1. PHT382
Lab. No.1

2. Microbiology:
It is the science that deals with the study of micro-organisms (very small organisms) that are invisible to the naked eye

3. Pathogenic micro-orgnisms:
Bacteria
Viruses
Fungi
Protozoa

5. Identification of unknown culture
1- Macroscopical Examination
(colony morphology):
Characters of colonies.
Hemolysis on blood agar.
Pigment production.
2- Microscopical Examination:
Examination of wet mount preparation.
Examination of stained preparation.

6. 3-Biochemical Tests:
(The ability to attack various substances e.g., carbohydrate breakdown;
or to produce particular metabolic products e.g., enzymes.
4-Additional Tests:
such as seriological tests

8. Classification of Bacteria

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

10. 2-Bichemical reactions:
1- catalase test.
2- O/F Test (Oxidation FermentationTest).

11. 1- 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 3
H2O2
Procedure: 1 2

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

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

14. 2- O/F Test (Oxidation FermentationTest)
To differentiate between Staphylococci and Micrococci.
Principle:
Saccharolytic bacteria attack carbohydrates either:
fermentatively ( in absence of oxygen) to yield relatively strong acids, or
oxidatively ( in presence of oxygen) to yield weak acids.
Oxidation process is much more easier than Fermentation process.

15. Procedure:
1 ml liquid paraffin O F

16. O-/F+ O-/F- O+/F+ O+/F- Results: Staphylococci Micrococci
Positive Test:
Results:
O-/F+
O-/F-
O+/F+
O+/F-
Non Saccharolytic
Fermentative
Oxidative
Staphylococci
Micrococci

17. O+/F+ O+/F- Catalase +ve Catalase -ve Staphylococci Micrococci
Gram’s +ve Cocci
Irregular Clusters
Chains or Pairs
Tetrads
Staphylococci
Micrococci
Streptococci
Catalase +ve
Catalase -ve
O+/F+
O+/F-

18. Identification of Staphylococci

19. Identification of Staphylococci
1- Microscopical Examination (Morphology):
Gram’s +ve cocci arranged in
irregular clusters, non motile,
non-sporeforming.
2- Macroscopical Examination
(cultural characteristics):
No special requirements for growth→ grow on simple nutrient media.
On nutrient agar → most strains of S.aureus produce golden yellow colonies.

20. 3- Biochemical reactions:
Coagulase Test.
2. Mannitol Fermentation Test.
3. Deoxyribonuclease (DNase) Test

21. 1. 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
Procedure: 2
1 ml rabbit plasma 1 3
Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

22. Results: Positive test: formation of visible clot. Coagulase -ve
S.epidermidis
Coagulase +ve
S.aureus

23. 2. Mannitol Fermentation Test:
Mannitol salt agar is a selective medium for Staphylococcus species (contains high conc. of salt (about 7.5%).
It is also a differential medium for S.aureus which is the only species of staphylococci that can ferment mannitol→ acid production → yellow color around the growth (due to change the color of the pH indicator).
N.B:
Test sugar: mannitol
pH indicator: phenol red (red in alkaline, yellow in acidic pH).

24. Procedure: 1. Inoculate MSA plate with the test organism by streaking.
Flam & Cool
Flam & Cool
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.

25. Results:
Mannitol fermentation is indicated by formation of yellow colour around the growth
Growth with yellow colour around the colonies
S.aureus
Growth without change in the colour of the medium
CONS

26. 3. 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.

27. 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.aureus
S.epidermidis

29. Antibiotic Sensitivity
1- Penicillin-resistant S.aureus:
95% of S.aureus & S.epidermidis strains are resistant to penicillin.
ttt: a- semisynthetic penicillins (penicillinase-stable)e.g., oxacillin & methicillin.
b- cephalosporins.

30. 2- methicillin-resistant S.aureus (MRSA):
ttt: vancomycin.
3- Vancomycin-resistant S.aureus (VISA/ VRSA):
ttt: Linezolid.

32. Practical Work Gram’s Stain (spots). Catalase test. O/F test.
Coagulase test.
MSA test.
DNase test.

33. Results Micrococci S.aureus S.epidermidis Gram’s +ve Cocci
Gram’s Stain
Catalase test
+ve
(air bubbles)
O/F test
O+/F-
O+/F+
Coagulase test -
(Visible clot)
-ve
(No clot)
MSA test
Growth with change in colour into yellow
Growth without any change in colour
DNase test
Clear zone around the growth while the rest of the plate is turbid
Turbidity in all the plate
Gram’s +ve Cocci
Tetrads
Clusters
Clusters

35. Thank you