Streptococci

Biochemical reactions
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University
Dr. Nabil El Aila
Diagnostic
Microbiology

Streptococci
Characters of Streptococci

Gram positive cocci

1m in diameter
Chains or pairs
Usually capsulated
Non motile

Non spore forming

Facultative anaerobes
Fastidious
Catalase negative (Staphylococci are catalase
positive)
Dr. Nabil
El Aila
Diagnostic Microbiology

Hemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis

Dr. Nabil El Aila

Diagnostic
Microbiology

Classification

Based on O2
Anaerobes

Aerobes

Peptostreptococci

Growth on BA
hemolysis

hemolysis

Incomplete hemolysis
(green color)

Complete hemolysis

hemolysis
/ / no hemolysis

Lancefield grouping

Strep. viridans

specific C carbohydrate Ag on cell wall

Strep. pneumoniae

18.05.09
Group A
S. pyogenes

Group B
S. agalactiae

Enterococcus fecalis

Group A U (21 groups)

Group C
S. equisimitis

Group D
Enterococcus

Other groups
(E-U)

Differentiation between -hemolytic

streptococci
The following tests can be used to differentiate between
-hemolytic streptococci
Lanciefield Classification
Bacitracin susceptibility Test
Specific for S. pyogenes (Group A)

CAMP test
Specific for S. agalactiae (Group B)

Dr. Nabil El Aila

Diagnostic Microbiology

Laboratory Diagnosis:
Group A Streptococcus
Colony morphology
Transparent, smooth,
and well-defined zone of
complete or b- hemolysis

Dr. Nabil El Aila

Laboratory Diagnosis:
Group A Streptococcus
Identification
Catalase-negative
Bacitracin-susceptible
PYR-positive
Bile-esculinnegative
6.5% NaCl-negative

Group A streptococci is susceptible to

Bacitracin disk (left); The right shows
resistance
Dr. Nabil El Aila

Bacitracin sensitivity
Principle:
Bacitracin test is used for presumptive
identification of group A
To distinguish between S. pyogenes
(susceptible to B) & non group A such as S.
agalactiae (Resistant to B)
Bacitracin will inhibit the growth of gp A
Strep. pyogenes giving zone of inhibition
around the disk

Procedure:
Inoculate BAP with heavy suspension of tested
organism
Bacitracin disk (0.04 U) is applied to
inoculated BAP
After incubation, any zone of inhibition around
the disk is considered as susceptible

Pyrrolidonyl arylamidase Test (PYR)

I.Principle
Some bacteria like group A streptococci and Enterococcu s species
produce pyrrolidonyl arylamidase which hydrolyzes the substrate Lpyrrolidonyl --naphthylamide to form -naphthylamine.
A pink to red color forms when p-dimethylaminocinnam-aldehyde (PYR
reagent) is added to -naphthylamine.
II. Inoculum
Strains are grown on blood agar plates overnight at 35C in CO 2 . More
than 1 day of incubation may be necessary for more fastidious genera such
as the gemellae, alloiococci, and helcococci. The strains to be tested are
grown on a blood agar plate until sufficient growth is seen to heavily
inoculate the disks.
III. Reagents and Materials
PYR disk (Remel) - PYR reagent
Loops
- Deionized Sterile water
Dr. Nabil El Aila

IV. Procedure

The procedure that is used in the Streptococcus laboratory is modified from the
package insert. The LAP test is usually done simultaneously.

1. Place the disks on blood agar plate in an area of little or no growth or on a

slide. The moisture from the plate is usually sufficient to rehydrate the disk. If
the disk is placed on a slide, then a tiny drop of sterile deionized water is added.
(DO NOT OVERSATURATE THE DISK).

2. Using a loop or wooden stick, inoculate the disks heavily. Using two or more
loop-fulls of culture is necessary for satisfactory results.

3. Leave the plates with the disks on the bench at room temperature for 10
minutes.

4. Add the detection reagent and read after 3 minutes.

V. Reading and Interpretation

The development of a red color within 3 minutes is positive. No change in color

or a yellow color is negative. The color develops immediately. Discard the test
after 10 minutes.

VI. Limitations

False negative reactions may result if too little inoculum is used.

