Isolation and Identification of Streptococci
and Enterococci
GENERAL DISCUSSION
The streptococci are gram-positive cocci (0.5-1.0µm in diameter) which
occur in pairs and chains of varying length.
They are usually classified based on their hemolytic properties on blood agar
and according to their serologic groups ( Lancefield grouping).
The streptococci are usually isolated on Sheep Blood agar ( BAP) ( Tryptic soy
agar with ( 5-10 ) % Sheep blood ).
Hemolysis refers to is the lysis of the red blood cells in the agar surrounding
bacterial colonies and is a result of bacterial enzymes called hemolysins.
Reactions on blood agar are said to be beta, alpha, gamma, or double-zone:
Hemolysis on sheep blood agar:
Beta hemolysis refers to a clear, colorless zone surrounding the colony,
where a complete lysis of the red blood cells by the hemolysins has
occurred.
Alpha hemolysis appears as a zone of partial hemolysis surrounding
the colony, often accompanied by a greenish or brown discoloration due to
oxidation of hemoglobin to met-hemoglobin by hydrogen peroxide
Gamma hemolysis refers to no hemolysis or discoloration of the agar
surrounding the colony.
Double-zone hemolysis refers to both a beta and an alpha zone of
hemolysis surrounding the colony.
Gram positive cocci
Gram Stain
Gram Positive cocci
Catalase
-
+
Staphylococci
Micrococci
streptococci
Growing on sheep blood agar ( hemolysis )
α
S. Pneumoniae
S. viridans
β
S.agalactiae
S. pyogenes
γ
Enterococcus spp.
(E. faecalis)
Non Enterococcus
(S.bovis)
Serological Typing of Streptococci ( Precipitin test )
Many of the streptococci can be placed into serological groups called Lancefield
groups based on carbohydrate antigens in their cell wall. Although there are 20
different Lancefield groups of streptococci, the groups A, B, C, D, F, and G are the
ones usually associated with human infections.
The Slidex Strepto-Kit® system is a commercial kit for typing the 6 Lancefield
groups of streptococci that commonly infect humans.
To make the reaction more visible, since the antigens for which one is testing are
only fragments of the bacterial cell wall, the known monoclonal antibodies have
been adsorbed to latex particles.
In this way, when the known monoclonal antibodies react with the streptococcal
cell wall antigens, agglutination of the latex particles will occur and can be easily
seen with the naked eye.
Dr. rebecca lancefield
L
L
Antibody
S
S
S
L
S
Fc
L
L
L
L
S
S
L=Latex particle
Strepto. antigen
Gram Stain
Gram Positive cocci
Catalase
-
+
Staphylococci
Micrococci
Streptococci
Lancefeild grouping ( A,B and D )
‫ــــــ‬
S. Pneumoniae
S. viridans
A
S. Pyogenes
(GAS)
B
S.agalactiae
(GBS)
D
Enterococcus spp.
(E. faecalis)
Non Enterococcus
(S.bovis)
β-hemolytic Sterptococci
S.agalactiae
S. pyogenes
Bacitracin Sensitivity Test:
Definitive test to differentiate between
S. pyogenes & Non group A β-hemolytic Streptococci ( S. agalactiae )
Principle:
A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth
of S.pyogenes giving a zone of inhibition around the disc.
Bacitracin Sensitivity Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
2. Aseptically apply Bacitracin disc onto
the center of the streaked area.
3. Incubate the plate at 37oC for 24 hrs.
B
Bacitracin Sensitivity Test:
Results:
Positive test: any zone of inhibition around the disc.
B
Bacitracin Resistant
S.agalactiae
B
Bacitracin Sensitive
S.pyogenes
Bacitracin Sensitivity Test:
Pyrrolidonyl-beta-naphthylamide (PYR) test
S.pyogenes posses the enzyme L-pyroglutamyl aminopeptidase which
hydrolyzes Pyrrolidonyl-beta-naphthylamide with the formation of free
β- naphthylamine, which combines with cinnamaldehyde reagent to form
a bright- red end product.
Method :
Rub a small amount of a colony to be tested on
a surface of a filter paper impregnated with PYR
( available commercially ).
Add one drop of freshly reconstituted detector
reagent N,N- dimethylaminocinnamaldehyde,
with detergent ( available commercially), and
observe for a red color within 5 minutes.
Hippurate hydrolasis test:
Group B streptococci contain the enzyme hippuricase , which can hydrolyze
hippuric acid . Other β streptococci lack this enzyme .
