1. PHT 313 Lab (1)
Staphylococci

2. Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve
Corynbacterium
Clostridum
Bacillus
Staphylococci
Streptococci
Micrococci
Neisseria
Enterobacteriaceae Pseudomonas.

3. Staphylococci Three mainly species that are human pathogens:
Staph. aureus
Staph. epidermidis
Staph. saprophyticus

4. Habitat: S. aureus: S. epidermidis: S. saprophyticus:
Nasal passages, skin, oral cavity and gastrointestinal tract.
S. epidermidis:
skin.
S. saprophyticus:
Rarely found in healthy humans.

5. Microscopic Morphology:
Gram positive cocci, arranged in grape like clusters, non-motile, non-spore forming.

6. Classification Coagulase positive e.g. S.aureus
According to Coagulase enzyme production
Coagulase positive e.g. S.aureus
Coagulase negative e.g.
S. epidermidis &
S. saprophyticus
According to Pigment production (endopigment)
Golden yellow colonies S .aureus
White colonies S. epidermidis
Yellow colonies S. saprophyticus

7. Culture Characteristics
Environment: Facultative anaerobe
Temp.: 37 ° C
PH: 7.2
Media:
Nutrient agar (Simple medium).
Blood agar (Enriched medium).
Mannitol salt agar (Selective & diffrential medium) .

8. Staphylococci on Nutrient Agar
Organism
S.aureus
Media
Nutrient agar
Appearance
Golden Yellow
colonies

9. Staphylococci on Nutrient Agar
Organism
S.epidermidis
Media
Nutrient agar
Appearance
White colonies

10. Staphylococci on Blood Agar
Organism
S. aureus
Media
Blood agar
Appearance
Beta heamolysis
(complete
haemolysis)

11. Staphylococci on Blood Agar
Organism
S.epidermidis
S saprophyticus
Media
Blood agar
Appearance
Non hemolytic

12. Mannitol Salt Agar (MSA)
The name: Mannitol salt agar
Appearance: Solid, opaque, pink.
Type: Selective & diffrential medium
Composition: Contains high conc. of salt (about 7.5%).
Test sugar: mannitol.
pH indicator: phenol red (red in alkaline, yellow in acidic pH).
Sterilization: autoclave.

14. S. aureus gives yellow colonies on MSA because of mannitol fermintation which leads to decrease in PH this turns the color of phenol red to yellow.
Note: MSA is differential for S.aureus

15. S. aureus
S.epidermidis
S saprophyticus

16. Deoxyribonuclease (DNase) Test:
Principle:
DNA
DNase enzyme
Nucleotides
Insoluble
In acid
soluble
In acid
Procedure:
1. Inoculate DNase agar plate with the test organism.
2. Incubate the plate at 35oC for 24 hrs.
3. Flood the plate with 1M HCl.

17. Results:
DNase activity is indicated by a clear zone around the growth after addition of Hcl
Clear zone around the growth while the rest of the plate appears cloudy
Cloudiness in all the plate
S.epidermidis
S.aureus

18. Biochemical Tests Catalase test:
Differentiative test to separate Staphylococci and Micrococci which are catalase +ve from Sterptococci which are catalase –ve.
Principle:
H2O2
Catalase enzyme
H2o O2
Air bubbles
Procedure:

19. Results: Positive test: Rapid appearance of gas bubbles. Catalase +ve
Staphylococci or Micrococci
Streptococci

20. Coagulase Test:
Definitive test to differentiate between S.aureus & other species of staphylococci (coagulase-negative staphylococci “CONS”) e.g. S.epidermidis
Principle:
Fibrinogen
Plasma
Coagulase enzyme
Fibrin
Visible Clot 1
Procedure: 2 3
Place at water bath at 37oC, observe for formation of
visible clot for up to 4 hrs.

21. Results: Positive test: Formation of visible clot. Coagulase -ve
S.epidermidis
Coagulase +ve
S.aureus

22. Diseases Due to invasion: Toxin mediated
Local lesions of skin e.g. boils, carbauncles, abscesses.
Systemic infections e.g. septicemia, meningitis.
Toxin mediated
Food poisoning
Toxic shock syndrome
Scalded skin syndrome

23. Diagnosis 1.The specimen: Swab from wounds, abscesses
Blood in case of septicemia
Urine in case of UTI
CSF in case of meningitis.
2. Direct examination :
Film is prepared and examined by gram stain .

24. 3.Culture : S. aureus: Nutrient agar: Golden yellow colonies
Blood agar: Beta hemolysis .
MSA: Yellow colonies (mannitol fermentation)

25. S. epidermidis:
Nutrient agar: White colonies
Blood agar: Non haemolytic
MSA: Non fermentative
S. saprophyticus:
Nutrient agar: Yellow colonies

26. 4. Biochemical reactions:
Catalase test : positive
Coagulase test: positive in case of s. aureus

27. 5-Typing Used for epidemiological purposes. Phage typing. Antibiogram.
Molecular typing (ribotyping or PCR).

28. Phage Typing
Staphylococci isolated from wounds, from nose and nail bed of attending personnel (doctors, nurses, ….) and from fomites are identified by phage typing to reveal the source of infection. Also it is used to trace the source of food poisoning.

29. Antibiogram
Helpful in tracing sources of staphylcoccal infections

30. Molecular typing (ribotyping or PCR)
Used to document the spread of epidemic disease-producing clones of S.aureus