Isolation and Identification of Gram Positive Cocci

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

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

Mohammed laqqan

Direct identification The mannitol fermentation test. The Coagulase Test The DNase Test Novobiocin (NB) disc Mohammed laqqan

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. Mohammed laqqan

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

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. Mohammed laqqan

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. 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 Mohammed laqqan

Identification of Gram-positive cocci Mohammed laqqan

types Of Streptococcus Mohammed laqqan

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 Mohammed laqqan

(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 Mohammed laqqan

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

Sensitivity to the antibiotic bacitracin 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. Mohammed laqqan

Bacitracin sensitivity Mohammed laqqan

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) Mohammed laqqan

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. 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 Not groupable by Lancefield groups No group specific CHO Streptococcus pneumoniae Primary human pathogen S.sanguis S.mitis S.mutans S.salivarius Mohammed laqqan

Mohammed laqqan

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 Mohammed laqqan

Mohammed laqqan

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 Mohammed laqqan

Mohammed laqqan

Enterococcus Mohammed laqqan

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 Mohammed laqqan

End of lecture Mohammed laqqan