Lab Pyogenic cocci
Transcript of Lab Pyogenic cocci
Lab Pyogenic cocci
Department of Microbiology
Faculty of Medicine, KKU
After completion of the laboratory, student could understand and describe as following:
1. Bacteriology of pyogenic cocci
2. Differentiation of pyogenic cocci
3. principle method and interpretation each result:
• Coagulase test, Catalase test, Bacitracin test, Optochin test, CTA sugar test
Learning objectives
Pyogenic cocci
Pyogenic = ท ำใหเ้กดิหนอง
Streptococcus spp.
Staphylococcus spp.
Neisseria spp.
Staphylococci
• Gram’s stain: Gram positive cocci (cluster)
• Colony morphology on blood agar
• Catalase test (+)
• Biochemical test in Plasma, PR mannitol, PR glucose
“Grape-like cluster”Staphylococcus aureusStaphyloccocus epidermidis
Staphylococcus spp. : Catalase test
“Positive”
Staphylococcus spp. : Biochemical test
Coagulation test PR- glucosePR-manitol
(+)
(-)
“Plasma”
(+)
Gram Positive cocci
Catalase test
+ -
Staphylococci
Plasma, PR-manitol, PR-glucose
อ่านผลเทียบในตาราง S
-strep. gr.A
Streptococci
• Gram’s stain
(How different between Streptococcus spp.
and Streptococcus pneumoniae)
• Catalase test (-)
• Hemolysis (, , )
- Bacitracin test
- Optochin test
- Bile esculin test and 6.5% NaCl
Streptococcus spp. : Gram’s stain
Streptococcus pneumoniae
Chain arrangement
“Diplococci lancet shape”
Streptococcus spp. : Catalase test
“Negative”
Streptococcus spp. : colony on blood agar
Staphylococcus spp. : β-hemolysis
-hemolysis
Bacitracin
S R
-strep. gr.A
S. pyogenes
-strep. non gr.A
S. agalactiae
A
Bacitracin sensitive (inhibition zone)
Bacitracin resistance
Staphylococcus spp. : α-hemolysis
-hemolysis
Optochin
S. pneumoniae -strep อืน่ๆ
Viridans
S R
Optochin sensitive (inhibition zone)
Optochin resistance
Gram Positive cocci
Catalase test
+ -
Staphylococci
Plasma, PR-manitol, PR-glucose
อำ่นผลเทียบในตำรำง
-hemolysis -hemolysis γ-hemolysis
Bacitracin
S R
-strep. gr.A
S. pyogenes
-strep. non gr.A
S. agalactiae
Optochin
S. pneumoniae -strep อืน่ๆ
Viridans
Bile esculin
+ -
6.5% NaCl
+ -
Gr.DEnterococci
Rule-out:
1. Gr.D non-enterococci 2. Viridans Streptococci
S R
Neisseria spp.
• Gram’s stain (Diplococci kidney shape)
• GC on chocolate blood agar in candle jar
• Neisseria spp. on blood agar
• Oxidase test
• Biochemical test – oxidation of glucose,
maltose, sucrose
Neisseria spp. : Gram’s stain
“Diplococci kidney shape”
Intracellular Gram negative diplococci
Neisseria gonorrhoeae: colony on chocolate agar
GC on chocolate agar in candle jar
“blood agar”
Neisseria gonorrhoeae
Neisseria sp.
Neisseria spp. :Oxidase test
Neisseria spp. :Biochemical test
oxidation of glucose, maltose, sucrose
Oxidase test
Neisseria
CTA glucose CTA maltose CTA sucrose
+ - - N. gonorrhoeae
+ + - N. meningitidis
+ + + N. sicca, N. mucosa
Gram negative
diplococci kidney shape
(+)
Pyogenic cocci identification
Unknown bacterial colony from clinical specimen
Gram’s staining
Clinical specimen
- Pus
- Sputum
- Blood
- CSF
etc.
