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

https://www.pinterest.com/pin/381680137149195687/?d=t&mt=login

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

37

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

38

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

43

E-test

MIC 0.25 ug/ml

44

• 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

47

Drug sensitivity test Broth tube dilution methodMinimun Inhibitory Concentration MIC = µg/mlMinimum Bactericidal Concentration MBC = µg/ml

48

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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Marut Laohaviroj

Department of MicrobiologyFaculty of Medicine, KKU