Global Salm-Surv: Laboratory Protocols Level 1 Training Course Agar Diffusion Using E-Test 4 Ed. April 2003

Global Salm-Surv
A global Salmonella surveillance and laboratory support project

of the World Health Organization
Laboratory Protocols
Level 1 Training Course
Agar diffusion using E-test
4th Ed. April 2003
EDITED BY: RENE S. HENDRIKSEN (DFVF)
Contents
Page
1. Susceptibility testing: Determination of phenotypic resistance...........................................3
1.2 Agar diffusion with E-test (determination of an approximate MIC-value) .........................5
2.
Composition and preparation of culture media and reagents.............................................10
Laboratory record sheets...........................................................................................................11
1. Susceptibility testing: Determination of phenotypic resistance

1) Agar diffusion with disk
2) Agar diffusion with E-test
3) MIC-determination using Agar dilution method.
Introduction
The MIC (Minimal Inhibitory Concentration) of a bacterium to a certain antimicrobial agent gives a
quantitative estimate of the susceptibility.
MIC is defined as the lowest concentration of antimicrobial agent required to inhibit growth of the
organism. The principle is simple: Agar plates, tubes or microtitre trays with two-fold dilutions of
antibiotics are inoculated with a standardised inoculum of the bacteria and incubated under
standardised conditions following NCCLS guidelines. The next day, the MIC is recorded as the
lowest concentration of antimicrobial agent with no visible growth.
The MIC informs you about the degree of resistance and might give you important information
about the resistance mechanism and the resistance genes involved. MIC-determination performed as
agar dilution is regarded as the gold standard for susceptibility testing.
Agar diffusion tests are often used as qualitative methods to determine whether a bacterium is
resistant, intermediately resistant or susceptible. However, the agar diffusion method can be used
for determination of MIC values provided the necessary reference curves for conversion of
inhibition zones into MIC values are available. After an agar plate is inoculated with the bacteria, a
tablet, disk or paper strip with the antimicrobial agent is placed on the surface. During incubation
the antimicrobial agent diffuses into the agar and inhibits growth of the bacteria if susceptible.
Diffusion tests are cheap compared to most MIC-determination methods. E-test is a diffusion test,
but has been developed to give an approximate MIC-value.
Well standardised methods are essential for all kinds of susceptibility testing, since the methods are
highly sensitive to variations in several factors, such as size of inoculum, contents and acidity of the
growth medium, time and temperature of incubation. The agar diffusion methods are also strongly
influenced by factors, such as agar depth, diffusion rate of the antimicrobial agent and growth rate
of the specific bacteria.
The MIC-determination and disk diffusion methods described in this protocol are in accordance
with the international recommendations given by the National Committee for Clinical Laboratory
Standards (NCCLS). The NCCLS describes how to perform the tests and sets international
guidelines for interpretation of the results. It should be noted that the WHO does not prescribe any
specific method for performance and interpretation of susceptibility tests.
Internal quality control should be regularly performed as recommended by NCCLS.
References
1. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial
disk susceptibility tests, 7th ed. Approved standard. M2-A7. NCCLS, Wayne, Pennesylvania,
2000.
2. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial
disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard.
M31-A, NCCLS, Wayne, Pennesylvania, 1998.
3. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. 5th ed. Approved standard. M7-A5,
NCCLS, Wayne, Pennesylvania, 2000.
1.2 Agar diffusion using E-test (determination of an approximate

MIC-value)
Materials
Equipment
E-test strips
McFarland standard 0.5
Scissors
Forceps
Loops (1 l and 10 l)
Bunsen burner
Small sterile cotton swabs
White paper with black lines on
Media
Sterile normal saline, 4 ml volumes in tubes
Mueller Hinton II agar plates (15 cm and a uniform agar depth of 4 mm)
Nutrient agar plates (9 cm)
Bacterial strains
Salmonella or other non-fastidious cultures plated out for single colonies
Strain for quality control: Escherichia coli ATCC 25922
Safety
Carry out all procedures in accordance with the local codes of safe practice.
Procedure
Theory / comments
Day 1
Prepare inoculum
Remove the E-test package from the freezer
(-20oC) at least 30 minutes before required.
With a loop, touch the top of 3 or 4 individual
colonies and transfer to a tube of saline.
Emulsify the inoculum on the inside of the
tube to avoid lumps.
Compare turbidity to that in the 0.5 McFarland
standard. Adjust turbidity of inoculum to
match that standard.
Inoculate agar plate
Swab plate within 15 minutes of preparing the
adjusted inoculum:
Ensure the agar surface is dry, but not overly

dry (all the constraints of disk diffusion test
apply to E-test).
Dip a sterile cotton swab into the inoculum

and pulling out slightly, rotate the swab
several times against the inside of the tube
above the fluid level to remove excess liquid.
Streak the swab over the entire surface of the
agar plate. Rotate the plate approximately 60o
then repeat streaking motion. Rotate 60o again
and repeat streaking. Complete inoculation by
running the swab around the rim of the agar.
Leave the lid of the plate ajar for 5 minutes
(no more than 15 minutes) to allow any excess
moisture to be absorbed before applying strips.
Apply E-test strips
Open E-test package by cutting along the
broken line.
Apply strips to agar surface using forceps (or
E-test applicator if available). Place the strip
with the E end at the edge of the plate and
with the scale visible. An example of
antimicrobial strips placed on a plate is shown
in figure 1.
If strips stick together, they may be pulled

apart by handling the section marked E.
Do not touch any other area of the strip.
Use templates to position 4 to 6 strips onto a
150 mm plate or one (seldom two) strips onto
a 90 mm plate. Do not remove a strip once it
has touched the agar.
Procedure
Theory / comments
Check purity of the inoculum:

Transfer inoculum from the tube onto a
nutrient agar plate using a 10 l loop.
Incubate plates at 35oC for 16 to 20 hours in
ambient air.
Otherwise the expiry date will not be the same
as for the original package.
Place any open packages in separate airtight

containers containing active desiccant and
store at -20oC.
Figure 1. Example of antimicrobials for Enterobacteriaceae susceptibility testing (ref. 2).

