– Inoculate a BAP with a heavy inoculum of β-
hemolytic Streptococcus
– Place the bacitracin disk in the center of the
streaked area
– Incubate @ 37°C for 18-24 hrs

• Results:
– Positive: any zone of inhibition around the
disk
– Negative no zone of inhibtion around the disk
CAMP (CHRISTIE, ATKIN, MUNCH-PETERSON) test
• Principle: to determine the ability of a group B streptococci to produce
the CAMP factor which acts synergistically with the beta toxin produced
by S. aureus on sheep BAP to produce an enhanced lytic activity
• Procedure:
– streak a β toxin producing S. aureus or place a disk containing β lysine
in a straight line across the center of BAP
– Streak the test organism in a straight line perpendicular to the
staphylococcus inoculum (lines must not touch)
– Incubate @ 35°C, 5-10 % CO2 for 18-24 hrs
– Examine the plates for an arrowhead zone of complete hemolysis
(enhanced lytic activity)
Flow chart for
identification of
ALPHA hemolytic
Streptococci Alpha hemolytic

Growth in presence of optochon

sensitive resistant

Streptococcus pneumoniae Group D

Viridans group
Nutritionally variant Strept
Optochin susceptibilty test
• Principle: to determine whether the growth of an organism is inhibited by
a low concentration of ethylhydroxycupriene hydrochloride (optochin)
The test differentiates between S. pneumoniae (+) from other α hemolytic
Streptococci

• Procedure:
– Inoculate a BAP with a heavy inoculum of α hemolytic Streptococci
– Place a 5 ug optochin disk in the center of the streaked area
– Incubate @ 35°C for 18-24 hrs in a CO2 incubator

• Results:
– Resistant: zone of inhibition < 14 mm
– Sensitive: zone of inhibition >= 14 mm
Flow chart for
identification of
Gamma hemolysis
Gamma hemolytic
Streptococci
Growth on Bile Esculin

+
Group D Strept

Growth in SF broth

+ -
Enterococcus Nonenterococcus
 Bile esculin agar
• Ingredients: esculin, ferric citrate, bile
• Purpose: group D Streptococci & Enterococci
can grow in the presence of 40% bile& can
also hydrolyze esculin to esculetin which
forms a black complex with ferric citrate
 Enterococcus selective media (SF)

• Principle: to determine the ability of

enterococcus to grow in the presence
of sodium azide & to ferment glucose
with the production of acid resulting
in pH change.
• Procedure: inoculation, incubation @
35°C for 18-24 hrs
• Results
– Positive: growth (turbidity) with a
yellow color
– Negative: no growth with a purple color
(original color of the media)
Biochemical Differentiation of some Medically
Important Gram-negative Bacilli
Flow chart for identification Gram negative bacilli
of Gram negative bacilli

Growth on MacConkey

Lactose fermenter Non-lactose fermenter

Enterobacteriaceae Oxidase

+ -

Pseudomonas aeruginosa Enterobacteriaceae

Enterobacteriaceae
• Gram negative rods
• Oxidase negative
• Glucose fermenters
• Lactose fermenters:
• E.coli
• Klebsiella spp
• Citrobacter freundii
• Enterobacter
• Non-lactose fermenters:
• Salmonella spp
• Shigella spp
• Proteus spp
• Providencia spp
• Morganella spp
• Serratia spp
• Yersinia spp
Oxidase test
• Principle: determine the presence of bacterial cytochrome
oxidase using the oxidation of the substrate 1 % tetramethyl-
p-phenylenediamine dihydrochloride to indophenol (a dark
purple-colored end product)

• Procedure:
1. Moisten filter paper with the substrate or select a
commercially available paper disk that has been impregnated
with the substrate
2. Use a wooden stick to remove a small portion of the bacterial
colony from the agar surface & rub the sample on the filter
paper or impregnated disk
3. Observe inoculated area of paper or disk for a color change

Results:
+ve: development of a dark purple
color within 10 seconds
-ve: absence of color
Biochemical Testing
1. Decarboxylase & dehydrolase tests
a) Lysine decarboxylase (LDC)
b) Ornithine decarboxylase (ODC)
c) Arginine dehydrolase (ADH)
2. Lysine Iron Agar test (LIA)
3. Kligler’s iron agar (KIA)/ Triple sugar iron agar (TSI)
4. Citrate test
5. Urease test
6. Indole test
7. Carbohydrate fermentation
8. Methyl red (MR)
Carbohydrate Fermentation Tests
Glucose w/ Durham Fermentation Tube
broth with 1% carbohydrates, phenol red
 IMViC Series
Indole Test
•Principle: to determine the ability of Add 1 drop of Kovacs indole or James reagent
an organism to produce the enzyme
tryptophanase which hydrolyses
tryptophan

broth with
tryptophan
IMViC Series
Methyl Red Test

Principle: to determine the ability of an organism to produce strong

acids from mixed fermentation of glucose. Acid production is detected
by the formation of a red color.
 IMViC Series
Voges-Proskauer Test
Leave uncapped for 15-20 minutes……..


Detects acetoin

MRVP broth
IMViC Series Citrate Utilization Test

- +

•Principle: to determine
the ability of an organism
to utilize citrate as the only
source of carbon with
production of ammonia
resulting in alkalinity of the
medium
 Urease Test

Positive Slow positive

Negative result Uninoculated
(acidic) urea agar result Principle: to determine the
(alkaline) ability of an organism to
produce urease.
Motility Test
Decarboxylase & dehydrolase tests
Lysine decarboxylase (LDC)
Ornithine decarboxylase (ODC)
Arginine dehydrolase (ADH)

Used to determine the

ability of an organism to
break down the a.as lysine,
ornithine & arginine with
the production of an
amine product resulting in
alkalinity of the media.
negative positive
 Kligler’s iron agar (KIA)/ Triple sugar
iron agar (TSI)
• Results:
color development slant/butt KIA TSI
Red/yellow (k/a) glucose utilization glucose utilization

Yellow/yellow (a/a) glucose& lactose utilization glucose&

lactose &/or sucrose
Red/red (k/k) neither neither
 Gas production: bubbles in the medium or splitting or cracking of medium
 H2S production: black ppt in the medium
Identification of Pseudomonas aeruginosa
• Oxidase-positive

• Characteristic grape-like odor

• TSI with an alkaline no change

reaction(k/k)

• Good growth @ 42̊ C

• Production of bright bluish-

green, yellow-green, red or
brown diffusible pigment on
Mueller-Hinton agar or
trypticase soy agar
 Automated:
GNI (VITEK system)
4-13 hrs incubation time
 Refer to the API20E
catalogue for
identification
API needs 24-48 hrs incubation time