PYR Negative

Dr. Nabil El Aila

PYR positive

Laboratory Diagnosis:
Group B -Hemolytic Streptococcus
Colony morphology
Grayish-white, mucoid,
creamy, narrow zone of bhemolysis
Presumptive Identification
tests
Catalase-negative
Bacitracin-resistant

Dr. Nabil El Aila

Laboratory Diagnosis:
Group B b-Hemolytic Streptococcus
Presumptive identification
tests
Bile-esculin-hydrolysis
negative
Does not grow in 6.5% NaCl
CAMP-testpositive
Hipppurate Hydrolysis

S. agalactiae shows the arrow-shaped

hemolysis near the staphylococcus
streak, showing a positive test for
CAMP factor
Dr. Nabil El Aila

CAMP Test
I. Principle
Some bacteria produce CAMP factor (a diffusible extracelluar
protein) that synergistically acts with the beta-lysin of
Staphylococcus aureus and enhances the lysis of red blood
cells. The purpose of the CAMP test is to aid in the
identification of nonhemolytic group B streptococci and other
-hemolytic streptococci.
II. Specimen
Growth from a blood agar plate or any solid media.
III. Reagents and Materials
TSA-sheep blood agar
Dr. Nabil El Aila

IV. Procedure
1. The CAMP test is performed on TSA-sheep blood agar. A single streak of -lysin
producing S. aureus made across the center of the plate. Strain SS-695 (Strep. Lab
number is a -lysin producing strain of S. aureus.
2. A single colony of the unknown strain (beta hemolytic streptococci) is picked up
with an inoculating loop and used to make a single streak perpendicular but not
touching the S. aureus streak. A 2-3 mm space should remain between the streaks.
3. Incubate the inoculated plate normal atmosphere overnight at 35C. Group B
streptococci and a few other beta-streptococci produce an enhancement of the lysin activity of the S. aureus strain.
V. Reading and Interpretation
This enhanced activity is in the shape of an arrowhead at the juncture of the two
streaks, with the widest portion of the arrowhead on the group B side.
VI. Limitations
Do not incubate in an anaerobic environment or under CO 2. Some S. pyogenes
strains will give a positive reaction when incubated in CO 2.

CAMP test

Dr. Nabil El Aila

Hipppurate Hydrolysis Test

I. Principle
Some bacteria like group B streptococci produce the enzyme hippurate
hydrolase which hydrolyzes sodium hippurate to form benzoic acid and
glycine. The addition of ferric chloride to benzoic acid forms an insoluble
brown ferric benzoate precipitate.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated at 35 C or a fresh
bacterial suspension in Todd Hewitt broth may be used as the inoculum. An
inoculating loopful of culture from a blood agar plate may also be used.
III. Reagents and Materials
1. Hippurate broth commercial suppliers.
2. Ferric choride (FeCl3) commercial supplies. Labeled as TDA if purchased
for bioMereiux

IV. Procedure
1. The hippurate broth is inoculated with one drop of a fresh (16-20 h) ToddHewitt broth culture.
2. The broth is incubated for up to 7 days or until turbid growth is seen at
35C.
3. The tube of broth is then centrifuged to sediment the bacteria.
4. Pipette 0.8 ml the clear supernatant to a small clear tube (13 x 100).
5. Add 0.2 ml of ferric chloride reagent to the supernatant. Mix well.
V. Results and Interpretation
A heavy precipitate that does not clear within 10 minutes indicates a positive
test. A clear golden-brown liquid indicates a negative test.
VI. Limitations
Growth should be turbid before testing. Some fastidious organisms may
show poor growth
Dr. Nabil El Aila

Laboratory Diagnosis:
Streptococcus Group D and
Enterococcus Species
Identification tests
Catalase: may produce a weak catalase reaction
Hydrolyze bile esculin
Differentiate Group D from Enterococcus sp. with
6.5% NaCl or PYR test

Dr. Nabil El Aila

Laboratory Diagnosis: Streptococcus

Group D and Enterococcus Species
Microscopic
morphology
Cells tend to elongate

Colony morphology
Most are nonhemolytic, although
some may show or,
rarelyhemolysis
Possess Group D
antigen

Dr. Nabil El Aila

Bile Esculin Test

I.Principle
A selective and differential medium used in the identification of
catalase-negative bacteria. The selective agent bile, inhibits most gram
positive bacteria. The enterococci and Streptococcus bovis will grow.
Esculin in the medium is hydrolyzed to esculetin and dextrose. The
esculetin reacts with ferric chloride in the media to form a black-brown
color.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated over night at 35
C or a fresh bacterial suspension in Todd Hewitt broth may be used as
the inoculum. An inoculating loopful of culture may also be used.
III. Reagents and Materials
1. Bile esculin slant (Remel)
Dr. Nabil El Aila

