Microbio Lab 4

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

Gram stain of pus from cervix – Gonorrhea

Culture of Microorganisms Different Streaking Methods Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method of Streaking: Simple Streaking Neisseria gonorrhea – Gram stain Illustration:

Neisseria gonorrhea – Stanford’s stain Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method of Streaking: 4 Quadrant Method of Streaking Note: This method of streaking is used for better isolation of the organism Illustration:

Clinical specimen: Urethral Discharge Staining used: Gram Staining Results: Gram Negative Cocci in Pairs Intracellularly located

Culture Media Used: Eosin Methylene Blue Organisms Used: Escherichia Coli Method of Streaking: Overlapping method of streaking Note: This method of streaking is used for sensitivity testing Illustration:

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 (complete hemolysis)

2.

Mannitol salt agar (MSA) – Yellow colonies (due to mannitol fermentation)

Demo slides: Gram stain of pus from an epyema cavity showing long and short chains of Gram-positive streptococci and large numbers of pus cells (stained red) x3,900.

Staphylococcus aureus. Gram stain of culture showing characteristic irregular clusters of gram-positive cocci. There are no spores or capsules.

Staphylococcus aureus on mannitol Salt Agar (MSA), differentiating S. aureus from other catalase positive gram posititve cocci. The medium contains 7.5% NaCl which inhibits the growth of many organisms. S. aureus ferments mannitol resulting to acid end product causing the phenol red indicator to change from pink to yellow.

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

Staphylococcus aureus on BAP colonies showing beta or complete type of hemolysis.

Mannitol Salt Agar (MSA) differentiates S. aureus from other catalase-positive gram-positive cocci. The medium contains 7.5%NaCl which inhibits the growth of many orgs.

Growth of staphylococcus on BAP with characteristic pigmentation (golden-yellow) and ‘oil paint’ appearance.

Streptococcus pneumoniae (+) results – fermentation of mannitol resulting to acid end product causes the phenol red indicator to change from pink to yellow (right half of the plate) (-) result – no fermentation, no chage of color (left half)

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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

Colonies of Staph. Aureus on MSA (close-up). surrounding colonies has turned yellow

Medium

Coagulase test confirms identiy as Staphylococcus aureus (coagulase positive) and Staphylococcus epidermidis (coagulase negative): 1.

Slide coagulase test – when a saline suspension of staphylococci is mixed with human or rabbit plasma on a slide, there is immediate clumping with coagulase positive staphylococci.

Streptococcus pyogenes gram positive cocci in chains catalase negative beta hemolytic/complete hemolysis on BAP resistant to optochin disk Optochin sensitivity of pneumococci in contrast with Streptococcus viridans which also produces alphahaemolytic colonies on blood agar.

Streptococcus viridans on BAP produces alpha hemolytic/incomplete hemolysis on BAP 2.

Tube coagulase test – when diluted plasma is inoculated with S. aureus, jellification occurs w/in 24 hours. This result is due to coagulase converting fibrinogen in the plasma to fibrin and a positive test confirms a positive slide test.

Growth of a beta-haemolitic stretptococcus on blood agar medium showing large zones of clear haemolysis around small transparent colonies.

Staphylococcus aureus on Blood Agar Plate (BAP) Showing beta hemolytic hemolysis; complete hemolysis

Growth of pneumococci on blood agar showing characteristic ‘draughtsman’ colonies with concentric surface rings.

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

Gram stain of Streptococci in broth culture (x1250). Gram stain of a positive blood culture broth demonstrating gram-positive cocci arranged in chains. Streptococci are normal microbiota in the upper respiratory tract and the gastrointestinal tract. For this reason, gram stains of specimens from these sites are not helpful in diagnosing infections caused by the pathogenic Streptococci such as S. pyogenes and S. pneumoniae.

Neisseria Gonorrhea Gram stain for N. Gonorrhea

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

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