cocci Practical No 9 GENERAL CHARACTERISTIC Gve cocci arrange in chains or pairs Some strains are capsulated Majority are facultative anaerobic few are obligatory anaerobic ID: 927005
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Slide1
Genus Streptococcus
Gram positive, Catalase negative cocci
Practical
No. 9
Slide2GENERAL CHARACTERISTIC:*
G+ve cocci , arrange in chains or pairs.* Some strains are capsulated* Majority are facultative anaerobic, few are obligatory anaerobic.* Catalase –ve
* Non motile.* Non spore forming* Fastidious microorganism
Slide3Members of the genus
Streptococcus are responsible for disease as well as being part of the
normal flora of humans. Among the diseases caused are:bacterial pneumonia
meningitis
Tonsillitis
Endocarditis
scarlet fever
Erysipelas
urinary tract infections.
Streptococcus
species are also
found
normally in the
mouth
and on the
skin surface
.
The streptococci are classified by
two major methods
:
1. hemolytic
activity
2. serologic
classification of Lancefield.
Slide4Slide5When grown on sheep blood agar, streptococci display one
of three types of hemolysis of the red blood cells in the agar.
Alpha hemolysis--The red blood cells in
the media are partially digested producing a greening of the
agar.
Beta
hemolysis-
-The red blood cells in the media are completely digested producing a clearing of the
agar.
Gamma
hemolysis-
-No change is noted in the agar. The red blood cells are not lysed.
Streptococcus
pyogenes
always beta
hemolylic
Streptococcus
agalactiae
usually beta hemolytic
Streptococcus
pneumoniae
and
Viridans
streptococci
are always alpha
hemolylic
Alpha hemolysis
on
blood
agar
(partial hemolysis).
Beta hemolysis
on blood agar
(complete hemolysis).
No hemolysis on blood agar
Hemolytic Streptococci
Alpha hemolysis (green discoloration)
(Beta hemolysis)
clear zone around colonies
Slide7DISEASES S. pyogenes
Slide8Lab dx.Specimens: sputum, throat swab, nasopharyngeal swab, blood, CSF…etc.
Gram stain: G+ve cocci, arrange in chains.Culture: on blood agar
pinpointed, Grayish white, translucent, matte or glossy colonies with large zone of β- hemolysis.
S.pyogenes on blood agar (beta hemolysis)
Slide9Bacitracin Susceptibility tests
Commercially available paper disks saturated with a solution containing Bacitracin will inhibit about 97% of all strains of Group A streptococci; other groups of beta-hemolytic streptococci will not be affected.
Streptococcus pyogenes
group
A is inhibited
by
small amount of
bacitracin in the disk other B hemolytic streptococci usually are not.Streptococcus pyogenes Strept. pyogenes Strept. agalactiae
Slide10A blood test that checks for a strep infection
. When you come into contact with harmful bacteria, your body produces antibodies to defend itself against these bacteria. Your body produces antibodies specific to the bacteria they fight.The
antistreptolysin O (ASO) titer testThe
ASO titer test
measures antibodies produced by your body in response to a toxin known as
streptolysin
O.
Streptolysin
O is a toxin produced by group A
Streptococcus (GAS) bacteria. Your body makes the antistreptolysin O antibodies when you have a strep infection caused by GAS bacteria.
Slide11A positive test usually is >200 units/mL, but normal ranges vary from laboratory to laboratory and by age. Normal
levels is
less than 160 unit per
milliliter.
Slide12S. agalactia
(Group B β- hemolytic):
Slide13Identification 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 act 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 - l
ysin. The two streak lines must not touch one another. The inoculated plate must be incubated with room temperature. Any Bacitracin-negative, CAMP-positive, bile-
esculin
-negative streptococcus can be reported as: Group B streptococcus
CAMP (Christie, Atkins and Munch – Peterson) test
Slide14CAMP test
Slide15The CAMP Test
Slide16The Hippurate test:
Is a qualitative procedure for determining the ability of bacteria to enzymatically hydrolyze sodium
hippurate into glycine and benzoic acid by action of hippuricase enzyme present in
bacteria
.
An
oxidizing agent
ninhydrin
is used as an indicator
.Ninhydrin react with glycine to form a deep blue or purple color.
