革兰氏阳性球菌的分离与鉴定
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General Microbiology Laboratory
Isolation and Identification of Gram Positive Cocci
Identified method for Staphylococci Gram-stain Isolation and culture Pure culture Direct identification
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
Bacitracin ቤተ መጻሕፍቲ ባይዱensitivity
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
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
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 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
S. agalactiae
Synergistic haemolysis observed between betahemolytic Staphylococcus aureus and group B streptococci. ������ Positive reaction: arrowhead haemolysis pattern
Mohammed laqqan
Isolation and Identification of group A beta hemolytic (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
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
Staphylococcus saprophyticus Growing on Blood Agar
Mohammed laqqan
Identification of Gram-positive cocci
Mohammed laqqan
types Of Streptococcus
Mohammed laqqan
Mohammed laqqan
Staphylococcus aureus Growing on DNase Agar
Mohammed laqqan
Blood agar with a novobiocin (NB) disc
Staphylococcus aureus Growing on Blood Agar
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
Test
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Mannitol fermentation
Positive
Negative
Usually positive
Mohammed laqqan
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
(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
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
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 mannitol fermentation test. Inoculate the bacteria into a mannitol microtube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.
Isolation and Identification of Gram Positive Cocci
Identified method for Staphylococci Gram-stain Isolation and culture Pure culture Direct identification
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
Bacitracin ቤተ መጻሕፍቲ ባይዱensitivity
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
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
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 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
S. agalactiae
Synergistic haemolysis observed between betahemolytic Staphylococcus aureus and group B streptococci. ������ Positive reaction: arrowhead haemolysis pattern
Mohammed laqqan
Isolation and Identification of group A beta hemolytic (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
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
Staphylococcus saprophyticus Growing on Blood Agar
Mohammed laqqan
Identification of Gram-positive cocci
Mohammed laqqan
types Of Streptococcus
Mohammed laqqan
Mohammed laqqan
Staphylococcus aureus Growing on DNase Agar
Mohammed laqqan
Blood agar with a novobiocin (NB) disc
Staphylococcus aureus Growing on Blood Agar
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
Test
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Mannitol fermentation
Positive
Negative
Usually positive
Mohammed laqqan
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
(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
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
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 mannitol fermentation test. Inoculate the bacteria into a mannitol microtube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.