PHT382 Lab. No.1

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

Pathogenic micro-orgnisms: Bacteria Viruses Fungi Protozoa

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.

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

Classification of Bacteria

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

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

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

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

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

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.

Procedure: 1 ml liquid paraffin O F

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

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-

Identification of Staphylococci

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.

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

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.

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

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).

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.

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

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.

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

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.

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

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

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

Thank you