1. PHT313
Lab. No. 3

2. Identification of Gram's +ve Cocci
Microscopical Appearance:(Gram’s Stain)
Gram’s +ve Cocci
Irregular Clusters
Chains or Pairs
Tetrads
Staphylococci
Micrococci
Streptococci

3. Catalase Test Catalase +ve Catalase -ve Staphylococci Micrococci
Gram’s +ve Cocci
Irregular Clusters
Chains or Pairs
Tetrads
Staphylococci
Micrococci
Streptococci
Catalase +ve
Catalase -ve

4. Catalase Test
Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve.
H2O2
Catalase enzyme
H2o O2
Air bubbles
Principle:
Procedure: 1 3 2

5. Catalase Test Results: Positive test: rapid appearance of gas bubbles.
Staphylococci or Micrococci
Catalase –ve
Streptococci
Catalase +ve

7. Identification of Sterptococci

8. Identification of Sterptococci
1- Microscopical Examination (Morphology):
Gram’s +ve cocci arranged in:
chains
pairs (S. Pneumonia)
Nonmotile, nonsporeforming.

9. 2-macroscopical examination (cultural characteristics):
Growth requires enriched medium containing blood or serum
e.g., sheep blood agar
→Transparent pin point colonies
Catalase negative.
3-Biochemical reactions:

10. Classification of streptococci:
Current classification of streptococci depends on 6 features; some of them are:
Hemolytic reactions on sheep blood agar
Lancefield serological groups (A-W with the exception of I and J).
N.B: S.pyogenes, S.agalatiae & S.pneumoniae are the most important species.

11. Growth on Blood Agar
Sterptococci are divided into three main groups accorging to its action on erythrocytes:
β-hemolytic Sterptococci.
α-hemolytic Sterptococci.
Non-hemolytic streptococci

12. β-hemolytic Sterptococci. α-hemolytic Sterptococci.
non-hemolytic Sterptococci.
β-hemolytic Sterptococci.
α-hemolytic Sterptococci.

13. α-hemolytic Sterptococci:

14. β-hemolytic Sterptococci:
It causes complete hemolysis to RBCs (caused by hemolysins) leading to formation of clear zone around the colonies
Example: S. pyogenes (group A β-hemolytic Strept.), S.agalactiae.

15. β-hemolytic Sterptococci
Definitive test to differentiate between
S.pyogenes & Non group A β-hemolytic streptococci
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

16. Bacitracin Sensitivity Test:
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

17. Results: Positive test: any zone of inhibition around the disc.
Bacitracin Sensitive
Bacitracin Resistant
Non group A β-hemolytic Streptococci
S.pyogenes

18. Antibiotic sensitivity
Penicillin:
still uniformly effective in treatment of S.pyogenes disease.

20. α-hemolytic Sterptococci:
It causes:
1. Zone of greenish discolouration around the colonies.
2. It is due to peroxidase release that changes haemoglobin to methemoglobin
Example: S.pneumonia, viridans streptococci.

21. α-hemolytic Sterptococci
Definitive test to differentiate between
S.Pneumoniae & Viridans Streptococci
1- Optochin sensitivity.
2- Bile solubility test.

22. 1. 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.

23. Procedure: 1. Inoculate blood agar plate with the test organism.
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. O

24. Viridans Streptococci
Results:
Positive test: inhibition zone more than 15 mm in diameter.
Optochin sensitive
S.pneumoniae
Optochin resistant
Viridans Streptococci

25. 2. 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.

26. 10 ml broth culture of the test organism
Procedure:
Add 1 ml 10% bile salt solution 2
10 ml broth culture of the test organism 1
3. Incubate at 37oC for 15 min .
4. Observe for the visible clearing of the turbid culture.

27. Results: Positive test: Visible clearance of the turbid culture.
Remain turbid
S.Pneumoniae
Viridans Streptococci

28. Antibiotic sensitivity
1-Penicillin-resistant pneumococci:
reported all over the world.
2- Vancomycin + extended-spectrum cephalosporins e.g., ceftriaxone
→ used in serious infections; e.g; meningitis.

30. Effect on blood agar: 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.
Effect on blood agar:
It has no effect on RBCs (Non hemolytic)

31. Definitive test for Enterococcus faecalis
Growth on MacConkey’s agar:
Definitive test for Enterococcus faecalis
Principle:
MacConkey’s agar is a selective medium for Gram’s –ve bacteria.
It contains bile salts and crystal violet to inhibit the growth of Gram’s +ve bacteria.
Enterococcus faecalis is the only gram-positive cocci which can grow on MacConkey’s agar giving pink colonies.

32. Growth on MacConkey’s agar:
Procedure:
1. Inoculate MacConkey’s agar plate with the test organism by streaking.
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.

33. Results: Sterptococci Enterococcus faecalis No Growth
Growth of pink colonies
Sterptococci
Enterococcus faecalis

34. Antibiotic sensitivity
Enterococci can be very resistant to antibiotics.
1- Penicillin + aminoglycosides
2- Vancomycin → used for penicillin-resistant strains.
3- VRE (vancomycin-resistant enterococci)→
ttt: Linezolid.

36. Practical Work Gram’s Stain (spots) Catalase test Blood agar plate.
Bacitracin & Optochin Sensitivity.
MacConkey’s agar plate. B O
Flam & Cool

37. Identification of Sterptococci
β-hemolytic Sterptococci
α-hemolytic Sterptococci
Enterococci
Gram’s Stain
Catalase test
Growth on blood agar
Gram’s +ve Cocci arranged in chains
- ve
- ve
- ve
Complete hemolysis
Non
hemolytic
Greenish discoloration

38. Identification of Sterptococci
β-hemolytic Sterptococci
α-hemolytic Sterptococci
Enterococci
Bacitracin sensitivity
Inhibition zone
No zone
Bacitracin sensitive
Bacitracin resistant - -
S.pyogenes
Non group A β-hemolytic Strept.

39. Identification of Sterptococci
β-hemolytic Sterptococci
α-hemolytic Sterptococci
Enterococci
Optochin sensitivity
Inhibition zone >15mm
No zone or <15mm - -
Optochin sensitive
Optochin resistant
Bile Solubility -
Visibile clearance
Remain turbid -
(+ve)
(-ve)
S.pneumoniae
Viridans Streptococci

40. Identification of Sterptococci
β-hemolytic Sterptococci
α-hemolytic Sterptococci
Enterococci
Growth on MacConkey’s Agar
No Growth
No Growth
Growth of pink colonies
Enterococcus faecalis

41. Thank you