1. Isolation and Identification of Staphylococci
Shadi Alashi

2. Gram Stain Gram Positive coccus Catalase Oxidase Coagulase_ +
Streptococci
Oxidase _ + R
Macrococci
Staphylococci
Bacitracin
Susceptibility test S
Micrococci
Coagulase _ +
Staphylococcus epidermidis
CNS
Staphylococcus haemolyticus
Staphylococcus aureus
Staphylococcus saprophyticus

3. Gram positive cocci

4. Catalase +ve

5. Oxidase -ve

6. Coagulase test Coagulase CNS Slide method Tube method _ +
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus aureus
CNS
Staphylococcus saprophyticus
Negative
Positive
Positive
Negative
Slide method
Tube method
Coagulase test

7. Non-hemolytic Staphylococcus species: Staphylococcus epidermidis

8. Staphylococcus saprophyticus: non-hemolytic, bright white, creamy colonies

9. Strains of Staphylococcus aureus produce a golden yellow pigment

10. Strains of Staphylococcus aureus not a golden yellow pigment producer

11. Staphylococcus haemolyticus on Blood Agar

12. Coagulase Negative Staph
Staph saprophyticus
Coagulase Negative Staph
Staph heamolyticus
Staph epidermidis
Novobiocin resistance Test:
Procedure:
1. Inoculate blood agar plate with the test organism.
2. Aseptically apply Novobiocin disc onto the center of the streaked area.
3. Incubate the plate at 37oC for 24 hrs. NB

13. Novobiocin test
A novobiocin disk will be placed on the plate, Novobiocin is an antibiotic that many Staphylococcus strains are sensitive to with the exception of one ,
Staph. saprophyticus that is risist to novobiocin antibiotic.

14. Staphylococcus epidermidis Growing on Blood Agar
Note there is no hemolysis (gamma reaction) on the blood agar and the organism is sensitive to the antibiotic novobiocin as shown by the zone of inhibition.

15. Staphylococcus saprophyticus Growing on Blood Agar
Note there is no hemolysis (gamma reaction) on the blood agar and the organism is resistant to the antibiotic novobiocin.

16. Staphylococcus aureus Growing on Blood Agar
Note beta hemolysis (complete lysis of the red blood cells around the colonies; see arrows) on the blood agar and the organism is sensitive to the antibiotic novobiocin.

17. MANNITOL SALT AGAR ( MSA )
INGREDIENTS
Peptone.
Beef Extract.
D-Mannitol %.
Sodium Chloride %.
Agar %.
Phenol Red. AS PH INDICATOR
Final pH 7.4 ± 0.2 at 25°C.

18. PRINCIPLE AND RESULTS
Mannitol Salt Agar is a nutritive medium due to its content of peptones and beef extract, which supply essential growth factors, such as nitrogen, carbon, sulfur and trace nutrients.
The 7.5% concentration of sodium chloride results in the inhibition of bacterial organisms other than staphylococci.
Mannitol fermentation, as indicated by a change in the phenol red indicator, aids in the differentiation of staphylococcal species.

19. Mannitol Salt agar

20. Mannitol Salt agar Staph. epidermidis Staph. heamolyticus
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staph. epidermidis
Staph. heamolyticus

21. Principles of the Procedure
DNase TEST AGAR
Media ingredients
Enzymatic Digest of Casein %
Enzymatic Digest of Animal Tissue %
Sodium Chloride %
Deoxyribonucleic Acid %
Agar %
Final pH: 7.3 ± 0.2 at 25°C
Principles of the Procedure
The nitrogen, vitamin, and carbon sources are provided by Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue. Sodium Chloride provides essential ions while maintaining osmotic balance. Deoxyribonucleic Acid enables the detection of DNase that depolymerize DNA. Agar is the solidifying agent.
Test Procedure
1. Inoculate plates by spotting or streaking a heavy inoculum of test organism.
2. Incubate plates at 35 ± 2°C for 18 – 24 hours and up to 48 hours.
3. Flood plates with 1 N HCl.
4. Observe for clearing around the spot or streak. Record results.
Results
A zone of clearing around the spot or streak indicates DNase activity.

22. Staphylococcus aureus Growing on DNase Agar
Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the bacterial growth where there is no longer any DNA left in the agar to precipitate out of solution after the 1N HCL was added.

23. Staphylococcus epidermidis Growing on DNase Agar
Note there is no breakdown of the DNA in the agar. After adding the 1N HCl, the entire plate turned cloudy as the DNA precipitated out of solution. There is no clear zone around the bacterial growth.

24. DNase Test Agar with Methyl Green
Methyl green forms a complex with intact (polymerized) DNA to form the green color of the medium. DNase activity depolymerizes the DNA, breaking down the methyl green-DNA complex, which results in the formation of colorless zones around colonies of the test organism. A negative test is indicated by the absence of a colorless zone around the colonies.

25. DNase Test Agar with Toluidine Blue
Toluidine blue forms a complex with intact (polymerized) DNA. In the intact DNA complex, the toluidine blue has the normal blue color. Dnase activity depolymerizes the DNA, breaking down the dye-DNA complex. In the presence of nucleotides produced from the DNase depolymerization, the dye takes on its metachromatic color, forming pink to red zones around bacterial growth. A negative test is indicated when the medium remains blue.

26. Protein A Latex Test L Protein A is found on the cell L IgG S S L S S
surface of about 95 % of human strains of S. aureus and has the ability to bind the Fc portion of immunoglobulin G (IgG) L
IgG S S L S S Fc L L S
The amount of protein A expressed by S. aureus is dependent on the medium, and other
growth conditions.
Rare strains of S. aureus that do not produce coagulase but still
express some protein A on their cell surface may yield a weak positive reaction with the
BBL™ Staphyloslide™ Latex Test. If such a strain is suspected, alternate identification
methods should be used.
S=S.aureus with Protein A
L=Latex particle S
Protein A S

27. Staphyloslide™ Latex Test for Staphylococcus aureus
Latex Test consists of latex particles coated with human fibrinogen and IgG. On mixing the latex reagent with colonies of staphylococci which have clumping factor or Protein A present, cross-linking will occur giving visible agglutination of the latex particles.
Such agglutination will occur notably with S. aureus.
If neither clumping factor nor Protein A are present, no agglutination will occur and the result will be regarded as negative.

28. The End