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

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 hemolyticS. viridansS. 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


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


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.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


Hippurate hydrolysis test :

hippuratehydrolase enzyme hydrolizehippurate benzoic acid + glycine

We add ninhydrin reagent which will react with glycine to give us purple color.


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 .

1. Make a suspension of the organism.2. Inoculate on a blood agar (0 time) 3. 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

1. Do gram stain and show it to the instructor

2. Do camp test with group A and B3. Do bacitracin sensitivity with group A4. Do esculin hydrolysis test with group D5. Do salt tolerance test with group D6. See instructors hippurate test

(Demonestration)7. See instructors rapid identification kit for

Beta-heamolytic stryptococci (Demonestration)

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

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