General Microbiology Laboratory Isolation and Identification of Gram Positive Cocci

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

Mohammed laqqan TestStaphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Mannitol fermentationPositiveNegativeUsually positive

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 37 0 C 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 S Staphylococcus saprophyticus Growing on Blood Agar R

Mohammed laqqan Types Of Streptococcus

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

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

Mohammed laqqan Isolation and Identification of (Streptococcus pyogenes) 1.on Blood agar. Streptococcus pyogenes produces a zone of beta hemolysis around 2-3mm 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 S. agalactiae Identification  Catalase negative  Bacitracin resistant  CAMP positive  Growth on Mac (weak)  Appearance on BA  Larger colonies than Group A  Small zone of hemolysis

Mohammed laqqan CAMP test  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.  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

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. 3: Gram stain: gram-positive, diplococci

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

Enterococcus

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