1 / 30

Isolation and Identification of Gram Positive Cocci

Isolation and Identification of Gram Positive Cocci. Identified method for Staphylococci. Gram-stain Isolation and culture Pure culture Direct identif ication. Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters. Direct identif ication.

odism
Télécharger la présentation

Isolation and Identification of Gram Positive Cocci

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Isolation and Identification of Gram Positive Cocci

  2. Identified method for Staphylococci • Gram-stain • Isolation and culture • Pure culture • Directidentification

  3. Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters

  4. Directidentification • The mannitol fermentation test. • The Coagulase Test • The DNase Test • Novobiocin (NB) disc

  5. The mannitol fermentation test. • Inoculate the bacteria into a mannitol micro-tube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.

  6. The Coagulase Test • Coagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. • It might be a virulence factor with the coagulated blood around the bacteria protecting them from the immune system. • Coagulase-negative strains are often as pathogenic as coagulase-positive strains.

  7. The DNase Test • Inoculate DNase agar plates with a loop so that the growth is in plaques about 1 cm in diameter.I • ncubate at 370C overnight. • Flood the plate with 1 N hydrochloric acid.Clearing around the colonies indicates DNase activity. • The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a cloudy precipitate. • A few other bacteria,e.g. Serratia,may give a positive reaction.

  8. Staphylococcus aureus Growing on DNase Agar

  9. Blood agar with a novobiocin (NB) disc Staphylococcus aureus Growing on Blood Agar Staphylococcus saprophyticus Growing on Blood Agar

  10. Identification of Gram-positive cocci

  11. types Of Streptococcus

  12. β haemolytic steps: Lancefield groups • Historically • A, B, C, F and G group specific antigens cell wall polysaccharide • group D and Enterococci lipoteichoic acid moiety Not always β haemolytic • Now: 18 different groups of strep: from A-H, and from K-T

  13. (β) Haemolytic Streptococcus groups • S. pyogenes • Group A • S. agalactiae • Group B (occasionally α or none) • Streptococcus equismilis • Group C (occasionally α or none) • Streptococcus bovis • Group D (α or none, occasionally β) • Streptococcus anginosus • Group F & G

  14. Gp A strep: S. pyogenes Laboratory ID • Gram +ve • Catalase • Haemolysis • small, round, grey colonies • β haemolysis • zone is large • Gp A antigen

  15. Isolation and Identification of group A beta hemolytic (Streptococcus pyogenes) • on Blood agar. Streptococcus pyogenes produces a zone of beta hemolysis around 2-3mm in diameter surrounding each colony • Sensitivity to the antibiotic bacitracin • Group A Strep is senstive, other b haemolytic streps are resistant.

  16. Bacitracin sensitivity

  17. Group B: S. agalactiae • S. agalactiae • Identification • Catalase negative • Appearance on BA • Larger colonies than Gp A • Small zone of hemolysis • Latex agglutination Gp B • Bacitracin resistant • CAMP positive • Growth on Mac (weak)

  18. Presumptive ID Tests - CAMP • The “CAMP” test (acronym for developers) is used to differentiate S. agalactiae (GBS), which is positive, from other beta hemolytic strep, all of which are negative • “CAMP factor” is a soluble hemolysin produced by GBS that combines in a synergistic way with a similar hemolysin of S. aureus to form an arrowhead zone of clearing. See image • The staph is streaked perpendicularly to the unknown strep. After appropriate incubation time an “arrowhead-shaped” clear zone of hemolysis will appear.

  19. S. agalactiae • Synergistic haemolysis • observed between betahemolytic • Staphylococcus • aureus and group B • streptococci. • 􀁺 Positive reaction: arrowhead haemolysis pattern

  20. α – Haemolytic streps: Viridans streps • Not groupable by Lancefield groups • No group specific CHO • Streptococcus pneumoniae • Primary human pathogen • S.sanguis • S.mitis • S.mutans • S.salivarius

  21. The Pneumococcus (Streptococcus pneumoniae) 1: Isolation on Blood agar • Pneumococci frequently require enriched media and increased CO2 tension for initial isolation. • They are usually isolated on Blood agar and incubated in a candle jar (a closed container in which a lit candle is placed to remove O2 and increase CO2 ) at 37C. On Blood agar, colonies appear small, shiny, and translucent. • They are surrounded by a zone of alpha hemolysis 2: Optochin sensitivity • Pneumococci are the only streptococci that are sensitive to the drug optochin(ethylhydrocupreine hydrochloride). 3: Gram stain:gram-positive, diplococci

  22. The Genus Enterococcus • Enterococci are gram-positive streptococci, typically occurring in pairs and short chains, that are normal flora of the intestinal tract. Enterococci responsible for a variety of opportunistic infections in humans, and serologically belong to Lancefield group D streptococci. • On Bile Esculin agar • Unlike most bacteria, the enterococci will grow in the presence of the bile salts in the medium. • They hydrolyze the esculin, producing esculetin which reacts with the iron salts in the medium turning the agar black

  23. Enterococcus

  24. Laboratory ID: Enterococcus • white colonies • alpha and no haemolysis (rarely beta) • Also grow on MacConkey • Resembles S. pneumoniae in Gram stains • Bile esculin positive • Grows on Mac

  25. End of lecture

More Related