Slide 1

Isolation and Identification of Staphylococci Shadi Alashi

Slide 2

Gram Positive coccus Catalase Oxidase Staphylococci Streptococci + _ Gram Stain _ + Bacitracin Susceptibility test Coagulase Staphylococcus aureus _ + CNS Staphylococcus epidermidis Staphylococcus saprophyticus Macrococci Micrococci S R Staphylococcus haemolyticus

Slide 3

Gram positive cocci

Slide 4

Catalase +ve

Slide 5

Oxidase -ve

Slide 6

Coagulase test Negative Positive Slide method Negative Positive Tube method Coagulase Staphylococcus aureus CNS Staphylococcus epidermidis Staphylococcus saprophyticus + _ Staphylococcus haemolyticus

Slide 7

Non-hemolytic Staphylococcus species: Staphylococcus epidermidis

Slide 8

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

Slide 9

Strains of Staphylococcus aureus produce a golden yellow pigment

Slide 10

Strains of Staphylococcus aureus not a golden yellow pigment producer

Slide 11

Staphylococcus haemolyticus on Blood Agar

Slide 12

Procedure: 1. Inoculate blood agar plate with the test organism. NB 2. Aseptically apply Novobiocin disc onto the center of the streaked area. 3. Incubate the plate at 37 o C for 24 hrs. Novobiocin resistance Test: Coagulase Negative Staph Staph saprophyticus Staph epidermidis Staph heamolyticus

Slide 13

Novobiocin test A 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.

Slide 14

Staphylococcus epidermidis Growing on Blood Agar Note 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.

Slide 15

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

Slide 16

Staphylococcus aureus Growing on Blood Agar Note 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.

Slide 17

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

Slide 18

PRINCIPLE AND RESULTS Mannitol 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.

Slide 19

Mannitol Salt agar

Slide 20

Staph. heamolyticusStaph. epidermidis Staphylococcus haemolyticus Staphylococcus epidermidis

Slide 21

Enzymatic Digest of Casein.................1.5% Enzymatic Digest of Animal Tissue.....0.5% Sodium Chloride...................................0.5% DNase TEST AGAR Media ingredients Principles 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 25C Results A zone of clearing around the spot or streak indicates DNase activity.

Slide 22

Staphylococcus aureus Growing on DNase Agar Note 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.

Slide 23

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.

Slide 24

DNase Test Agar with Methyl Green Methyl 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.

Slide 25

DNase Test Agar with Toluidine Blue Toluidine 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.

Slide 26

26 Protein A Latex Test S S SS S L L L L L IgG S=S.aureus with Protein A L=Latex particle Protein A S Fc S Protein A is found on the cell surface of about 95 % of human strains of S. aureus and has the ability to bind the Fc portion of immunoglobulin G (IgG)

Slide 27

Staphyloslide Latex Test for Staphylococcus aureus Latex 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.

Slide 28

The End