Antimicrobial
susceptibility test
(LAB-1)
Antimicrobial susceptibility test
1. Preparation of bacterial isolates
& inoculum
2. Preparation of growth media
3. Preparation of antibiotics
4. AST
4.1 Disk diffusion assay
4.2 Dilution method
 Agar dilution method
 Broth microdilution method
Antimicrobial susceptibility test
Day 1 Preparing materials
(Growth media, antibiotics, bacterial culture
etc.)
Day 2 AST
Day 3 Reading results
Bacterial isolates and
inoculum
Bacterial isolates
1. Quality control strains
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 29213
Pseudomonas aeruginosa ATCC 27853
2. Bacterial samples
Streak to get single isolates on MHA,
35oC (37C) overnight
Antimicrobial susceptibility test
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
A BaSO4 turbidity standard
McFarland Densitometer
Bacterial cells in inoculum
Initial
1x108
CFU/ml
1x108
CFU/ml
1x108
CFU/ml
Used conc.
1x108
CFU/ml
1x106
CFU/ml
1x107
CFU/ml
1x104
CFU/well
1x104
CFU/spot
Final
-
Growth media
Growth media
Mueller-Hinton media (agar or broth)
 pH
 Cation conc.
 Blood and serum suppl.
 Thymidine content
 Thickness
Growth media
MHA
or
Autoclave
(121oC, 1.5 psi,
15 min)
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) =
Volume(ml).Concentration(/ml)
Potency(g/mg)
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
Example
To prepare 100 ml of a stock solution containing 1280 g/ml
of streptomycin with streptomycin powder with the potency
of 990 g/mg.
Weight = Volume(ml).Concentration(/ml)
Potency(g/mg)
= 100*1280/990
= 129 mg
Strepmycin should be weighed 129-150 mg.
Example
If the actual weight is 145 mg the volume of diluent needed
is as follows:
Volume (ml)
=
Weight mg). Potency(g/mg)
Concentration (g/ml)
=
145*990/1280
=
112 ml
Antimicrobial susceptibility test
Day 1 Preparing materials
(Growth media, antibiotics, culture etc.)
Day 2 AST
Day 3 Reading results
Disk diffusion method
Disk diffusion method
1. Preparation of agar plates
2. Preparation of bacterial isolates &
inoculum
3. Inoculating agar plates
4. Applying disk
5. Interpretation
Day 1 Preparation of growth media
autoclave
MHA
Day 1 Preparation of bacterial isolates
QC strains:
Samples:
E. coli ATCC 25922
S. aureus ATCC 29213
P. aeruginosa ATCC 27853
Salmonella S……
Streak the bacterial strain on MHA,
Incubate at 35oC (37C) for 16-20 h
Day 2 Preparation of inoculum & inoculated
agar plates
Sterile cotton swab
60 ₒ
60 ₒ
saline/broth
Pick 4-5 single
colonies
Re-suspend the bacterial
Inoculate Muller Hinton
colonies and adjust turbidity agar (MHA) plate using
to 0.5 McFarland
sterile cotton swab
Day 2 Application of antimicrobial disk
SXT
AMC
GEN
CIP
Apply antibiotic disk onto MHA
using sterile forceps or applicators
ₒ
Incubate at 35 C (37oC), for 16-18 hr
Agar dilution method
Multiple inoculators
Agar dilution method
1. Preparation of bacterial isolates
2. Prepare agar plates with antibiotics
3. Preparation of the bacterial suspension
& plate inoculation
4. Reading results & Interpretation
Day 1 Prepare agar plates with antibiotics
2 ml
256 g/ml
2 ml
128 g/ml
2 ml
64 g/ml
2 ml
32 g/ml
2 ml
16 g/ml
Total volume = 20 ml per plate (18 + 2)
Day 1 Prepare antibiotic stock solution
Antibiotic stock solution
 Tetracycline 10 mg/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
C1V1
= C2V2
C1(2ml) = 256 g/ml(20ml)
C1
= 2,560 g/ml
Day 1 Prepare agar plates with antibiotics
Method A
C1V1
= C2V2
C1(2ml) = 256 g/ml(20ml)
C1
= 2,560 g/ml
Add 2 ml of antibiotic solution (2,560 g/ml) into
18 ml of MHA separately into plates
Day 1 Prepare agar plates with antibiotics
Method B
Mix 2 ml of antibiotic solution &
18 ml of MHA separately in tubes (1:10)
and pour into plates
Day 1 Prepare agar plates with antibiotics
256 g/ml
8 g/ml
128 g/ml
64 g/ml
32 g/ml
4 g/ml
2 g/ml
1 g/ml
16 g/ml
0 g/ml
Control
plates
Day 1 Preparation of bacterial isolates
Control strains:
Samples:
E. coli ATCC 25922
S. aureus ATCC 29213
P. aeruginosa ATCC 27853
Salmonella S1-S10
Grow the bacterial strains on MHA, 37C overnight
Day 2 Preparation of inoculum
 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 Inoculating the agar plates
Pipette 50 l of dilution into wells of a microtiter plate
 Place the replicator into the microtiter plate and
transfer it onto the agar plate
 1 l/spot (1x104 CFU/spot)
 Incubate the plates at 35-37C for 16–20 hrs.
Broth microdilution method
Broth microdilution method
1. Preparation of bacterial isolates
2. Preparation of broth with a serially-diluted
antibiotic
3. Preparation of the bacterial suspension
4. Inoculation of bacterial suspension
5. Reading results & Interpretation
Day 1 Preparation of growth media
Cation adjusted MHB (CAMHB)
autoclave
Day 1 Preparation of growth media
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
Label the plates
256 128 64 32 16
8
4
2
1
0.5
0
µg/ml
Day 2 Preparation of broth with a serially-diluted
antibiotics
Antibiotic stock solution
 Tetracycline 10 mg/ml
512 g/ml
C1V1 = C2V2
10mg/ml V1 = 512 g/ml(10ml)
V1 = 512 l
 Add 50 µl of CAMHB in the
microtitre plate
(Except the first column)
 Do it in triplicate
Day 2 Preparation of broth with a serially-diluted
antibiotics
 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)
Day 2 Preparation of broth with a serially-diluted
antibiotics
256 128 64 32 16
8
4
2
1
0.5
0
µg/ml
Day 1 Preparation of bacterial isolates
Control strains:
Samples:
E. coli ATCC 25922
S. aureus ATCC 29213
P. aeruginosa ATCC 27853
Salmonella S….
Grow the bacterial strains on MHA, 35-37C overnight
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
5 x 105 CFU/ml
104 CFU/well
Seal with parafilm and incubate the plates at 35-37◦C for
16-20 hours

FR-1 CCL Catalogue (1-9)

The SCS Instrument

Document 3127619

Prof. Steven M. Jacobs NAU Schedule for Spring 2015

PDFで表示