IV. Procedure
1. Inoculate tube with 1 drop of inoculum allowing drop to run down slant.
Alternatively, the slant may be inoculated with a loopful of growth from a
blood agar plate.
2. The slant is then incubated at 35C for 2 days in ambient air. Fastidious
organisms may be held up to 14d.
V. Reading and Interpretation
The bile esculin test is positive when a black color forms over one-half or
more of the slant. If no blackening occurs the test is negative.
VI. Limitations
Do not incubate medium in a carbon dioxide atmosphere. The increase in C0 2
will cause the viridans streptococci to grow better and increase the likelihood
of a positive BE reaction. Streptococcus bovis and enterococci do not require
C02 for good growth.

Dr. Nabil El Aila

Identification Schema

Schema to differentiate Group A and B from other

b-hemolytic streptococci

Identification Schema

Schema to differentiate Enterococcus and Group

D streptococci from other nonhemolytic
streptococci

Laboratory Diagnosis:
Streptococcus pneumoniae
Colony
morphology
Smooth,
glistening, wetlooking, mucoid
-Hemolytic
CO2enhances
growth

Dr. Nabil El Aila

Laboratory Diagnosis:
Streptococcus pneumoniae
Identification
Catalase negative
Optochinsusceptibility-test
susceptible
Bile-solubility-test
positive

Dr. Nabil El Aila

Differentiation between -hemolytic

streptococci
The following definitive tests used to differentiate between S.
pneumoniae & viridans streptococci
Optochin Test
Bile Solubility Test
Inulin Fermentation

Dr. Nabil El Aila

Optochin Susceptibility Test

Principle:
Optochin (OP) test is presumptive test that is used to identify
S. pneumoniae
S. pneumoniae is inhibited by Optochin reagent (<5 g/ml)
giving a inhibition zone 14 mm in diameter.
Procedure:
BAP inoculated with organism to be tested
OP disk is placed on the center of inoculated BAP
After incubation at 37oC for 18 hrs, accurately measure the
diameter of the inhibition zone by the ruler
14 mm zone of inhibition around the disk is considered as
positive and 13 mm is considered negative
S. pneumoniae is positive (S) while S. viridans is negative (R)

Optochin Susceptibility Test

Optochin resistant
S. viridans
Optochin susceptible
S. pneumoniae

Dr. Nabil El Aila

Bile Solubility Test

I.Principle
The purpose of the bile solubility test is to aid in the differentiation
of S. pneumoniae from all other alpha-hemolytic streptococci.
Sodium deoxycholate (2%) acts on the cell wall of pneumococci
resulting in lysis.
II. Inoculum
An overnight culture grown on blood agar incubated 35C in CO2.
III. Reagents and Materials
1. 2% deoxycholate (CDC Central Services Laboratory, formula #5333)
2. physiologic saline pH 7.0
3.13 X 100mm glass tube

Dr. Nabil El Aila

IV. Procedure
1. Make a 1.0 ml saline suspension of cells from growth on an agar plate. A
turbidity equal to that of 1.0 to 2.0 McFarland density standard should be
used.
2. After a satisfactory density is achieved, divide the suspension into 2 tubes
with approximately 0.5 ml in each.
3. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube and 0.5 ml
saline to the other tube. Mix by vigorous shaking.
4. Incubate the tubes at 35-37C for up to 2 h.
V. Reading and Interpretation
Examine for clearing of the turbidity periodically.
A clearing of the turbidity in the bile tube but not in the saline control tube
indicates a positive test, i.e., the pneumococcal cells have lysed
("solubilized").
If the tube containing the cells and bile have not cleared the test is
negative.
VI. Limitations
The turbidity must be sufficient to detect a difference in the saline
control tube.

Dr. Nabil El Aila

Dr. Nabil El Aila

Identification Schema

Schema to differentiate S.
pneumoniae from other hemolytic streptococci

Dr. Nabil El Aila

Differentiation between -hemolytic streptococci

CAMP test

Bacitracin
sensitivity

Hemolysis

Negative

Susceptible

S. pyogenes

Positive

Resistant

S.
agalactiae

Differentiation between -hemolytic

streptococci
Inulin
Fermentatio
n

Bile
solubilit
y

Optochi Hemolysi
n
s
sensitivi
ty

Not ferment

Soluble

Sensitive
( 14
mm)

Ferment

Insoluble

Resistant

S.
pneumoniae

Outline of differentiation
between Gram-Positive cocci

e.g. S. epidermidis