Isolation and Identification of Staphylococci

Gram Positive coccusCatalaseOxidaseStaphylococciStreptococci+_Gram Stain_+MicrococciCoagulase

Staphylococcus aureus_+

CNS Staphylococcus epidermidis Staphylococcus saprophyticus

Gram positive cocci

Catalase +ve

Oxidase -ve

Coagulase testNegativePositiveSlide methodNegativePositiveTube methodCoagulase

Staphylococcus aureusCNS Staphylococcus epidermidis Staphylococcus saprophyticus+_

Non-hemolytic Staphylococcus species: Staphylococcus epidermidis

Staphylococcus saprophyticus: non-hemolytic, bright white, creamy colonies

Strains of Staphylococcus aureus produce a golden yellow pigment

Strains of Staphylococcus aureus not a golden yellow pigment producer

MANNITOL SALT AGAR ( MSA )INGREDIENTSPeptone.Beef Extract.D-Mannitol .............. 1.0%.Sodium Chloride ...... 7.5%.Agar ......................... 1.5%.Phenol Red. AS PH INDICATORFinal pH 7.4 0.2 at 25C.

PRINCIPLE AND RESULTSMannitol Salt Agar is a nutritive medium due to its content of peptones and beef extract, which supply essential growth factors, such as nitrogen, carbon, sulfur and trace nutrients.

The 7.5% concentration of sodium chloride results in the inhibition of bacterial organisms other than staphylococci.

Mannitol fermentation, as indicated by a change in the phenol red indicator, aids in the differentiation of staphylococcal species.

Mannitol Salt agarStaph. aureusStaph. epidermidis

Mannitol Salt agar

Procedure:1. Inoculate blood agar plate with the test organism.2. Aseptically apply Novobiocin disc onto the center of the streaked area.3. Incubate the plate at 37oC for 24 hrs.4. Accurately measure the diameter of the inhibition zone around the disc.Novobiocin resistance Test:Coagulase Negative StaphStaph saprophyticus

Staph epidermidis

Novobiocin testA novobiocin disk will be placed on the plate, Novobiocin is an antibiotic that many Staphylococcus strains are sensitive to with the exception of one ,Staph. saprophyticus that is risist to novobiocin antibiotic.

Staphylococcus epidermidis Growing on Blood AgarNote there is no hemolysis (gamma reaction) on the blood agar and the organism is sensitive to the antibiotic novobiocin as shown by the zone of inhibition.

Staphylococcus saprophyticus Growing on Blood AgarNote there is no hemolysis (gamma reaction) on the blood agar and the organism is resistant to the antibiotic novobiocin.

Staphylococcus aureus Growing on Blood AgarNote beta hemolysis (complete lysis of the red blood cells around the colonies; see arrows) on the blood agar and the organism is sensitive to the antibiotic novobiocin.

Enzymatic Digest of Casein.................1.5% Enzymatic Digest of Animal Tissue.....0.5% Sodium Chloride...................................0.5% DNase TEST AGARMedia ingredientsPrinciples of the Procedure The nitrogen, vitamin, and carbon sources are provided by Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue. Sodium Chloride provides essential ions while maintaining osmotic balance. Deoxyribonucleic Acid enables the detection of DNase that depolymerize DNA. Agar is the solidifying agent.Test Procedure 1. Inoculate plates by spotting or streaking a heavy inoculum of test organism.2. Incubate plates at 35 2C for 18 24 hours and up to 48 hours. 3. Flood plates with 1 N HCl. 4. Observe for clearing around the spot or streak. Record results. Deoxyribonucleic Acid.............0.2% Agar........................................1.5%Final pH: 7.3 0.2 at 25CResults A zone of clearing around the spot or streak indicates DNase activity.

Staphylococcus aureus Growing on DNase AgarNote there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the bacterial growth where there is no longer any DNA left in the agar to precipitate out of solution after the 1N HCL was added.

Staphylococcus epidermidis Growing on DNase Agar

Note there is no breakdown of the DNA in the agar. After adding the 1N HCl, the entire plate turned cloudy as the DNA precipitated out of solution. There is no clear zone around the bacterial growth.

DNase Test Agar with Methyl GreenMethyl green forms a complex with intact (polymerized) DNA to form the green color of the medium. DNase activity depolymerizes the DNA, breaking down the methyl green-DNA complex, which results in the formation of colorless zones around colonies of the test organism. A negative test is indicated by the absence of a colorless zone around the colonies.

DNase Test Agar with Toluidine BlueToluidine blue forms a complex with intact (polymerized) DNA. In the intact DNA complex, the toluidine blue has the normal blue color. Dnase activity depolymerizes the DNA, breaking down the dye-DNA complex. In the presence of nucleotides produced from the DNase depolymerization, the dye takes on its metachromatic color, forming pink to red zones around bacterial growth. A negative test is indicated when the medium remains blue.

*Protein A Latex TestSSSSS

L

LL

LL

IgGS=S.aureus with Protein AL=Latex particleProtein ASFcSProtein A is found on the cellsurface of about 95 % of human strains of S. aureus and has the ability to bind the Fc portion of immunoglobulin G (IgG)

Staphyloslide Latex Test for Staphylococcus aureusLatex Test consists of latex particles coated with human fibrinogen and IgG. On mixing the latex reagent with colonies of staphylococci which have clumping factor or Protein A present, cross-linking will occur giving visible agglutination of the latex particles. Such agglutination will occur notably with S. aureus.If neither clumping factor nor Protein A are present, no agglutination will occur and the result will be regarded as negative.

The End

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