Lab. No. 3. 1. Microscopical Appearance:(Gram’s Stain) Gram’s +ve Cocci Irregular ClustersChains...

Lab. No. 3

1. Microscopical Appearance:(Gram’s Stain)1. Microscopical Appearance:(Gram’s Stain)

Gram’s +ve Cocci

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

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

Gram’s +ve Cocci

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

Catalase TestCatalase Test

Catalase +ve Catalase -ve

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

Catalase TestCatalase TestResults:

Positive test: rapid appearance of gas bubbles.

Staphylococci or Micrococci

Catalase –ve

Streptococci

Catalase +ve

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.

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


Example: Example: Strept. Pyogenes Strept. Pyogenes (group A (group A ββ-hemolytic-hemolytic Strept.)Strept.)

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.


Example: Example: Strept. Pneumonia, viridans Strept. Pneumonia, viridans Streptococci.Streptococci.

ββ-hemolytic Sterptococci:-hemolytic Sterptococci:

αα-hemolytic Sterptococci:-hemolytic Sterptococci:


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.


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:



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.

B



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

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:



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.



Optochin sensitive

S.pneumoniae

Optochin resistant

Viridans Streptococci

Results:Positive test: inhibition zone more than 15 mm in diameter.


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.



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



Results:Positive test: Visible clearance of the turbid culture.

Visible clearance

S.Pneumoniae

Remain turbid

Viridans Streptococci

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.




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




Results:

Enterococcus faecalis

Growth of pink colonies

No Growth

Other Strain of Sterptococci

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

Identification of Identification of 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




γγ-hemolytic -hemolytic SterptococcSterptococc

ii

Inhibition zone

Bacitracin

sensitiveS.pyogenes

No zone

Bacitracin

resistantNon group

A β-hemolytic

Strept.

- -

BacitraciBacitracin n sensitivitsensitivityy


ββ--hemolytic hemolytic SterptococSterptococ

cici


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)


ββ--hemolytic hemolytic SterptococSterptococ

cici


γγ--hemolytic hemolytic SterptococSterptococ

ciciGrowth on MacConkey’s Agar

No Growth No Growth Growth of pink colonies

Enterococcus faecalis

Lab. No. 3. 1. Microscopical Appearance:(Gram’s Stain) Gram’s +ve Cocci Irregular ClustersChains...

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