Disk diffusion susceptibility tests - ChulaDisk diffusion susceptibility test-2 Interpretation...
Transcript of Disk diffusion susceptibility tests - ChulaDisk diffusion susceptibility test-2 Interpretation...
Disk diffusion susceptibility test
Objective : To determine antimicrobial susceptibility of bacterial strains
Preparation of inoculum
Pick 4-5 single colonies
Re-suspend the bacterial
colonies and adjust turbidity to 0.5 McFarland
Inoculate Muller Hinton
agar (MHA) plate using sterile cotton swab
Sterile cotton swab
Testing procedure
Incubate at 35ₒC
for 16-18 hr
Apply antibiotic disk onto
MHA using forceps or applicator
Am
CL
CT
GM
NA
DC
Measure the diameters of the zone of complete inhibition (mm.)
60 ₒ 60
ₒ
saline/broth
Am
CL
CT
GM
DC
NA
Inhibition (clear) zone
Bacterial growth
Disk diffusion susceptibility test-1
Disk diffusion susceptibility test-2
Interpretation
Examples of criteria for interpretation of susceptible, intermediate or resistant E.coli and
other enteric bacteria
Antimicrobial agent
Disk content Zone diameter (mm) MIC interpretive
standard (µg/ml)
S I R S I R
Ampicillin 10 µg ≥17 14-16 ≤13 ≤8 16 ≥32
Tetracycline 30 µg ≥15 12-14 ≤11 ≤4 8 ≥16
Gentamicin 10 µg ≥15 13-14 ≤12 ≤4 8 ≥16
Antimicrobial agent
Disk content
Disk diffusion testing Acceptable limit (mm)
MIC testing Acceptable limit (µg/ml)
A B C A B C
Ampicillin 10 µg 16-22 - - 2-8 - 0.5-2
Tetracycline 30 µg 18-25 - - 0.5-2 8-32 0.12-1
Gentamicin 10 µg 19-26 16-21 - 0.25-1 0.5-2 0.12-1
A = E.coli ATCC® 25922
B = Pseudomonas aeroginosa ATCC ® 27853 C = Staphylococcus aureus ATCC ® 29213
Disk diffusion test and MIC test - Acceptable limit for quality control strains used to
monitor accuracy
CLSI, 9th edition
Antimicrobial agent Disk content Zone diameter (mm) MIC interpretive
standard (µg/ml)
S I R S I R
Ampicillin 10 µg ≥29 - ≤28 ≤0.25 - ≥0.5
Tetracycline 30 µg ≥19 15-18 ≤14 ≤4 8 ≥16
Gentamicin 10 µg ≥15 13-14 ≤12 ≤4 8 ≥16
Examples of criteria for interpretation of susceptible, intermediate or resistant S.aureus
E- test
Objective: To determine of an approximate-MIC value
Preparation of inoculum
60 ₒ 60
ₒ
saline/broth
Apply E-test strips onto MHA
MIC
Bacterial growth
Pick 4-5 single colonies
Re-suspend the bacterial
colonies and adjust turbidity to 0.5 McFarland
Inoculate Muller Hinton
agar (MHA) plate using sterile cotton swab
Testing procedure
Incubate at 35ₒC for 16-18 hr
Agar Dilution Method
Objective : To determine Minimal Inhibitory Concentration (MIC) of
antimicrobial agents
Streak the test bacterial strains on Muller
Hinton Agar (MHA) to get single colonies,
37 C overnight
II. Preparation of agar plates with a serially-diluted antibiotic
I. Preparation of test bacteria
MHA
Antibiotic
stock solution
Prepare a serial two-fold dilution of
antibiotics
Add 2 ml of antibiotic solution to 18 ml
of MHA
Agar Dilution Method-1
Pour into petridish and leave MHA
solidified
Standard strains:
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
ATCC27853
ATCC 27853
ATCC 29213
III. Preparation of the bacterial suspension
Interpretation
The MIC is the lowest concentration of an antimicrobial agent that
completely inhibits visible growth of the bacterial isolate tested.
4 g/ml 0.5 g/ml 1 g/ml 2 g/ml 0 g/ml
MIC = 2 g/ml
Agar Dilution Method-2
Transfer the growth into saline solution.
