Gram Positive Cocci · β-Hemolytic streptococci on sheep blood agar β-Hemolytic streptococci produce hemolysins that completely lyse sheep erythrocytes, resulting in a clearing

Gram Positive Cocci

I- Staphylococcus spp.

Prof. Dr.

AbD El-GAwAD M. HAsHEM

I- Basic Characters

Morphology Shape: gram positive cocci Arrangement: Grape shape clusters Non spore forming, non motile

Culture Characteristics N. Agar: Round, regular, opaque, smooth , shiny hard

colored colonies. S. aureus: golden yellow S. albus : white S. citrus: Lemon yellow

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Basic Characters cont.

Blood agarBeta-heamolytic strains: colonies are surrounded with

a zone of complete blood hemolysis, e.g. S. aureus, Non- heamolytic strains: No hemolytic zone is

observed surrounding the colonies.

Mannitol Salt AgarMannitol Fermentors: e.g. S. aureus, colonies are

surrounded with yellow color due to acid production.Non- mannitol fermentors: e.g. S. albus, colonies are

surrounded with pink color.

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II- Microscopic examination

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Staphylococcus Gram Stain

This mixture consists of the gram-positive Staphylococcus albus(purple-stained spheres arranged in grape-like clusters), and the gram-negative Escherichia coli (red-stained rods scattered singly throughout the field).

Shape: gram positive cocci, Bunch (cluster), Non spore forming, non motile

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Gram Stain of Pus from abscess

Overlying the pus cell is atypical cluster of Staphylococcus aureus, looking like a small bunch of grapes. Paired and single cocci are also present.

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III- Culture characteristics.

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Colonies of two species of staphylococcia- S. aureus :The larger colonies note that the

distinctive golden pigment is most intense in the centre of the colony.

b- S. albus : Small white colonies c- S. citrus: Small yellow colonies

a

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Colonies of S. aureus on blood agar

Growth of Staphylococcus aureus on blood with characteristic hemolysis and golden yellow pigmentation.

Prof. Dr.


IV-Biochemical Reactions

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Staphylococci on Mannitol salt Agar

Mannitol salt agar (MSA)NameMannitol + 7.5% NaclConstituent

Phenol redIndicator only Staph. Can grow in presence of 7.5% Nacl- only Staph. aureus (pathogenic) can utilize mannitol → mannic acid ∴ind. → yellow

Principle

Selective & differential Type

- isolation of Staph.- differentiation bet. Pathogenic Staph. aureus & non- pathogenic

Use

Prof. Dr.


Catalase testThe test is performed by placing drops of 3% hydrogen peroxide to the

suspected colonies of S. aureus. Immediate and vigorous bubbling owing to the production of oxygen gas indicates a positive test

Catalase testName

Nutrient agar + 1%glucoseConstituent

upon adding H2O2 Catalase converts it to H2O + O2 (froth)

Principle

Differentiate bet. Staph. & Strept. SpeciesUse

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Test for Phosphatase

Phosphatase testNameNutrient agar + ph.Ph.diphosphateConstituentPhosphatase enzyme liberates free ph.ph.+ NH3 vapour Pink

Principle

Test for the ability of Staph. sp. to produce phosphataseUse

+ve

-ve-ve +ve

Prof. Dr.


Nutrient agar containing phenolphthalin diphosphate is streaked with the test M.O. and incubated. After incubation the growth is exposed to ammonia vapor ز

Tube Coagulase testIn this test, extracellular coagulase produced by S. aureus complexes with a component

in plasma, called coagulase-reacting factor. This complex, in turn, reacts with fibrinogen to form fibrin and, consequently, the development of a visible clot.

Tube coagulase testNameCitrated plasmaConstituentFibrinogen (soluble) coagulase Fibrin (clot)Principle

Identification & differentiation of Staphylococcus aureusfrom non-pathogenic Staphylococci

Use

Prof. Dr.


