Antimicrobial susceptibility test (LAB-1)

Antimicrobial

susceptibility test

(LAB-1)

Antimicrobial susceptibility test

Agar dilution method

Broth microdilution method

4.1 Disk diffusion assay

4.2 Dilution method

2. Preparation of growth media

3. Preparation of antibiotics

4. AST

1. Preparation of bacterial isolates

& inoculum


Day 2 AST

Day 3 Reading results

Day 1 Preparing materials

(Growth media, antibiotics, bacterial culture

etc.)

Bacterial isolates and

inoculum

Streak to get single isolates on MHA,

35oC (37C) overnight

1. Quality control strains

Escherichia coli ATCC 25922

Staphylococcus aureus ATCC 29213

Pseudomonas aeruginosa ATCC 27853

2. Bacterial samples

Bacterial isolates

Routine inoculum preparation

Pure culture, 4-5 isolated colonies,

16-24 hrs old

Standardized inoculum size

using turbidity standard

(McFarland standard)

0.5 McFarland = 1.5 x 108 CFU/ml

Adjust by eye or using instrument


Turbidity and McFarland

McFarland Densitometer A BaSO4 turbidity standard

Used conc. 1x108 1x106 1x107

CFU/ml CFU/ml CFU/ml

Bacterial cells in inoculum

Final - 1x104 1x104

CFU/well CFU/spot

Initial 1x108 1x108 1x108

CFU/ml CFU/ml CFU/ml

Growth media

Mueller-Hinton media (agar or broth)

pH

Cation conc.

Blood and serum suppl.

Thymidine content

Thickness

Growth media

Growth media

MHA

Autoclave

(121oC, 1.5 psi,

15 min) or

Antibiotics

Antimicrobial agents

DO NOT use pharmacy stock or other clinical preparation

Store as recommended by the manufacturers

Warm to room temperature before opening

If possible, weigh more than 100 mg.

Potency = (Assay purity).(Active fraction).(1-water content)

Weight = Volume(ml).Concentration(/ml)

Potency(g/mg)

Volume (ml) = Weight(mg). Potency(g/mg)

Concentration (g/ml)

Stock solutions

Prepare stock solution at concentration at least

1000 g/ml or 10 times the highest concentration tested

Filter them through a membrane filter

Store the aliquots of sterile stock at -70oC or colder

(6 months)

Consult Vet01-S2 Tables 2A and 2B for number of

concentrations tested

To prepare 100 ml of a stock solution containing 1280 g/ml

of streptomycin with streptomycin powder with the potency

of 990 g/mg.

Example

Weight = Volume(ml).Concentration(/ml)

Potency(g/mg)

= 100*1280/990

= 129 mg

Strepmycin should be weighed 129-150 mg.

Example

Volume (ml) = Weight mg). Potency(g/mg)

Concentration (g/ml)

= 145*990/1280

= 112 ml

If the actual weight is 145 mg the volume of diluent needed

is as follows:


Day 2 AST

Day 3 Reading results

Day 1 Preparing materials

(Growth media, antibiotics, culture etc.)

Disk diffusion method

Disk diffusion method

1. Preparation of agar plates

2. Preparation of bacterial isolates &

inoculum

3. Inoculating agar plates

5. Interpretation

4. Applying disk

MHA

autoclave

Day 1 Preparation of growth media

Day 1 Preparation of bacterial isolates

Streak the bacterial strain on MHA,

Incubate at 35oC (37C) for 16-20 h

QC strains: E. coli ATCC 25922

S. aureus ATCC 29213

P. aeruginosa ATCC 27853

Samples: Salmonella S……

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

60 ₒ 60

ₒ

saline/broth

Day 2 Preparation of inoculum & inoculated

agar plates

Incubate at 35ₒC (37oC), for 16-18 hr

Apply antibiotic disk onto MHA

using sterile forceps or applicators

SXT

GEN

CIP

AMC

Day 2 Application of antimicrobial disk


Multiple inoculators



2. Prepare agar plates with antibiotics

3. Preparation of the bacterial suspension

& plate inoculation

4. Reading results & Interpretation

256 g/ml 128 g/ml 16 g/ml 32 g/ml 64 g/ml

2 ml 2 ml 2 ml 2 ml 2 ml

Day 1 Prepare agar plates with antibiotics

Total volume = 20 ml per plate (18 + 2)