The production pf hippuricase results in the hydrolysis of sodium hippurate
With the formation of sodium benzoate and glycine .
The hydrolysis of hippurate can be detected by one of two methods:
 Standard test for benzoate using ferric chloride
 Rapid test for glycine using ninhydrin reagent
Standard test for benzoate using ferric chloride
Media and reagent:
Sodium hippurate medium
Brain Heart infusion broth.
1% Sodium hippurate.
Ferric chloride reagent
Fecl3 12g.
2% aqueous HCl 100 ml.
Procedure :
Incubate a tube of sodium hippurate medium with the organism.
Incubate for 20 hours or longer at 37 OC.
Centrifuge the medium to pack the cells and pipette 0.8 ml of the
clear supernatant in to a clean test tube.
Add 0.2 ml of the ferric chloride reagent and mix.
Result:
A heavy precipitate will for upon addition of Fecl3 reagent.
Rapid test for glycine using ninhydrin reagent
Media and reagent:
Sodium hippurate medium
1% Sodium hippurate.
Distilled water.
Ninhydrin reagent
Ninhydrin 3.5 g.
1: 1 Acetone/butanol 100 ml.
Procedure :
Incubate a large loop of a β streptococci culture in to 0.4 ml of the
sodium hippurate medium and incubate for 2 hours at 37 OC.
Add 0.2 ml of the ninhydrin reagent.
Result:
The presence of a deep purple color after the adition of ninhydrin
reagent within 10 minutes indicate the presence of glycine.
CAMP test :
The laboratory identification of group B beta hemolytic Streptococci has been
simplified implementation of the CAMP test, which is easy perform and well
within the capabilities of small laboratories.
The CAMP phenomenon was first reported in 1944 by Cristie, Atkins, and
Munch-Peterson, whose contribution is acknowledged in the acronym
Principle:
The hemolytic activity of Staphylococcal beta –lysin on erythrocytes is
enhanced by an extracellular factor produced by group B streptococci, called
the CAMP factor. Therefore, where the two reactants overlap in a sheep blood
agar plate, accentuation of the beta hemolytic reaction occurs.
Media and reagent:
Sheep or bovin blood agar must be used.
Procedure:
The CAMP test is performed by making a single streak of the beta hemolytic
Streptococcus perpendicular to a strain of Staphylococcus aureus that is
known to produce beta-lysin .
The two streak lines must not touch one another then incubate the agar in to
candle jar to accelerate the hemolysis.
Results:
The zone of increased lysis assumes the shape of an arrow-head at the junction
of the two streak lines.
CAMP test :
S.agalactiae
Beta-hemolytic Staphylococcus aureus
Beta hemolytic
Streptococci not
S.agalactiae
α-hemolytic Sterptococci
S.pneumoniae
S. viridans
Optochin Sensitivity Test:
Definitive test to differentiate between
S. pneumoniae & viridans Streptococci
Principle:
S.pneumoniae is inhibited by less than 5 µg/ml Optochin reagent
(ethylhydroxycupreine hydrochloride ) giving a zone of inhibition.
Optochin Sensitivity Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
2. Aseptically apply Optochin disc onto
the center of the streaked area.
3. Incubate the plate at 37oC for 24 hrs.
4. Accurately measure the diameter of
the inhibition zone around the disc.
O
Optochin Sensitivity Test:
Results:
Sensitive with inhibition zone equal or more than 14 mm when disk with diameter
equal 6 mm or 16 when 10 mm disk is used.
Bile solubility test
The bile solubility test is used to determine the ability of bacterial cells
to lyse in the presence of bile salts, within a specified time .
S. pneumoniae possesses an autolytic enzyme which lyses the cell’s own
wall during division. The addition of bile salts (sodium deoxycholate)
activates the autolytic enzyme and the organisms rapidly autolyse. Other
α haemolytic streptococci do not possess such an active system and
therefore do not dissolve in bile.
 The bile solubility can be detected by one of two methods:
 Broth test.
 Agar blate test.
Bile solubility Broth test :
Media and reagent:
10% sodium deoxycholate or 10% sodium taurocholate.
Pure culture ( broth ) of the tested organism incubate at 37 OC for 24 hours.
Sterile normal saline.
Test tubes.