Test for caseinase Plates containing casein agar are streaked with the test M.O. and

growth plates were flooded with acetic acid. Clear zone around growth indicates positive test

Caseinae testNameNutrient agar +1% caseinConstituent

Casein around the growth is hydrolyzed by caseinase Transparent zone.

Principle

Test for M.O. producing the enzymeExamples are Staphylococci & Bacilli

Use

+ve -ve

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Test for DNAase

DNAase testNameDNA agar Constituent

Enzyme breaks DNA into smaller nucleotides-Add 1N HCl to ppt. DNA except around the growth

Principle

Confirmatory test for Staph. aureus after coagulase test Use

Plates containing DNA agar are streaked with the test M.O. and growth plates were flooded with 1N HCl.

Clear zone around growth indicates positive test

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Oxidation and fermentation of glucose

Duplicate tubes of Hugh and Leifson medium are stab-inoculated by the tested culture. One of the tubes is covered with 2-3ml of sterile liquid paraffin (for anaerobiosis).Incubate both tubes at 37°C for 10-15days. 4. Organisms that utilize glucose oxidatively will produce acid ( indicated by the production of a yellow color ) in the aerobic culture tube only.

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Oxidation and fermentation of glucose

Oxidation & fermentation of glucosea) +ve oxid. & -ve ferm. b) +ve ferm. & -ve oxid. c) +ve oxid. & +ve ferm

a b c

Oxidation and fermentation of glucose testLeifson medium [1% Glucose+0.5% agar (semisolid )+ tryptone ]ConstituentBromocresol purpleIndicator Staph. Facultative Anaerobe (Both tubes yellow)Ps., Micrococcus Obligate aerobes - if m.o grows it changes ind. From violet → yellow

Principle

DifferentialtypeTest for metabolic activity of M.O. according to O2 requirementUse

Staphylococcus

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Special Tests

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Novobiocin sensitivityNovobiocin sensitivity testName

Blood agar Constituent

Differentiate between Staphylococcus epidermidis & Staphylococcus saprophyticus

Principle

Confirmatory test for Staph. aureus after coagulase test Use

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API STAPH

For identification of staphylococci and micrococci

The strip is inoculated with an organism suspension and is incubated overnight.

Interpretation of the reactions generates a biotype number that is used, along with a computer-assisted database, to identify the organism.

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Phage typing of a strain of S. aureus The entire surface of the plate was sown with a broth culture of the Staphylococcus

sp. When the inoculum had dried, a series of 24 phages were spotted on the plate. After overnight incubation the phage type of the staphylococcus is determined by the

pattern of lysis shown.

Prof. Dr.


Gram Positive Cocci

II- Streptococcus spp.

Prof. Dr.


Prof. Dr.


I- Basic Characters

Morphology Shape: gram positive cocci Arrangement: Chains Non spore forming, non motile

Culture Characteristics N. Agar: Weak growth Blood Agar: classified according to type of hemolysis into

α- hemolytic streptococcus: e.g. S. viridins & S. pneumonia β- hemolytic streptococcus: e.g. S. pyogenus δ- hemolytic streptococcus: e.g. peptostreptococcus

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II- Microscopic examination

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Gram stain of streptococci growing in a broth culture

As their name suggests, streptococci characteristically grow in chains. These chain forms are most frequently seen when the organisms are grown in broth. On Gram-stained smears prepared from growth on agar media, the organisms usually appear in pairs or in shorter chains.

Prof. Dr.


Streptococci in a smear of pus

Long and short chains and a few pairs of gram-positive cocci are present. The pus cells are disintegrating.

The smear was prepared from an empyema (pus in the thoracic cavity). The streptococcus cultivated from the pus was anaerobic.

Prof. Dr.


Pneumococci in blood film

There are many pairs of lanceolate diplococci. Most are gram-positive, but some, especially intracellular pairs, are gram-negative.

Prof. Dr.


Gram stain of S. pneumoniae

This photograph shows the typical appearance of pneumococci in blood culture broth.

