Download - PHT 313 Lab (1) Staphylococci. Bacteria Gram’s Stain Gram’s +ve Cocci Bacilli Gram’s -ve Cocci Bacilli Staphylococci Streptococci Micrococci Neisseria.

PHT 313Lab (1)

Staphylococci

StaphylococciStreptococciMicrococci

NeisseriaCorynbacteriumClostridumBacillus

Enterobacteriaceae Pseudomonas.

Staphylococci

• Three mainly species that are human pathogens:

• Staph. aureus

• Staph. epidermidis

• Staph. saprophyticus

Habitat:

• S. aureus:– 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.

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(completehaemolysis)

Staphylococci on Blood Agar

Organism S.epidermidisS 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.epidermidisS saprophyticus

Deoxyribonuclease (DNase) Test: Principle:

DNADNase enzyme

NucleotidesInsoluble

In acidsoluble 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 TestsCatalase test: Differentiative test to separate Staphylococci

and Micrococci which are catalase +ve from Sterptococci which are catalase –ve.

H2O2

Catalase enzymeH2o + O2

Air bubbles

Principle:

Procedure:

Results:Positive test: Rapid appearance of gas bubbles.

Staphylococci or Micrococci

Catalase –ve

Streptococci

Catalase +ve

Coagulase Test:• Definitive test to differentiate between S.aureus &

other species of staphylococci (coagulase-negative staphylococci “CONS”) e.g. S.epidermidis

Principle:

FibrinogenPlasma

Coagulase enzymeFibrin

Visible Clot

Procedure:2

3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

1

Results:

Positive test: Formation of visible clot.

Coagulase +ve

S.aureus

Coagulase -ve

S.epidermidis

Diseases• Due to invasion:

– 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• Blood agar: Non haemolytic• MSA: Non fermentative

4. Biochemical reactions:

• Catalase test : positive

• Coagulase test: positive in case of s. aureus