lab. no. 3. 1. microscopical appearance:(gram’s stain) gram’s +ve cocci irregular clusterschains...
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Lab. No. 3
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1. Microscopical Appearance:(Gram’s Stain)1. Microscopical Appearance:(Gram’s Stain)
Gram’s +ve Cocci
Irregular Clusters Chains or PairsTetrads
Staphylococci Micrococci Streptococci
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Identification of Identification of SterptococciSterptococci Gram’s Stain:Gram’s Stain:
Gram’s +ve cocci arranged in: Gram’s +ve cocci arranged in:
chainschains pairs (pairs (S. PneumoniaS. Pneumonia))oror
Macroscopical Examination:Macroscopical Examination:Transparent pin point coloniesTransparent pin point colonies
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Gram’s +ve Cocci
Irregular Clusters Chains or PairsTetrads
Staphylococci Micrococci Streptococci
Catalase TestCatalase Test
Catalase +ve Catalase -ve
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Catalase TestCatalase Test Differentiative test to separate Differentiative test to separate Staphylococci Staphylococci
and Micrococci which are catalase +veand Micrococci which are catalase +ve from from Sterptococci which are catalase –ve.Sterptococci which are catalase –ve.
H2O2
Catalase enzymeH2o + O2
Air bubbles
Principle:
Procedure:
1
3 H2O2
2
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Catalase TestCatalase TestResults:
Positive test: rapid appearance of gas bubbles.
Staphylococci or Micrococci
Catalase –ve
Streptococci
Catalase +ve
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Growth on Blood AgarGrowth on Blood Agar
Sterptococci are divided into three Sterptococci are divided into three main groups accorging to its action main groups accorging to its action on erythrocytes:on erythrocytes:
1.1. ββ-hemolytic Sterptococci.-hemolytic Sterptococci.
2.2. αα-hemolytic Sterptococci.-hemolytic Sterptococci.
3.3. γγ-hemolytic Sterptococci.-hemolytic Sterptococci.
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ββ-hemolytic Sterptococci:-hemolytic Sterptococci:
It causes It causes complete hemolysiscomplete hemolysis to to RBCs leading to formation of clear zone RBCs leading to formation of clear zone around the coloniesaround the colonies
Growth on Blood AgarGrowth on Blood Agar
Example: Example: Strept. Pyogenes Strept. Pyogenes (group A (group A ββ-hemolytic-hemolytic Strept.)Strept.)
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αα-hemolytic Sterptococci:-hemolytic Sterptococci:
It causes:It causes:1. 1. Partial hemolysisPartial hemolysis to RBCs. to RBCs.2. Act enzymatically on blood pigment leading 2. Act enzymatically on blood pigment leading to to green discolourationgreen discolouration around the around the colonies.colonies.
Growth on Blood AgarGrowth on Blood Agar
Example: Example: Strept. Pneumonia, viridans Strept. Pneumonia, viridans Streptococci.Streptococci.
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ββ-hemolytic Sterptococci:-hemolytic Sterptococci:
αα-hemolytic Sterptococci:-hemolytic Sterptococci:
Growth on Blood AgarGrowth on Blood Agar
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It has It has no effectno effect on RBCs on RBCs
(Non hemolytic Sterptococci)(Non hemolytic Sterptococci) Example: Example: Enterococcus Enterococcus
faecalisfaecalis
γγ-hemolytic Sterptococci:-hemolytic Sterptococci:
ββ-hemolytic Sterptococci.-hemolytic Sterptococci.αα-hemolytic Sterptococci.-hemolytic Sterptococci.
γγ-hemolytic Sterptococci.-hemolytic Sterptococci.
Growth on Blood AgarGrowth on Blood Agar
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Growth on Blood AgarGrowth on Blood Agar
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ββ-hemolytic -hemolytic SterptococciSterptococci
Definitive test to differentiate betweenDefinitive test to differentiate between
S.PyogenesS.Pyogenes & Non group A & Non group A ββ-hemolytic Streptococci-hemolytic Streptococci
A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth of S.pyogenes giving a zone of inhibition around the disc
Principle:
Bacitracin Sensitivity Test:Bacitracin Sensitivity Test:
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ββ-hemolytic -hemolytic SterptococciSterptococci
Bacitracin Sensitivity Test:Bacitracin Sensitivity Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
B
2. Aseptically apply Bacitracin disc onto the center of the streaked area.3. Incubate the plate at 35oC for 18 hrs.
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B
ββ-hemolytic -hemolytic SterptococciSterptococci
Bacitracin Sensitivity Test:Bacitracin Sensitivity Test:
Results:Positive test: any zone of inhibition around the disc.
B
Bacitracin Resistant Bacitracin Sensitive
S.PyogenesS.PyogenesNon group A Non group A ββ-hemolytic Streptococci-hemolytic Streptococci
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αα-hemolytic -hemolytic SterptococciSterptococci
Definitive test to differentiate betweenDefinitive test to differentiate between
S.Pneumoniae & Viridans StreptococciS.Pneumoniae & Viridans Streptococci
S.Pneumoniae is inhibited by less than 5 µg/ml Optochin reagent giving a zone of inhibition more than 15 mm in diameter.