These bacteria characteristically grow in pairs in which the cells have a slightly elongated “lanceolate” morphology.

With some cells in this frame, a clear area or “halo” may be observed surrounding the organism pairs, indicating the presence of the polysaccharide capsule of S. pneumonrae.

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Quelling test for identification of S. pneumoniae

This microscopic “precipitin test” can be used to identify or to determine capsular subtypes of pneumococci.

Reaction of anticapsular antibody with the carbohydrate material of the capsule causes the capsular appears to “swell.”

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III- Culture characteristics.Streptococci initially may be classified on the basis of

their hemolytic properties on sheep blood agar.

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α-Hemolytic streptococci on sheep blood agar

Partial hemolysis of the erythrocytes results in a “greening” of the agar medium surrounding the colonies (α-hemolysis).

Streptococci that are α-hemolytic include S. pneumoniae, the viridans group of streptococci, and most Enterococcus (formerly Streptococcus) species

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β-Hemolytic streptococci on sheep blood agar

β-Hemolytic streptococci produce hemolysins that completely lyse sheep erythrocytes, resulting in a clearing of the medium surrounding the colonies.

The group A streptococcus shown here demonstrates this type of hemolysis.

More intense β-hemolysis is noted in areas where the medium has been “stabbed,” pushing some of the bacteria under the medium surface. The β -hemolysis in these areas is due to both streptolysin S on the surface (oxygen stable) and streptolysin O below the surface (oxygen-labile)

Prof. Dr.


S. pneumoniae colonies on sheep blood agar

At left is shown a typical α-hemolytic mucoid strain of S. pneumoniae, an appearance that is due to large amounts of capsular polysaccharide.

Prof. Dr.


IV-Biochemical Reactions

Prof. Dr.


Test for DNAase

DNAase testNameDNA agar Constituent

Enzyme breaks DNA into smaller nucleotides-Add 1N HCl to ppt. DNA except around the growth

Principle

Confirmatory test Streptococcus pyogenesUse

Plates containing DNA agar are streaked with the test M.O. and growth plates were flooded with 1N HCl.

Clear zone around growth indicates positive test

Prof. Dr.


Bile solubility tests for pneumococcus

The two tubes on the left contain broth cultures of test microorganism To one tube was added 0.1 ml sodium deoxycholate ; to the other 0.1 ml

distilled water. Within three minutes the pneumococcus was shown to be bile-soluble; the

alpha-streptococcus was not.

Prof. Dr.


Bile solubility tests for pneumococcus

Bile solubility testName

Culture in N. Broth then incubate add bile salt then incubate

Constituent

Bile salts (surface active agents) cause lysis of sensitive m.o. as Streptococcus Pneumoniae

Principle

Differentiate and test for haemolytic activitya) Streptococcus pneumonia +ve b) Streptococcus Viridans –ve

Use

a b

Prof. Dr.


Optochin sensitivity of pneumococci An optochin disc was placed on the blood agar plate which was sown with

test organism. After overnight incubation optochin inhibited pneumococci.

Optochin sensitivity testName

Blood AgarConstituent

Alkaloid “Optochin” [ethyl hydrocuperin.HCl] inhibit the growth of some M.O.

Principle

Differentiate between Streptococci causing α-haemolysis S. Pneumonia + ve & S. viridans -ve

Use

Streptococcus Streptococcus

Pneumoniae viridans

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Bacitracin sensitivity tests

The organism on the left is presumptively identified as a group A streptococcus by its inhibition by bacitracin.

The bacitracin resistant streptococcus on the other half of the plate was found to belong to Group G.

Bacitracin disc was placed on the blood agar plate which was sown with test organism.

After overnight incubation bacitracin inhibited group A streptococci.

gpA streptococci

Prof. Dr.


Documents

Gram Positive Cocci · β-Hemolytic streptococci on sheep blood agar β-Hemolytic streptococci produce hemolysins that completely lyse sheep erythrocytes, resulting in a clearing