Adjust turbidity to reach 0.5 McFarland Standard
Dilute bacterial suspension 1:10 (107 CFU/ml)
Transfer 50 l of each bacterial dilution into
a microtitre plate
Inoculate onto MHA with antibiotics and incubate
the plates at 37C for 18–24 h
IV. Inoculation of test bacteria 107 CFU/ml
108 CFU/ml
Broth Microdilution Method
I. Preparation of test bacteria
Objective: To determine antibiotic susceptibility of bacterial isolates in
terms of MIC
Streak bacterial strain on Mueller-Hinton agar at 37◦C, overnight
II. Preparation of broth with a serially-diluted antibiotic
Antibiotic
stock solution
Add 50 µl of antibiotic stock solution to the first column
Mix suspension thoroughly and transfer 50 µl of suspension to the next column Repeat until reach the last wells
Add 50 µl of Mueller-Hinton broth in the microtitre plate
Two-fold antibiotic dilution
Mueller-Hinton broth (MHB)
2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 20
Broth Microdilution Method-1
20 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 +VE
Minimal inhibitory concentration (MIC)
III Preparation of bacterial suspension
Re-suspend at least 3-4 single colonies from a pure culture into sterile
normal saline and adjust turbidity to 0.5 McFarland Dilute bacterial suspension 1:10 (107 CFU/ml)
IV Inoculation of bacterial suspension
Transfer 50 µl of bacterial suspension into microtitre plate Seal with parafilm and incubate the plate at 37 ◦C for 18-22 hours
MIC is recorded as the lowest concentration that inhibits visible growth of bacteria
Interpretation
20 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10
Positive control
Bacterial suspension
Bacterial suspension
Broth Microdilution Method-2
Phenol Coefficient Method
Test organisms:
Staphylococcus aureus ATCC 6538
Salmonella Choleraesuis ATCC 10708
Pseudomonas aeruginosa ATCC 15442
Test culture preparation
Nutrient agar (NA), 37◦C, 22-26 hr
Nutrient broth (NB)
37◦C 22-26 hr
At least 4 consecutive daily transfers
1 loopful
Day 5
Disinfectant & phenol preparation
Disinfectant
Add 0.5 ml stock inoculum
(S. aureus ATCC 6538) to
5 ml Disinfectants or Phenol 5 ml
5 min 10 min 15 min
10 ml NB in triplicates
Test procedure
Incubate at 37◦C for 48 hr
1 ml disinfectant
99 ml sterile distilled water
1:100 1:3001:200
5% (w/v) phenol stock
1:70 1:80 1:90
Day 4Day 3Day 2Day 1
Objective : To measure the efficacy of antiseptics and disinfectants by relating to a disinfecting power of phenol
Phenol Coefficient Method -1
Interpertation
Growth (+)No Growth (-)
Phenol Coefficient =
Example:
Phenol Coefficient = 200/80
= 2.5
Only the phenol coefficient ≥0.05 will be used in next step.
Dilution 5 min 10 min 15 min
1:100 - - - - - - - - -
1:200 + + + - - - - - -
1:300 + + + + + + + + +
Dilution 5 min 10 min 15 min
1:70 - - - - - - - - -
1:80 + + + - - - - - -
1:90 + + + + + + + + +
Disinfectant X
5% Phenol
Highest dilution of disinfectant killing test
organism in 10 min but not in 5 min
Highest dilution of Phenol killing
test organism in 10 min but not in 5 min
Calculation:
Phenol Coefficient Method -2
Carrier preparation
Soak in 1% asparagine
overnight
Incubate 37◦C,
30-60 min
Sterile membrane filter
10 min
Soak in S. aureus ATCC6538
inoculum, 15 min
60 carriers
Diluted disinfectants (1:50)
1 Carrier/ tube
NB
Incubate 37◦C, 48 hr
Interpretation
Growth(+)No Growth (-)
If no growth is found in 59-60 carriers (≤2 positive carriers), pass the test.
If growth is present in 59-60 carriers (≥2 positive carriers), retest to confirm the results.