Antibiotic stock solution

Tetracycline 10 mg/ml

C1V1 = C2V2

C1(2ml) = 256 g/ml(20ml)

C1 = 2,560 g/ml

Two-fold antibiotic dilution

C1V1 = C2V2

10 mg/ml V1 = 2,560 g/ml(10ml)

V1 = 2,560 l

Highest concentration = 256 g/ml

Day 1 Prepare antibiotic stock solution

Add 2 ml of antibiotic solution (2,560 g/ml) into

18 ml of MHA separately into plates

C1V1 = C2V2

C1(2ml) = 256 g/ml(20ml)

C1 = 2,560 g/ml

Method A


Mix 2 ml of antibiotic solution &

18 ml of MHA separately in tubes (1:10)

and pour into plates

Method B


256 g/ml 128 g/ml

8 g/ml

16 g/ml 32 g/ml 64 g/ml

4 g/ml 2 g/ml 1 g/ml 0 g/ml

Control

plates



Grow the bacterial strains on MHA, 37C overnight

Control strains: E. coli ATCC 25922



Samples: Salmonella S1-S10

Transfer 3-5 colonies of a overnight culture into 2 ml of 0.85%NaCl

Adjust turbidity to 0.5 McFarland Standard (1 to 2 x108 CFU/ml)

Dilute the bacterial suspensions 1:10 in 0.85%NaCl (107 CFU/ml)

(depend on the size of the pin)

Day 2 Preparation of inoculum

Day 2 Inoculating the agar plates

Pipette 50 l of dilution into wells of a microtiter plate

Incubate the plates at 35-37C for 16–20 hrs.

Place the replicator into the microtiter plate and

transfer it onto the agar plate

1 l/spot (1x104 CFU/spot)




2. Preparation of broth with a serially-diluted

antibiotic

3. Preparation of the bacterial suspension

4. Inoculation of bacterial suspension

5. Reading results & Interpretation

autoclave


Cation adjusted MHB (CAMHB)

Cation adjusted MHB (CAMHB)

20–25 mg Ca2+/L

10–12.5 mg Mg2+/L

10 mg/ml

CaCl2

10 mg/ml

MgCl2


For 200 ml MHB,

Ca2+ = 450 l

Mg2+ = 225 l

Day 2 Preparation of broth with a serially-diluted antibiotics

256 128 64 32 16 8 4 2 1 0.5 0 µg/ml

Label the plates

Add 50 µl of CAMHB in the

microtitre plate

(Except the first column)

Do it in triplicate

Antibiotic stock solution

Tetracycline 10 mg/ml

C1V1 = C2V2

10mg/ml V1 = 512 g/ml(10ml)

V1 = 512 l

512 g/ml


Add 50 µl of antibiotic stock solution (2X, 512) to the first

column

Add 50 µl of antibiotic stock solution (2X, 512) to the second

column

Mix suspension thoroughly and transfer 50 µl of

suspension to the next column

Repeat until finish (except for control the last column)


256 128 64 32 16 8 4 2 1 0.5 0 µg/ml



Grow the bacterial strains on MHA, 35-37C overnight

Control strains: E. coli ATCC 25922



Samples: Salmonella S….

Day 2 Preparation of the bacterial suspension

Transfer 4-5 colonies of a culture into

5 ml of 0.85%NaCl

Adjust turbidity to 0.5 McFarland Standard 0.5

(1 to 2 x 108 CFU/ml)

Dilute the bacterial suspensions

1:10 in 9 ml of CAMHB (107 CFU/ml)

Dilute the bacterial suspensions

1:10 in 9 ml of CAMHB

(106 CFU/ml)

Day 2 Inoculation of bacterial suspension

Transfer 50 µl of bacterial suspension into microtiter plate

Seal with parafilm and incubate the plates at 35-37◦C for

16-20 hours

5 x 105 CFU/ml

104 CFU/well

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Antimicrobial susceptibility test (LAB-1)