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.