General Microbiology Laboratory Isolation and Identification of Gram

General Microbiology Laboratory Isolation and Identification of Gram Positive Cocci

Identified method for Staphylococci v. Gram-stain v Isolation and culture v Pure culture v Direct identification Mohammed laqqan

Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters Mohammed laqqan

Direct identification v. Coagulase Test v. Mannitol fermentation test. v. DNase Test v Novobiocin (NB) disc Mohammed laqqan

Test Staphylococcus aureus Staphylococcus epidermidis Mannitol fermentation Positive Negative Mohammed laqqan Staphylococcus saprophyticus Usually positive

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

Staphylococcus aureus Growing on DNase Agar Mohammed laqqan

Blood agar with a novobiocin (NB) disc Staphylococcus aureus Growing on Blood Agar Staphylococcus saprophyticus Growing on Blood Agar S R Mohammed laqqan

Types Of Streptococcus Mohammed laqqan

(β) Haemolytic Streptococcus groups v S. pyogenes • Group A v S. agalactiae • Group B (occasionally α or none) v Streptococcus bovis • Group D (α or none, occasionally β) Mohammed laqqan

Group A strep: S. pyogenes v Gram +ve cocci v Catalase (+ve) v Small, round, grey colonies v β-haemolysis v zone is large v Group A antigen Mohammed laqqan

Isolation and Identification of (Streptococcus pyogenes) 1. on Blood agar. Streptococcus pyogenes produces a zone of beta hemolysis around 2 -3 mm in diameter surrounding each colony 2. Sensitivity to the antibiotic bacitracin 3. Group A Strep is senstive, other b haemolytic streps are resistant. Mohammed laqqan

Bacitracin sensitivity Mohammed laqqan

Identification of Streptococci Mohammed laqqan

Group B: S. agalactiae Identification v Catalase negative v. Bacitracin resistant v CAMP positive v Growth on Mac (weak) v. Appearance on BA v Larger colonies than Group A v Small zone of hemolysis Mohammed laqqan

CAMP test v 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 v “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. v The staph is streaked perpendicularly to the unknown strep. After appropriate incubation time an “arrowhead-shaped” clear zone of hemolysis will appear. Mohammed laqqan

S. agalactiae §Synergistic haemolysis observed between betahemolytic Staphylococcus aureus and group B streptococci. �� Positive reaction: arrowhead haemolysis pattern Mohammed laqqan

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

Mohammed laqqan

The Pneumococcus (Streptococcus pneumoniae) 1: Isolation on Blood agar • Pneumococci frequently require enriched media and increased CO 2 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 O 2 and increase CO 2 ) at 37 C. 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. 3: Gram stain: gram-positive, diplococci Mohammed laqqan

Mohammed laqqan

The Genus Enterococcus v 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. v On Bile Esculin agar v Unlike most bacteria, the enterococci will grow in the presence of the bile salts in the medium. v They hydrolyze the esculin, producing esculetin which reacts with the iron salts in the medium turning the agar black Mohammed laqqan

Mohammed laqqan

Enterococcus Mohammed laqqan

Enterococcus v. White colonies v Alpha and no haemolysis (rarely beta) v Also grow on Mac. Conkey v Resembles S. pneumoniae in Gram stains v Bile esculin positive v Grows on Mac Mohammed laqqan

End of lecture Mohammed laqqan