Test procedure
2.5 x 20 = 50 Dilution Disinfectant 1 : 50
Calculation of test dilution
Use Dilution Method
Dry on sterile membrane filter
sterilization
Carriers
(Stainless steel cylinders)
Phenol coefficient x 20
Phenol Coefficient Method -3
100 µl
cell suspension
Plate 1. Escherichia coli, pH 7.2
Re-suspend in normal saline
and adjust to 2 MacFarland
Incubate at 37 °C
for 18 hours
Add trimethoprim
to 0.05 ppm
100 ml
Assay agar,
pH 7.2
Plate 2. Micrococcus luteus, pH 8
Incubate at 37 °C
for 18 hours
100 ml Assay agar 8.0 200 µl cell suspension
European six plate test (ESPT)
Pipette 8 ml into petridish
Objective: To determine the presence of antimicrobial residues
using microbial inhibition plate assay based techniques
E. coli M. luteus
I. Preparation of test bacteria
Escherichia coli ATCC 25922 (cell suspension) Micrococcus luteus ATCC 9341 (cell suspension) Bacillus cereus ATCC 11778 (spore suspension) Bacillus subtilis ATCC 6633 (spore suspension)
Resuspend in MSS
& adjust to 2 MacFarland
Pipette 8 ml into petridish
European six plate test -1
Incubate at 37°C
for 3-5 days
100 ml
Assay agar 6.0
Plate 3 : Bacillus cereus, pH 6
100 µl
spore suspension
Pipette 8 ml into petridish
Plate 4-6 : Bacillus subtilis, pH 6, 7.2 & 8
Incubate at 37°C
for 3-5 days
spore suspension
100 µl 100 µl
100 ml
Assay agar 6.0
100 ml
Assay agar 8.0
100 µl
Pipette 8 ml into petridishes
B. cereus
B. subtilis
Re-suspend in normal saline
and adjust to 2 MacFarland
Re-suspend in normal saline
and adjust to 2 MacFarland
Add trimethoprim
to 0.05 ppm
100 ml
Assay agar, pH 7.2
European six plate test -2
Positive = The inhibition zone measure from the edge of meat > 2 mm
Negative = No inhibition zone
Meat Sample Serum / Secretions
Cut inside meat sample into 4x4x4 mm and put on assay agar
Sterile outside meat sample with flame
“Use aseptic technique”
Meat sample
Serum / Secretion sample
Incubate at 37 °C, 18 hours
Incubate at 37 °C, 18 hours
Dip serum / secretion sample and put on assay agar
European six plate test -3
II. Testing procedure
III. Interpretation
pH 6
Tetracyclines : Oxytetracycline, Chlortetracycline,
Tetracycline, Deoxycycline
Chloramphenical, Thianphenical
Nitrofurantoin, Nitrofurazone, Furazolidone
Peniciline G, Cloxacillin
Oxolinic acid
pH 7.2
Sulfaquinoxaline, Sulfadiazine, Sulfadimethoxine,
Sulfamethazine, Sulfamonomethoxine,
Sulfapyridine, Sulfamerazine Fluoroquinolones : Enrofloxacin, Ciprofloxacin,
Flumequine
pH 8
Tylosin, Erythomycin, Neomycin, Streptomycin
European six plate test -4
IV. Possible antimicrobial groups recovering from European Six Plate Test (ESPT)
AM-TEST and CM-TEST
Objective : To determine antimicrobial residues in milk, meat and other food of
animal origin
Detection method for AM-Test & CM-Test
Interpretation
- Negative. Color of the medium changes to yellow.
+ Positive. Color of the medium remains purple.
- +
Cut the test
tube including
negative control & label
Drop 0.1 ml
of milk sample
Seal the tubes
Wrap ~10 g
meat sample
with cloth & squeeze
Soak paper
disk with meat juice
Place into
the tube & seal
AM-Test:
Incubate at 65±1oC
for 2-3 hours
CM-Test:
Incubate at 65±1oC
for 2.5-3.5 hours
II. For AM-Test
II. For CM-Test
I. For AM-Test & CM-Test
Ctrl Ctrl Negative control. Color of the medium changes to
yellow. yellow.