Abbreviations are shown in table 4.
DC
GM
TS
CI
AB
Procedure
Theory / comments
Day 2: Results
Read MIC at the point where ellipse intersects
the scale. If a MIC value between two twofold dilutions is seen, always round up to the
highest value.
Read results only if a good inhibition ellipse

is visible, e.g. inoculum is sufficient.
Remember to read the MIC value at complete

inhibition of all growth including isolated
colonies.
If the intersect differs on either side of the
strip, read the MIC as the greater value.
However, remember that sulfonamide and

trimethoprim should be read at 80% of
growth and that swarming of Proteus should
be ignored.
Ignore any growth at the edge of the strip.

Refer to E-test reading guide if necessary.
This is caused by organisms growing in a

tunnel of water.
Consult the appropriate application sheet to

find breakpoint values telling at which MIC
values the bacteria should be interpreted as S
(Susceptible), I (Intermediate) or R
(Resistant). An example for
Enterobacteriaceae according to the NCCLS
guideline is shown in table 4.
Table 4. An example of criteria for interpretation of susceptible, intermediate or resistant bacteria

and the accepted range of MIC for the E. coli ATCC 25922 reference strain.
Antimicrobial
AM
CL*
NCCLS MIC interpretive criteria (g/ml) NCCLS QC ranges

E. coli ATCC 25922. MIC (g/ml)
S
I
R
16
2-8
8
32
16
2-8
8
32
Ampicillin
Chloramphenicol
CI*
Ciprofloxacin
2
1
4
NA
Nalidixic acid
16
32
SU
Sulphadiazine
256
512
TC
Tetracycline
8
4
16
* Notice the difference between these two abbreviations.
0.004-0.016
1-4
8 - 32
0.5 - 2
References
1. Etest technical guide 3B. Etest for MIC determination. AB Biodisk.
2. Etest Gram negative application sheet. AB Biodisk.
2. Composition and preparation of culture media and reagents

If no reference is given, it is the procedure used at DVL.
The media and reagents are available from several companies including Oxoid, Merck and Difco.
The composition of the dehydrated media given below is an example and may vary a little among
the different manufacturers. Also, the media should be prepared according to the manufacturers
description if it differs from the description given here. Refer to Appendix 2 for a colour
presentation of growth of Salmonella on selective agar media and positive and negative reactions of
biochemical tests.
Mueller Hinton II agar (e.g. from BBL)

Beef extract
Acid hydrolysate of casein
Starch
Agar
Distilled water
2.0 g
17.5 g
1.5 g
17.0 g
1000 ml
Preparation:
Dissolve the dehydrated medium in water by heating if necessary. Adjust pH to 7.2 - 7.4, transfer
into bottles and autoclave at 110oC for 20 min.
Saline solution
Sodium chloride
Water
8.5 g
1000 ml
Preparation:
Dissolve the sodium chloride in the water by heating if necessary. Adjust pH to 7.0 after
sterilisation. Dispense the solution into tubes so 4 ml is obtained after autoclaving at 121oC for 20
min.
References
1. Post D. E. (1997) Food-borne pathogens monograph number I Salmonella. Oxoid limited,
Hampshire, England.
2. ISO 6579 :1993(E) 3rd ed. Microbiology - General guidance on methods for the detection of
Salmonella.
nd
3. NMKL method no. 71, 2 ed., 1999: Salmonella. Detection in food. Nordic committee on food
analysis.
10
Date:
Initials:
Record sheet:
Disk diffusion susceptibility testing
Agar diffusion with E-test
Strain: DVL #13

Antimicrobial
MIC (g/ml)
Interpretation
(R-I-S)
Ampicillin
Chloramphenicol
Ciprofloxacin
Nalidixic acid
Sulphonamides
Tetracycline
Strain: ATCC 25922

Antimicrobial
MIC (g/ml)
Within the
QC range
Ampicillin
Chloramphenicol
Ciprofloxacin
Nalidixic acid
Sulphonamides
Tetracycline
11

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Global Salm-Surv

A global Salmonella surveillance and laboratory support project

Composition and preparation of culture media and reagents.............................................10

Laboratory record sheets...........................................................................................................11

1. Susceptibility testing: Determination of phenotypic resistance

1.2 Agar diffusion using E-test (determination of an approximate

Ensure the agar surface is dry, but not overly

Dip a sterile cotton swab into the inoculum

If strips stick together, they may be pulled

Check purity of the inoculum:

Place any open packages in separate airtight

Figure 1. Example of antimicrobials for Enterobacteriaceae susceptibility testing (ref. 2).

Read results only if a good inhibition ellipse

Remember to read the MIC value at complete

However, remember that sulfonamide and

Ignore any growth at the edge of the strip.

This is caused by organisms growing in a

Consult the appropriate application sheet to

Table 4. An example of criteria for interpretation of susceptible, intermediate or resistant bacteria

NCCLS MIC interpretive criteria (g/ml) NCCLS QC ranges

2. Composition and preparation of culture media and reagents

Mueller Hinton II agar (e.g. from BBL)

Strain: DVL #13

Strain: ATCC 25922

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