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PHT 313
Lab (3)
Streptococci
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Staphylococci
Streptococci
Enterococci
Gram’s -ve
Bacilli
Cocci
Corynbacterium
Clostridum
Bacillus
Neisseria
Bacilli
Enterobacteriaceae
Pseudomonas.
Streptococci
Classification Schemes (Taxonomy):
Based on Hemolysis on blood agar plates
– β-hemolytic : S. pyogenes
– α-hemolytic : Viridans streptococci (incomplete,)
– γ-hemolytic : Enterococci (no hemolysis)
β-hemolytic Sterptococci
α-hemolytic Sterptococci
Based on O₂ requirements
Aerobic streptococci
Anaerobic streptococci (Peptostreptococci):

These are normal flora in bowel and vagina.
 They are often participate in mixed anaerobic
infections (Abscesses) in the abdomen, brain, pelvis,
lung and uterus.

They
are
sensitive
to
penicillin
and
metronidazol.
Based on serology: Lancefield grouping based on
group specific carbohydrate antigens.
Lancefield serological groups (A-W with the exception of I & J).
Major pathogens:
Group A Streptococci: Streptococcus pyogenes
Group B Streptococci: S. agalactiae
Enterococcus faecalis was previously classified as Group
D streptococci .
N.B. Streptococci lacking group-specific antigens
(Viridans streptococci & S.pneumoniae )
Gamma or alpha-hemolysis
streptococci
Microscopic morphology:
- Gram positive cocci,
- arranged in chains or pairs,
- non-motile,
- non-spore forming.
Staphylococcus
Streptococcus
Culture Characters:
Environment: Facultative anaerobes
Temp.: 37 °C
pH: 7.2
Media: Blood agar (Fastidious organism):
Complex nutritional requirements, Require blood- or serumenriched media;
1-β-hemolytic Sterptococci (S. pyogenes ).
2-α-hemolytic Sterptococci (S.pneumonia, viridans streptococci).
3-Non-hemolytic streptococci.
β-hemolytic
α-hemolytic
Non-hemolytic
B.R.:
Catalase: - ve
Fermentation of glucose without the production of gas,
produces mainly lactic acid, ethanol, acetate
Blood culture
Definition:
It is microbiological culture of blood. It is employed to
detect infections that are spreading through the
bloodstream (Bacteremia & Septicemia). A sample of
blood is put in a special laboratory preparation and is
incubated in a controlled environment for 1 to 7 days.
Uses:
The detection of microorganisms in a patient’s blood has diagnostic
and prognostic importance. Blood cultures are done to detect the
presence of bacteria or yeasts, which may have spread from a
specific site in the body into the bloodstream.
Procedures:
Blood is drawn from a person and put directly into a blood culture
bottle containing a nutritional broth (Aerobic and anaerobic). After
the laboratory receives the blood culture bottle, several processes
must be completed:
• provide an environment for the bacteria to grow
• detect the growth when it occurs
• identify the bacteria that grow
• test the bacteria against certain antibiotics to determine which
antibiotic will be effective
Collecting blood cultures: by vein puncture
Fill the anaerobic bottle first, so that any air in the syringe
is not inoculated into that bottle.
Blood culture bottles should be placed in any ward
incubator until collected by the porters for transport to
the laboratory.
The type of system used may
be,
manual or automated
Manual technique
The bottles are placed in an incubator and kept at body
temperature.
They are watched daily for signs of growth, including cloudiness or a
color change in the broth, gas bubbles, or clumps of bacteria.
When there is evidence of growth, the laboratory does a gram stain
and a subculture.
To do the gram stain, a drop of blood is removed from the bottle
and placed on a microscope slide.
To do the subculture, a drop of blood is placed on a culture plate,
spread over the surface, and placed in an incubator.
If there is no immediate visible evidence of growth in the bottles,
the laboratory looks for bacteria by doing gram stains and
subcultures. These steps are repeated daily for the first several days
and periodically after that.
Automated technique
The blood culture machine (BACTEC 9000) are designed
for the rapid detection of microorganisms in clinical
specimens. The sample to be tested is inoculated into the vial
which is entered into the Blood culture instrument for
incubation and periodic reading. Each vial contains a sensor
which responds to the concentration of CO2 produced by the
metabolism of microorganisms or the consumption of oxygen
needed for the growth of microorgnisms. The sensor is
monitored by the instrument every ten minutes for an increase
in its fluorescence, which is proportional to the increasing
amount of CO2 or the decreasing amount of O2 present in the
vial.
