PHT 313 Lab (1) Staphylococci

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

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

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.

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

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

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) .

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

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

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

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

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.

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

S. aureus S.epidermidis S saprophyticus

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.epidermidis S.aureus

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:

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

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.

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

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

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 .

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

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

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

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

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.

Antibiogram Helpful in tracing sources of staphylcoccal infections

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