LAB 4 - Streptococcus

Pyogenic bacteria
staphylococcus ssp
streptococcus ssp
Neisseria ssp `
By M.SC Marwa.A.Ahmed
Subject
• Introduction
• Type of strep. Ssp
• Cater media
• Biochemical test
Type of Streptococci ssp.
Classification system is based on hemolysis reactions

• Beta - hemolytic – A, B, C, D and G (S. pyogenes & s. agalactea) .
• Alpha - hemolytic – S. pneumoniae and s. viridians
• Gama – hemolytic – S. Faecalis
Lance field classification of B - hemolytic streptococci based on
presence of carbohydrate antigen in cell wall – 17 groups (A, B, C,….
Streptococci ssp.
• Gram-positive spherical/ovoid cocci arranged in long chains;
commonly in pairs. Non-spore-forming, nonmotile.
• Can form capsules Facultative anaerobes Most parasitic forms are
fastidious and require enriched media.
• Small, non pigmented colonies.
• Sensitive to drying, heat, and disinfectants.
• Catalase test: All Streptococcus spp. are catalase negative.
1. S.Pyogenic
Most serious streptococcal pathogen.
It a inhabits throat, nasopharynx and
skin.
Morphology
• Gram-positive
• Spherical/ovoid cocci arranged in
long chains.
• Nonmotile
.• Can form capsules
Cultural characteristics
• The organism is cultured on

blood agar with an added
bacitracin antibiotic disk to
show beta-hemolytic colonies
and sensitivity (zone of
inhibition around the disk) for
the antibiotic.
Cultural characteristics
Microscope examination
• The diagnosis is suggested

by the finding of gram-
positive bacteria cocci in
chains in the sample.
2. S.pneumonia
• These are commonly seen in the nasopharynx
of healthy persons.
• It will not cause any illness itself unless a
viral infection or other factors.
• Morphology They are gram-positive cocci.
• Measures 0.5 – 1.25 µm Non-motile and
nonsporting Capsulated Small, oval-shaped
cells arranged in pairs and short chains.
• S. Pneumococci are frequently isolated from
samples such as sputum, blood, wound, CSF
Culture s.pneumonia
• Culture requires blood or chocolate

agar.
• Growth improved by 5-10% CO2.
• Facultative anaerobes.
• Best growth achieved temperature 25
- 40⁰C.
• Colonies are surrounded by greenish
hemolysis under aerobic conditions
and clear under anaerobic conditions.
3. S. Viridians
• It is alpha-hemolytic streptococci.
• It is oval in shape and found in short chains.
• It has six species groups (viridans group); S. mutans, S. oralis, S. salivarus S.
sanguis, S. milleri, S. mitis
• Found in gums and teeth, oral cavity, and also in the nasopharynx, genital
tract, and skin. Not very invasive; dental or surgical procedures facilitate
entrance.
• It causes dental caries, sepsis, and endocarditis.
• Persons with preexisting heart conditions should receive prophylactic
antibiotics before surgery or dental procedures
4. S. Faecalis
• It is also known as enterococci.

• It is a Gram-positive
• Oval cocci in pairs or short chains.
• Identified in McConkey agar. Colonies are magenta in color and pinpoint.
• It can grow in the range of 10 to 45°C and survive at temperatures of 60°C for 30 min.
• It is a non-motile, facultative anaerobic microbe.
• It ferments glucose and does not produce catalase.
• It is associated with urinary tract infections, biliary tract infections, septicemia,
endocarditis, and intra-abdominal abscess.
• Aminoglycosides are the drug of choice.
Biochemical test
1. Catalase test
• Principle: tests for the enzyme catalase
H2O2 2 H2O + O2
• Procedure
• Smear a colony of the organism to a slide.
• Add drop H2O2( hydrogen peroxide) onto
smear
• All Streptococcus ssp give a negative result
for the catalase test.
• differentiation between staphylococcus ssp
and streptococcus ssp.
2. Bile Esculin hydrolysis
• Ability to grow in 40% bile and hydrolyze Esculin are

features of streptococci that possess Group D antigen.
• The organism can hydrolyze esculin and produce black color
in the media.
• differentiation between Enterococcus faecalis
(positive)and streptococcus pyogenic (negative)
3. Bacteriocin test
• used to determine the effect of a small amount of bacitracin

(0.05 IU not higher) on an organism.
• Resistance differentiates S. pyogenes (sensitive) from
similarly B-hemolytic S. agalactiae (resistant).
4. Optochin test
• Optochin test: S. pneumonia strains are sensitive to

optochin.
• used to differentiate (S. pneumoniae) sensitivity from
other alpha-hemolytic streptococci alpha (s. viridans) is
resistant.
\
5. CAPM test (Christie, Atkins, and Munch – Peterson) test
• Identification of group B streptococci. (S. agalactiae) The CAMP Test
The laboratory identification of Group B hemolytic streptococci.
• Streptococcus agalactiae produce CAMP factor (a diffusible extracellular
protein) that synergistically acts with the beta–lysin of Staphylococcus
aureus and enhances the lysis of RBC.
• The CAMP test is performed by making a single streak of the
streptococcus perpendicular to a strain of Staphylococcus aureus that is
known to produce ß - lysin.
• The two streak lines must not touch one another.
• The inoculated plate must be incubated at room temperature.
6.The Hippurate test
• Is a qualitative procedure for determining the ability of bacteria to

enzymatically hydrolyze sodium Hippurate into glycine and benzoic acid
by the action of the hippuricase enzyme present in bacteria.
• Oxidizing agent ninhydrin is used as an indicator.
• Ninhydrin reacts with glycine to form a deep blue or purple color
Principle Hippurate test
• Suspend a loop full of bacteria in 0.5 ml of sodium Hippurate

solution. Incubate the suspension at 37ºC for 2 h in a water bath.
• Then carefully add 0.2 ml of ninhydrin solution without mixing.
• Incubate for another 10 min at 37ºC before reading the result.
• Differentiation between s.agalactiae positive result and other
-alpha hemolysis(s. pneumonia) give a negative result.
7. The Quelling test
• The Quelling reaction to differentiate S. pneumoniae from S. viridians'
Quelling reaction (capsular swelling reaction) is a biochemical reaction
in which antibodies bind to the capsule of Streptococcus.
• Pneumonia (antibody-antigen reaction), and thus allow them to be
visualized under a microscope.
• The protocol has three main steps:
• 1) preparation of a bacterial cell suspension
• 2) mixing of cells and antisera on a glass slide
• 3) Read the Quelling reaction using a microscope.
• If the reaction is positive, the capsule becomes opaque and appears to
enlarge.
Microscop examination for streptococcuc pyogenic

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Pyogenic bacteria

Classification system is based on hemolysis reactions

• The organism is cultured on

• The diagnosis is suggested

• Culture requires blood or chocolate

• It is also known as enterococci.

• Ability to grow in 40% bile and hydrolyze Esculin are

• used to determine the effect of a small amount of bacitracin

• Optochin test: S. pneumonia strains are sensitive to

• Is a qualitative procedure for determining the ability of bacteria to

• Suspend a loop full of bacteria in 0.5 ml of sodium Hippurate

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