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Microbio Lab 4

Microbio Lab 4

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Published by nkivc

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Published by: nkivc on Nov 16, 2009
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MICROBIOLOGY LABORATORY 4USTMED ’07 Sec C – AsM; Photos provided by JV.N & MeaM.Culture of MicroorganismsDifferent Streaking Methods
Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking:
Simple Streaking
Illustration:Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking:
4 Quadrant Method of Streaking
Note: This method of streaking is used for better isolation of the organismIllustration:Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking:
Overlapping method of streaking
Note: This method of streaking is used for sensitivity testingIllustration:
Demo slides:
Gram stain of pus from an epyema cavity showing long andshort chains of Gram-positive streptococci and large numbersof pus cells (stained red) x3,900.Staphylococcus aureus. Gram stain of culture showingcharacteristic irregular clusters of gram-positive cocci. Thereare no spores or capsules.Gram stain of pus from cervix – GonorrheaNeisseria gonorrhea – Gram stainNeisseria gonorrhea –Stanford’s stainClinical specimen: Urethral DischargeStaining used: Gram StainingResults: Gram Negative Cocci in Pairs Intracellularly located
Staphylococcus aureus
Gram Positive Cocci in clustersCatalase Test – Positive(best single test for the pathogenecity of Staph. Aureus)Colonies on plated media:1.Blood agar plate (BAP) – Beta hemolytic (completehemolysis)2.Mannitol salt agar (MSA) – Yellow colonies (due tomannitol fermentation)Staphylococcus aureus on mannitol Salt Agar (MSA),differentiating S. aureus from other catalase positive gramposititve cocci. The medium contains 7.5% NaCl which inhibitsthe growth of many organisms. S. aureus ferments mannitolresulting to acid end product causing the phenol red indicatorto change from pink to yellow.
Staphylococcus aureus on Mannitol Salt Agar (MSA)Showing yellow colonies due to Mannitol fermentationMannitol Salt Agar (MSA) differentiates S. aureus from othercatalase-positive gram-positive cocci. The medium contains7.5%NaCl which inhibits the growth of many orgs.(+) results – fermentation of mannitol resulting to acid endproduct causes the phenol red indicator to change from pink toyellow (right half of the plate)(-) result – no fermentation, no chage of color (left half)Colonies of Staph. Aureus on MSA (close-up). Mediumsurrounding colonies has turned yellowCoagulase test confirms identiy as
Staphylococcus aureus
(coagulase positive) and
Staphylococcus epidermidis
(coagulasenegative):1.Slide coagulase test – when a saline suspension of staphylococci is mixed with human or rabbit plasmaon a slide, there is immediate clumping withcoagulase positive staphylococci.2.Tube coagulase test when diluted plasma isinoculated with S. aureus, jellification occurs w/in 2-4 hours. This result is due to coagulase convertingfibrinogen in the plasma to fibrin and a positive testconfirms a positive slide test.Staphylococcus aureus on Blood Agar Plate (BAP)Showing beta hemolytic hemolysis; complete hemolysisStaphylococcus aureus on BAPcolonies showing beta orcomplete type of hemolysis.Growth of staphylococcus onBAP with characteristicpigmentation (golden-yellow)and ‘oil paint’ appearance.
Streptococcus pneumoniae
-gram positive cocci in pairs-catalase negative-alpha hemolytic/incomplete hemolysis on BAP-Sensitive to Optochin Disk Test as seen in ChocolateAgar Plate (CAP)
Streptococcus viridans
-gram positive cocci in chain-catalase negative-alpha hemolytic/incomplete hemolysis on BAP-Resistant to OPtochin Disk Test as seen in CAP
Streptococcus pyogenes
-gram positive cocci in chains-catalase negative-beta hemolytic/complete hemolysis on BAP-resistant to optochin diskOptochin sensitivity of pneumococci in contrast with
Streptococcus viridans
whichalso produces alpha-haemolytic colonies on bloodagar.Streptococcus viridans on BAP-produces alpha hemolytic/incomplete hemolysis onBAPGrowth of a beta-haemoliticstretptococcus on blood agarmedium showing large zonesof clear haemolysis aroundsmall transparent colonies.Growth of pneumococci on blood agar showing characteristic‘draughtsman’ colonies with concentric surface rings.

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