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MICROBIOLOGY LABORATORY 4USTMED 07 Sec C AsM; Photos provided by JV.N & MeaM.

Culture of Microorganisms

Different 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 ofthe organism

Illustration:

Culture Media Used: Eosin Methylene BlueOrganisms Used: Escherichia ColiMethod of Streaking: Overlapping method of streaking

Note: This method of streaking is used for sensitivity testing

Illustration:

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 Gonorrhea

Neisseria gonorrhea Gram stain

Neisseria gonorrhea Stanfords stain

Clinical specimen: Urethral DischargeStaining used: Gram StainingResults: Gram Negative Cocci in Pairs Intracellularly located

Staphylococcus aureus

Gram Positive Cocci in clusters

Catalase 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.

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Staphylococcus aureus on Mannitol Salt Agar (MSA)Showing yellow colonies due to Mannitol fermentation

Mannitol 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 end

product 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 yellow

Coagulase test confirms identiy as Staphylococcus aureus(coagulase positive) and Staphylococcus epidermidis (coagulasenegative):

1. Slide coagulase test when a saline suspension ofstaphylococci 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 hemolysis

Staphylococcus 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 Chocolate

Agar 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 disk

Optochin sensitivity ofpneumococci in contrast withStreptococcus viridans whichalso produces alpha-haemolytic colonies on bloodagar.

Streptococcus viridans on BAP- produces alpha hemolytic/incomplete hemolysis on

BAP

Growth of a beta-haemoliticstretptococcus on blood agarmedium showing large zonesof clear haemolysis aroundsmall transparent colonies.

Growth of pneumococci on blood agar showing characteristicdraughtsman colonies with concentric surface rings.

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Colonies of group A Strep on 5% sheep blood agar. Colonies ofgroup A Streptococci on 5% sheep blood agar are small andsurrounded by wide zones of beta hemolysis.

Gram stain of Streptococci in broth culture (x1250). Gram stainof a positive blood culture broth demonstrating gram-positivecocci arranged in chains. Streptococci are normal microbiotain the upper respiratory tract and the gastrointestinal tract.For this reason, gram stains of specimens from these sites arenot helpful in diagnosing infections caused by the pathogenicStreptococci such as S. pyogenes and S. pneumoniae.

Neisseria Gonorrhea

Gram stain for N. Gonorrhea

Culture of N. gonorrhea on selective media (35oC in 5% carbondioxide environment)

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