Procedure :
Transfer approximately 0.5 ml of an 24 hours broth culture to each of two test tubes
Add 0.5 ml of 10% sodium deoxycholate to one of the two test tubes ( marked test)
Add 0.5 ml of sterile normal saline to the second test tube ( marked control )
Gently agitate broth test tubes and palce them in a 37 OC incubator or water bath
for 3 hours, checking hourly.
Result:
Bile soluble, visible clearing of the turbid culture
within 3 hours.
The saline control tube should remain turbid.
Bile solubility Agar test :
Media and reagent:
2% sodium deoxycholate.
Isolated colonies of the tested organism on agar incubate at 37 OC for 24 hours.
Procedure :
To well isolated colony of the test organism on 5% sheep blood agar plate, add a
drop of 2% sodium deoxycholate without inverted the blate incubate in 37 OC
incubator for 30 minutes.
Result:
Bile solublecolony dissapear leaving hemolyzed area.
Colony insoluble in bile remain intact.
Quellung Reaction
Around the turn of the century (1900) German physician and bacteriologist
Friedrich Neufeld discovered that antibodies against specific pneumococcal
capsular antigens could be produced and used for typing Streptococcus
pneumoniae organisms.
These antibodies, when bound to the cell wall antigen produced a clearing
around each individual cell with the appearance of the capsule having swollen.
The word “Quellung” is German for ‘swelling’ however this is a misnomer as the
capsule does not swell but simply appears enlarged with the clear zone produced
by the bound antibodies.
The clearing is best visualized by using a stain to enhance contrast between the
clear zone of bound antibody and the surrounding material.
Procedure:
Colony isolate, is placed on a clean microscope slide and allowed to dry.
A drop of *Polyvalent Quellung antisera is applied to the slide and mixed
with a drop of methyene blue stain.
A coverslip is place onto the mixture and allowed to incubate/react at room
temperature for 15-20 minutes.
The slide is then examined under the microscope for distinct clearing around
the cells.
One may have to search for a microscopic field which best shows the
clearing. A distinct zone of clearing indicates a positive Quellung reaction and
confirms the identification as Streptococcus pneumoniae.
*Polyvalent Antisera- contains all known serotypes to identify any
pneumococcus. Specific or individual antisera can be employed for scientific
study of individual serotypes.
Quellung Reaction
Fluorescent antibody (FA) staining
Streptococcus pneumoniae, FA stain showing its
antiphagocytic capsule (CDC).
Streptococcus faecalis broth (SF broth) :
Media and reagent:
Tryptone
D-glucose
Monopotassium phosphate
Dipotassium phosphate
Sodium azide
Sodium chloride
Bromocresol purple
pH= 6.9
Principle:
The tryptone and dextrose supply the nutrients required for the growth of
enterococci. Sodium azide exhibits a bacteriostatic effect on gram-negative
bacteria through its inhibitory action on enzymes in the electron transport
system. Bromcresol purple serves as a pH indicator.
Bile esculin test :
Media and reagent:
Beef Extract.
Peptone.
Esculin.
Oxgall ( 1- 4 )%.
* sodium azide.
Ferric Ammonium Citrate.
Agar 1.5%
Final pH: 6.6 ± 0.2 at 25 OC
Principle:
Enterococci and certain streptococci hydrolyze the glycoside esculin to
esculetin and dextrose. Esculetin reacts with an iron salt to form a dark brown
or black complex.
Ferric citrate is incorporated into the medium as an indicator of esculin
hydrolysis and resulting esculetin formation.
Oxgall is used to inhibit gram-positive bacteria other than Enterococci.
Bile esculin test :
Positive result
Negative result
6.5% NaCl medium
Enterococcus spp. Grow well in 6.5% NaCl medium(6.5%NaCl in brain
heart infusion broth ) but S.bovis will not.
Negative after 24hrs
S.bovis
Positive after 24hrs
Enterococcus spp.
Pyrrolidonyl-beta-naphthylamide (PYR) test
Enterococcus spp. Posses the enzyme L-pyroglutamyl aminopeptidase
which hydrolyzes Pyrrolidonyl-beta-naphthylamide with the formation
of free β- naphthylamine, which combines with cinnamaldehyde reagent
to form a bright- red end product.
Method :
Rub a small amount of a colony to be tested on
a surface of a filter paper impregnated with PYR
( available commercially ).
Add one drop of freshly reconstituted detector
reagent N,N- dimethylaminocinnamaldehyde,
with detergent ( available commercially), and
observe for a red color within 5 minutes.