Slide17Slide18Slide19Non-beta hemolytic (alpha- and non hemolytic) streptococci
The non-beta hemolytic streptococci include the S.
pneumoniae, Viridans
streptococci
, Group D streptococci and enterococci.
Identification of group D Streptococci:
Lancefield Group D streptococci are divided into two groups
:
(1)
Enterococci (2) Non enterococci. Enterococcus
faecalis and Enterococcus faecium are the species of Enterococcus
.
Strep
.
bovis
and
Strep.
equinus
are species of "Group-D streptococci, not Enterococci"
Slide20Bile-Esculin Hydrolysis Test:
The purpose of this test is to determine the ability of an organism to hydrolyze the glycoside esculin to
esculatin and glucose in the presence of bile (10 - 40%). This test aids in the differentiation of group D streptococci from other "not group D streptococci". Procedure:
1. Inoculate the organism to be tested into the bile
esculin
medium. Incubate at 37
o
C for 24
hours.
Positive
Test: Presence of a black to dark brown color on the slant -(Enterococcus faecalis) Negative Test: No blackening of the medium - (Streptococcus agalactiae or Streptococcus pyogenes)
Slide21Bile
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.
Bile-Esculin Hydrolysis Test:
Slide22Viridans streptococcus
Slide23S. pneumoniae
(Pneumococci,
Diplococcus pneumoniae)
Capsular polysaccharide
Gram +VE diplococcus
Slide24S.
pneumoniae
Slide25Identification of
S. pneumonia and viridans
streptococci The alpha-hemolytic streptococci, or viridans
(
green
)
group, are
normal inhabitants of the
mouth,
nasopharynx
, and respiratory tract. Alpha-hemolytic streptococci may be isolated by blood culture in cases of bacterial endocarditis. Specimen sputum, blood, throat swab, ear swab, CSF
Slide261-Gram stain of Streptococcus
pneumoniae and
viridans Streptococci: Gram stain of
S.
pneumoniae
and a
viridans
streptococcus obtained from the
blood
agar
plate.
Capsular polysaccharide
Gram +VE
diplococcus
Strept
.
pneumoniae
gram-positive
diplococci
. Notice the characteristic elongated shape of the
diplococci
Slide272-The
Quellung reaction to differentiate S. pneumoniae
from viridans streptococci.
The
Quellung
reaction
(capsular swelling reaction)
is a
biochemical
reaction in which antibodies bind to the capsule of Streptococcus pneumoniae, Klebsiella
pneumoniae, Neisseria meningitidis and
Haemophilus
influenzae
and thus allow them to be visualized under a microscope
.
If the reaction is positive, the capsule becomes
opaque
and appears to enlarge
.
Slide28Optochin disc for S. pneumococci
3. Susceptibility test
Optochin
“P”
disk
Differentiates
S.
pneumoniae
from other
alpha-hemolytic streptococci (
Viridans
group
)
S. pneumococci
Slide29To determine
the ability of bacterial cells to lyse in the presence of bile salts.
4. Bile solubility test
The
test
is primarily used to
differentiate
bile
soluble
Streptococcus pneumoniae from bile insoluble alpha- hemolytic streptococci.
Slide305-Inulin fermentation test
Pneumococci
there will be a change in the color of the media due to acid formation.
Procedure
:
Inoculate a test tube containing nutrient broth + inulin + the organism, (red
color).
Then incubate over night, if the
color
remains red the organism is
Viridans
Streptococcus, If the organism has fermented the inulin resulting in acid production. The acid lowers the pH of the media and the indicator (phenol red) turns yellow.
Slide31Observe for the presence of a
yellow
color (center tube), indicative of acid formation from the fermentation of inulin.
Red
indicates no inulin fermentation (left tube; right tube is
uninoculated
control).
Inulin
fermentation test
Slide32CHARACTER
PNEUMOCOCCI
VIRIDANS STREPTOCOCCIMorphology
Ovoid or
lanceolate
diplococci
Rounded
cocci
in short or long chains.
Capsule
PresentAbsentOptochin sensitivity +ve-veBile solubility
+ve-veCapsular swelling test (Quelling reaction
)
+ve
-ve
Virulence in mice
+
ve
-
ve
Differentiation of alpha and non-hemolytic streptococci
Slide33Hemolysis Aids in Classification
Slide34Thank you