A positive reading indicates the presumptive presence of
viable microorganisms in the vial.
When a bottle is registered as “positive”, it is removed from the
machine and a portion is placed on a glass slide, gram-stained
and examined under the light microscope.
It’s also used to inoculate agar plates on which most bacteria
will grow. The inoculum is also spread over a susceptibility agar
plate, on which are placed antibiotic discs appropriate for the
organism seen on the gram stain. This will give the “direct”
sensitivity results.
Gram’s +ve Cocci
Catalase test
+ ve
Staphylococci
- ve
Streptococci
Streptococci
Growth on Blood agar
α-Haemolysis
β-Haemolysis
Optochin Test
Bacitracin Test
S
S.pneumoniae
R
Viridans
Streptococci
S
S. pyogenes
R
S. agalactiae
β-hemolytic Sterptococci
Example:
S. pyogenes (group A β-hemolytic Strept.)
S.agalactiae (group B β-hemolytic Strept.
= Non group A β-hemolytic strept.)
To differentiate between S.pyogenes & Non group
A β-hemolytic streptococci,…………………………..
Bacitracin Sensitivity Test:
Bacitracin Sensitivity Test
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
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 35oC for 18 hrs.
B
Results:
Positive test: any zone of inhibition around the disc.
B
Bacitracin Sensitive
S.pyogenes
B
Bacitracin Resistant
β-hemolytic Streptococci
Non group A
CAMP test
CAMP reaction: The hemolysis is enhanced in the vicinity
of the ß -lysin producing S.aureus (Arrow head-shaped
zone of ß-hemolysis).
Group B streptococci (S.agalactiae. ) produce a factor
that increases beta hemolysis of an S. aureus indicator
strain.
CAMP+ve
S.agalactiae
CAMP-ve
α-hemolytic Sterptococci
It causes:
1. Zone of greenish discolouration around the colonies.
2. It is due to peroxidase release.
Example: S.pneumonia, viridans streptococci.
To differentiate between S.pneumonia, viridans
streptococci, ………
1) Optochin sensitivity.
2) Bile solubility test
Optochin Sensitivity Test
Principle:
S.pneumoniae is inhibited by less than 5µg/ml Optochin
reagent giving a zone of inhibition more than 15 mm in
diameter.
Procedure:
1. Inoculate blood agar plate with the test organism.
2. Aseptically apply Optochin disc onto the
center of the streaked area.
O
3. Incubate the plate at 35oC for 18 hrs.
4. Accurately measure the diameter of the
inhibition zone around the disc.
Results:
Positive test: inhibition zone more than 15 mm in diameter.
Optochin sensitive
S.pneumoniae
Optochin resistant
Viridans Streptococci
Bile Solubility Test:
Principle:
S.pneumoniae produces a self-lysing enzyme to
depress the growth of old colonies. The presence of
bile salt accelerate this process.
Procedure:
1) Add 1 ml 10% bile salt solution to 10 ml broth culture of the test
organism
2) Incubate at 37oC for 15 min
3) Observe for the visible clearing of the turbid culture
Results
Visible clearance
S.Pneumoniae
Remain turbid
Viridans Streptococci
Enterococci
 Gram-positive cocci, arranged singly, in pairs, and in short chains.
 Catalase negative
 Most strains react with Lancefield group D antisera.
 E.faecalis is the most important species.
 It has no effect on RBCs (Non hemolytic)
 Enterococcus faecalis is the only gram-positive cocci which
can grow on MacConkey’s agar giving pink colonies.
Enterococcus faecalis
Identification of Sterptococci
β-hemolytic
Sterptococci
α-hemolytic
Sterptococci
Enterococci
Gram’s
Stain
Gram’s +ve Cocci arranged in chains
Catalase
test
- ve
Growth on
blood agar
Complete
hemolysis
- ve
Greenish
discoloration
- ve
Non
hemolytic
Identification of Sterptococci
β-hemolytic
Sterptococci
Optochin
sensitivity
-
α-hemolytic
Sterptococci
Enterococci
Inhibition zone No zone or
>15mm
<15mm
-
Optochin
sensitive
Bile
Solubility
-
Visibile
clearance
(+ve)
S.pneumoniae
Optochin
resistant
Remain
turbid
(-ve)
Viridans
Streptococci
-
Identification of Sterptococci
β-hemolytic
Sterptococci
Growth on
MacConkey’s
Agar
No Growth
α-hemolytic
Sterptococci
No Growth
Enterococci
Growth of
pink colonies
Enterococcus
faecalis