CLINICAL BACTEROLOGY (LABORATORY)

LESSON 10: IDENTIFICATION OF STREPTOCOCCUS AND ENTEROCOCCUS SPECIES

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2nd SEMESTER I S.Y. 2021-2022
TRANCRIBED BY: JEAN HERSHEY REYES

Introduction bacterial suspension of S. pyogenes or S.

agalactiae. Always practice aseptic
 Streptococci are gram positive occurring in pairs or in chains, technique.
non-motile, facultative anaerobes and catalase negative organism. 2. Stain with Gram stain.
Some species are well known to cause pyogenic infection to 3. Focus on oil immersion objective
man. But some species have been noted to be part of the normal
4. Observe the morphology of the organisms
flora that resides in the skin, oral cavity, nasopharynx and genital
tract.

 Streptococci are classified on their hemolytic properties; Alpha-

hemolysis causes partial hemolysis that develops a greenish
color on blood agar; Beta-hemolysis, causes complete
hemolysis that makes the blood agar clear; Gamma-hemolysis,
there has been no hemolysis took place in blood agar.

Pre-Analytical Phase

 Sterilization of working area is very necessary by using Lysol or

bleach to wipe the tabletops. The laboratory scientist should
consider all specimens as infectious and must always wear
personal protective equipment inside the laboratory.

Materials

 Blood agar plate

 Bacitracin disk
 6.5% NaCl
 Wire loops
 Petri dish
 3% H2O2
 Gram stain reagents
 Alcohol lamps
 Glass slides
 Forceps  Colonies of Streptococci on most enriched
 Cotton swab media are grayish, translucent to
 Bacterial broth of the following: slightly opaque, circular, pinpoint
o E. faecalis colonies. Colonies may vary from the
o B-hemolysin-producing strain of S. aureus mucoid or smooth type to the matte or
o S.pyogenes rough form.
o S.agalactiae Colonial
Morphology
Procedures 1. Using cotton swab, collect sample in the
throat region of one of the group’s
 Cell Morphology member.
 Colonial Morphology 2. On a blood agar plate inoculate the swab
 Test for Identification: and the bacterial suspension of other
o Catalase Test streptococcus organism.
o Hemolysis 3. After incubation, observe for hemolysis
o Bacitracin Disk Test
o Camp Test
o Growth On 6.5% NaCl
o Sodium Hippurate Hydrolysis
o Bile Esculin Hydrolysis

 The Streptococci are Gram-positive cocci

normally arranged in chains of varying
Cell Morphology lengths. Cells are about 0.5um.

1. Prepare a bacterial smear from the

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 A. Beta Hemolytic Streptococcus Materials:
• Group A : S. pyogenes 1. Isolated colonies of S. agalactiae on BAP.
• Group B: S. agalactiae 2. Beta lysine producing S.aureus on
• Some Group D: Enterococcal and non- sheep’s blood.
enterococcal Strep Procedure:
1. Inoculate a streak beta-toxin producing S.
Hemolysis B. Alpha Hemolytic Streptococcus aureus straight on the center of sheep’s
• S. pneumoniae BAP.
• Viridans Strep 2. Inoculate straight line of S. agalactiae at
• Some Group D Streptococcus right angle to the Staphylococcal streak
without touching the colonies of
C. Gamma Hemolytic Streptococcus Staphylococcus.
• Some Group D Streptococcus 3. Incubate at 35°C for 18 hours in a candle
• Some Viridans group jar.
4. Observe an arrow-head shaped zone of
enhanced hemolysis at the junction of the
colonies of Streptococcus and
Staphylococcus.

Interpretation:
 Presence of arrowhead hemolysis –
POSITIVE
 No presence of arrowhead hemolysis –
NEGATIVE

 Principle: Group D streptococci can grow

in the presence of 40% bile and
subsequently hydrolyze esculin to
esculetin and glucose. Esculetin diffuses
into the agar and combines with ferric
 Sample should not be from the blood citrate in the medium to give a black
agar plate because it will produce a false complex.
positive result. Esculin ---- bacterial enzyme -----
esculetin+glucose
1. Emulsify a colony using applicator stick in
1 drop of 3% H2O2 in a glass slide.
Catalase Test Esculetin + Fe2 ------------- black complex
2. Observe for the production of gas bubbles.

Materials:
Interpretation: Bile-Esculin
 Gas bubbles - POSITIVE 1. Isolated colonies on BAP of Group D
Hydrolysis
 No gas bubbles - NEGATIVE Streptococcus.
2. Bile-esculin agar.
 Principle: This test is used to differentiate
Procedure:
group A streptococci form other groups of
1. Inoculate one to two colonies on a bile-
beta-hemolytic streptococci, based on the
esculin agar slant or streak in a line on the
sensitivity of S.pyogenes to low
surface of a bile-esculin agar plate.
concentration of Bacitracin.
Materials: 2. Incubate at 35 C for 18-24 hours.
3. Observe for blackening of agar slant or
1. 18-24-hour broth culture of S. pyogenes.
blackening around line of growth.
2. Blood agar plate
Bacitracin Disk 3. 0.04 units of Bacitracin (TAXO-A)
Test Interpretation:
 Blackening of growth or medium –
Procedure:
POSITIVE
1. Dip a cotton swab in broth suspension;
 No blackening of growth or medium –
express excessive liquid from the swab
NEGATIVE
against the side of the tube.
2. Streak the surface of the BAP.
 Principle: Enterococci can withstand a
3. Aseptically apply and incubate for 18
higher salt concentration than non-
hours at 35 C.
4. Observe for zone of inhibition. enterococci.
Growth on a
6.5% NaCl
Materials:
 Principle: S. agalactiae produces a
1. 18-24-hour broth suspension of
substance known as CAMP factors which
CAMP Test Enterococci
enhances the hemolysis of sheep RBC by
2. 6.5% NaCl broth
Staphylococcal beta lysine.
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 Procedure:
1. Inoculate a loopful of Enterococci to 6.5%
NaCl broth.
2. Incubate at 35 C for 18 hours.
3. Observe for turbidity.

Interpretation:
 Visible turbidity – POSITIVE
 No visible turbidity– NEGATIVE

 Principle: Based on the presence of

enzyme hippuricase which is able to
hydrolyze hippuric acid to glycine and
benzoic acid. Glycine when mixed with
ninhydrin reagent is reduced giving a color
change or purple.

Materials:
1. Isolated colonies of S. agalactiae.
2. Sodium Hippurate substrate.
3. Ninhydrin reagent.
Sodium
Hippurate
Hydrolysis Procedure:
1. Inoculate on a Sodium Hippurate
substrate a suspension of S. agalactiae to
produce a milky suspension.
2. Incubate at 35 C for 2 hours.
3. Add 0.2 ml of Ninhydrin reagent and
further incubate for 15 minutes.
4. Observe for the appearance of deep
purple color.

Interpretation:
 Formation of deep purple color–
POSITIVE
 No color change– NEGATIVE

Post-Analytical Phase

 After the experiment, all the glassware, materials that have been in
contact with bacteria like inoculating loop and including the
working station, should be sterilized properly. The student must
prepare 10% bleach or Lysol for disinfection. Materials like
inoculating wire can also be utilized by flaming. All disposable
materials should be placed properly in correct trash bags.

 PPEs should be disposed in infectious trash bag and should not be

worn outside the laboratory.

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