Streptococcus Basmah almaarik Lab # 6.

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Streptococcus Basmah almaarik Lab # 6

Streptococci All are Gram Positive Cocci arranged in chains (short or long) All are Catalase -ve + ve - ve

Gram Stain Staphylococci Streptococci

Culture Fastidious organism  no growth on NA Grows well on BAP Facultative anaerobe Grow at 37C need 24 to 48 hour .

Streptococcus Classification Alpha hemolytic S. viridans S. pneumoniae Beta hemolytic: Group A, B, C, G & D Lancefield grouping according to antigens in their cell wall Non-hemolytic: Enterococcus (formally known as Group D)

Hemolytic Streptococci Alpha hemolysis on BAP (partial hemolysis). Beta hemolysis on BAP (complete hemolysis). No hemolysis on BAP

Beta Hemolytic Streptococci Lancefield grouping depending on antigens in their cell wall, they are classified into groups : A, B, C, D, F, and G. Facultative anaerobes, grows at 37˚C for 24h. Need 5-10% CO2

Gram Stain

Beta Hemolytic Streptococci Group A (S. pyogenes). Bacitracin Group B (S. agalactiae) CAMP test. Hippurate hydrolysis. Group D (Enterococcus) Grow in high salt conc. Esculin hydrolysis Tolerate high tempreture

Beta Hemolytic Streptococci S. Pyogenes(Group A Strep) S. Agalactiae(Group B Strep) Catalase –ve. Bacitracin sensitive. Catalase –ve. Bacitracin resistant .

S. agalactiae(Group B Strep) CAMP test: Streak a Staph.aureus across a blood agar plate. Inoculate test organism at right angles to it. The tested organism must not touch the staphylococcal inoculum. 37˚C X 24 hr

S. agalactiae(Group B Strep) S.aureus produce beta- lysin. S.agalactiae produce extracellular protein CAMP factor. Both react together synergeticly to produce enhanced haemolysis arrow shape haemolysis.

CAMP test +ve and -ve

S. agalactiae(Group B Strep) Hippurate hydrolysis test : hippuratehydrolase enzyme hydrolizehippurate benzoic acid + glycine We add ninhydrin reagent which will react with glycine to give us purple color.

S. agalactiae(Group B Strep) Bacterial suspenstion in 1% sodium hippurate Incubate 2 hours at 37˚C Add ninhydrine reagent. Incubate for 10 min. Purple color  +ve No purple color  -ve

Enterococcus (Group D Strep) Grow at wide range of temperature 10˚C - 45˚C. Mainly non hemolytic on blood agar but can show alpha or beta-haemolysis. Non-fastidious  can grow on nutrient agar.

Grow in high slat concentration Inoculate the organism in a tube of broth containing 6.5% NaCl. Incubate overnight at 37oC. Turbid  growth  can tolerate high salt conc  +ve Clear  no growth  can’t grow in high salt conc.  - ve

Esculin Hydrolysis The organism can hydrolyze esculin and produce black color in the media. It can also grow in the presence of 40% bile salt.

Esculin Hydrolysis Black color  esculin hydrolysis +ve

Growth in high heat. Make a suspension of the organism. Inoculate on a blood agar (0 time) Incubate organism suspension in water bath 45˚C X 10 min  sub-culture to BAP Repeat step 3 tow more times. Then incubate the BAP 37 ˚C x 24 hr.

Rapid identification test for beta-heamolytic streptococci

YOUR WORK FOR TODAY Do gram stain and show it to the instructor Do camp test with group A and B Do bacitracin sensitivity with group A Do esculin hydrolysis test with group D Do salt tolerance test with group D See instructors hippurate test (Demonestration) See instructors rapid identification kit for Beta-heamolytic stryptococci (Demonestration)