Principle:
1. Optochin Sensitivity Test:1. Optochin Sensitivity Test:
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αα-hemolytic -hemolytic SterptococciSterptococci
1. Optochin Sensitivity Test:1. Optochin Sensitivity Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
O
2. Aseptically apply Optochin disc onto the center of the streaked area.3. Incubate the plate at 35oC for 18 hrs.
4. Accurately measure the diameter of the inhibition zone around the disc.
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αα-hemolytic -hemolytic SterptococciSterptococci
1. Optochin Sensitivity Test:1. Optochin Sensitivity Test:
Optochin sensitive
S.pneumoniae
Optochin resistant
Viridans Streptococci
Results:Positive test: inhibition zone more than 15 mm in diameter.
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αα-hemolytic -hemolytic SterptococciSterptococci
2. Bile Solubility Test:2. Bile Solubility Test:
Principle:
S.Pneumoniae produce a self-lysing enzyme to depress the growth of old colonies. The presence of bile salt accelerate this process.
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αα-hemolytic -hemolytic SterptococciSterptococci
2. Bile Solubility Test:2. Bile Solubility Test:
Procedure:
3. Incubate at 37oC for 15 min .
4. Observe for the visible clearing of the turbid culture.
10 ml broth culture of the test organism
1
Add 1 ml 10% bile salt solution 2
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αα-hemolytic -hemolytic SterptococciSterptococci
2. Bile Solubility Test:2. Bile Solubility Test:
Results:Positive test: Visible clearance of the turbid culture.
Visible clearance
S.Pneumoniae
Remain turbid
Viridans Streptococci
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γγ-hemolytic -hemolytic SterptococciSterptococci
Definitive test for Definitive test for Enterococcus faecalisEnterococcus faecalis
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Principle:
MacConkey’s agar is a selective medium for Gram’s –ve bacteria.It contains bile salt and crystal violet to inhibit the growth of Gram’s +ve bacteria.Enterococcus faecalis is the only Streptococcus species which can grow on MacConkey’s agar giving pink colonies.
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γγ-hemolytic -hemolytic SterptococciSterptococci
Definitive test for Definitive test for Enterococcus faecalisEnterococcus faecalis
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Procedure:
1. Inoculate MacConkey’s agar plate with the test organism by streaking.
2. Incubate the plate at 35oC for 24 hrs.
Flame & Cool
Flame & Cool
Flame & Cool
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γγ-hemolytic -hemolytic SterptococciSterptococci
Definitive test for Definitive test for Enterococcus faecalisEnterococcus faecalis
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Results:
Enterococcus faecalis
Growth of pink colonies
No Growth
Other Strain of Sterptococci
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Practical WorkPractical Work
1.1. Gram’s Stain (spots)Gram’s Stain (spots)
2.2. Catalase testCatalase test
3.3. Blood agar plate.Blood agar plate.
4.4. Bacitracin & Optochin Bacitracin & Optochin Sensitivity.Sensitivity.
5.5. MacConkey’s agar plate.MacConkey’s agar plate.
O
B
Flame & Cool
Flame & Cool
Flame & Cool
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Identification of Identification of SterptococciSterptococci
ββ-hemolytic -hemolytic SterptococciSterptococci
αα-hemolytic -hemolytic SterptococciSterptococci
γγ-hemolytic -hemolytic SterptococcSterptococc
ii
Gram’s Gram’s StainStain
Catalase Catalase testtest
Growth Growth on blood on blood agaragar
Gram’s +ve Cocci arranged in chains
- ve - ve- ve
Complete hemolysis
1. Partial hemolysis
2. Green pigmentation
Non hemolytic
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Growth on Blood AgarGrowth on Blood Agar
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Identification of Identification of SterptococciSterptococci
ββ-hemolytic -hemolytic SterptococciSterptococci
αα-hemolytic -hemolytic SterptococciSterptococci
γγ-hemolytic -hemolytic SterptococcSterptococc
ii
Inhibition zone
Bacitracin
sensitiveS.pyogenes
No zone
Bacitracin
resistantNon group
A β-hemolytic
Strept.
- -
BacitraciBacitracin n sensitivitsensitivityy
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Identification of Identification of SterptococciSterptococci
ββ--hemolytic hemolytic SterptococSterptococ
cici
αα-hemolytic -hemolytic SterptococciSterptococci
γγ--hemolytic hemolytic SterptococSterptococ
ciciInhibition zone >15mm
Optochin
sensitive
S.pneumoniae
No zone or
<15mm Optochi
n resistan
t
Viridans Streptococc
i
Optochin Optochin sensitivitsensitivityy
Bile Bile SolubilitSolubilityy
Visibile clearance
Remain turbid
-
-
-
-
(+ve) (-ve)
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Identification of Identification of SterptococciSterptococci
ββ--hemolytic hemolytic SterptococSterptococ
cici
αα-hemolytic -hemolytic SterptococciSterptococci
γγ--hemolytic hemolytic SterptococSterptococ
ciciGrowth on MacConkey’s Agar
No Growth No Growth Growth of pink colonies
Enterococcus faecalis