Culture on agarGram’s stain
Gram’s stain result
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Gram Positive cocci
Catalase test
Catalase result
Positive
Negative
Unknown bacterial strains
Gram’s staining
Gram Positive cocci
Catalase test
+ -
Staphylococcus Strephylococcus
- Plasma- PR glucose- PR manitol
Staphylococcus aureus
Catalase result
Positive
Negative
Unknown bacterial strains
Gram’s staining
Gram Positive cocci
Catalase test
+ -
Hemolysis
Staphylococcus Strephylococcus
- β-hemolysis --> Bacitracin- α-hemolysis --> Optochin
Bacitracin sensitive (inhibition zone)
S. pyogenes
Optochin sensitive (inhibition zone)
S. pneumoniae
Pyogenic cocci- unknown identification
Staphylococci
Staphylococcus aureus
Gram’s stain result
Gram Negative diplococci kidney shape
Oxidase test
Gram Negative diplococci kidney shape
Neisseria spp. : Identification
Oxidase test
Neisseria
CTA glucose CTA maltose CTA sucrose
+ - - N. gonorrhoeae
+ + - N. meningitidis
+ + + N. sicca, N. mucosa
Gram negative
diplococci kidney shape
(+)
N. gonorrhoeae
Pyogenic cocci identification
Unknown bacterial strains
Gram’s staining
Gram Positive cocci
Catalase test
+ -
Gram Negative diplococci kidney shape
Neisseria
Oxidase test
- CTA glucose
- CTA maltose- CTA sucroseHemolysis
Staph. Strep.
- Plasma- PR glucose- PR manitol - β-hemolysis --> Bacitracin
- α-hemolysis --> Optochin
Gram Negative diplococci kidney shape
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It is used to determine which antimicrobials will inhibit the growth of the bacteria causing a specific infection.
The results from this test will help a doctor determine which drugs are likely to be most effective in treating a person's infection.
Drug suscetibility tests
1. Agar disk diffusion method
2. Dilution method
• Broth tube dilution method
• Agar plate dilution method
Drug susceptibility test
• Kirby- Bauer method
• A qualitative method: susceptible,
intermediate susceptible, resistant
• Used with rapid growing bacteria
: Enterobacteriaceae, Staphylococcus
• Effective for most routine testing
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Agar disk diffusion method
Bacterial culture
S. aureus (S)
E. coli (S)
Pseudomonas aeruginosa (S)
Klebsiella pneumoniae (S)
Salmonella typhi (S)
39Streak 3 timesPlace antibiotics disc
Antibiotic discs
AMP = Ampicillin
C = Chloramphenicol
CN = Gentamicin
K = Kanamycin
P = PenicillinT = Tretacycline
NN = Tobramycin
Mc Farland No. 0.5- BaSO4 (BaCl2 + H2SO4)
1.5x 108 bacterial cells/ ml
Agar disk diffusion method
R IS S 41
Tobramycin (NN) 10 mg; 11 or less; 12-13 ; 14 or more
Inhibition zone
S = Sensitive
IS = Intermediate Sensitive
R = Resistant
Zone of inhibition
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disc R IR S Zone size
Interprete
AMP 11 12-13 14 0 R
C 12 13-17 18 0 R
CN 13 16 S
K 13 14-17 18 0 R
P 11 12-21 22 0 R
T 14 15-18 19 7 R
NN 11 12-13 14 21 S
P. aeruginosa
Inhibition zone
S = Sensitive
IS = Intermediate Sensitive
R = Resistant
Antibiotic discs
AMP = Ampicillin
C = Chloramphenicol
CN = Gentamicin
K = Kanamycin
P = PenicillinT = Tretacycline
NN = Tobramycin
Agar disk diffusion method
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E-test
MIC 0.25 ug/ml
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• A quantitative method:
MIC = Minimal inhibitory concentration
: The lowest concentration that inhibits visible growth of an organism
MBC = Minimal Bactericidal concentration
: The lowest concentration that kill an organism
A quantitative susceptibility test: need for proper management of antimicrobial therapy
e.g. bacteremia, endocarditis
Broth tube dilution method
AntibioticSolution 4 ml(Ampicillin 32
mg/ml)
16 8 4 2 1
MHB - 2 2 2 2 2 2(ml)
(1) (2) (3) (4) (5) (6) (7)
2ml 2ml 2ml2ml 2ml
45เชือ้ + + + + + + +
2ml
Control
Broth tube dilution method
MIC = Minimal inhibitory concentration
4 ug/ml8 ug/ml 2 ug/ml
MBC = Minimal bactericidal concentration
MIC = mg /ml
MBC = mg /ml
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2
4
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Drug sensitivity test Broth tube dilution methodMinimun Inhibitory Concentration MIC = µg/mlMinimum Bactericidal Concentration MBC = µg/ml
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32 16 8 4 2 1 Control
Drug susceptibility test : broth dilution
Microtiter plate
Drug susceptibility test: microdilution
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Drug susceptibility test : interpretation
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Chulapan engchanilSakawrat kanthawongUmaporn Yordpratum
Marut Laohaviroj
Department of MicrobiologyFaculty of Medicine, KKU