SIST EN ISO 20776-1:2020

SLOVENSKI STANDARD
oSIST prEN ISO 20776-1:2018
01-september-2018
3UHVNXVREþXWOMLYRVWLSRY]URþLWHOMHYLQIHNFLMQDGHORYDQMHDQWLPLNUREQRREþXWOMLYLK
QDSUDYGHO5HIHUHQþQDPHWRGD]DSUHVNXVDNWLYQRVWLLQYLWURDQWLPLNUREQLK
SRY]URþLWHOMHYQDYSOLYEDNWHULMSULQDOH]OMLYLKEROH]QLK,62',6
Susceptibility testing of infectious agents and evaluation of performance of antimicrobial
susceptibility test devices - Part 1: Broth micro-dilution reference method for testing the
in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved
in infectious diseases (ISO/DIS 20776-1:2018)
Labormedizinische Untersuchungen und In-vitro-Diagnostika-Systeme -
Empfindlichkeitsprüfung von Infektionserregern und Evaluation von Geräten zur
antimikrobiellen Empfindlichkeitsprüfung - Teil 1: Referenzmethode zur Testung der In-
vitro-Aktivität von antimikrobiellen Substanzen gegen schnell wachsende aerobe
Bakterien, die Infektionskrankheiten verursachen (ISO/DIS 20776-1:2018)
Sensibilité in vitro des agents infectieux et évaluation des performances des dispositifs
pour antibiogrammes - Partie 1: Méthode de référence pour la détermination de la
sensibilité in vitro aux agents antimicrobiens des bactéries aérobies à croissance rapide
impliquées dans les maladies infectieuses (ISO/DIS 20776-1:2018)
Ta slovenski standard je istoveten z: prEN ISO 20776-1
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
oSIST prEN ISO 20776-1:2018 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 20776-1:2018

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oSIST prEN ISO 20776-1:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20776-1
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
2018-06-26 2018-09-18
Susceptibility testing of infectious agents and evaluation of
performance of antimicrobial susceptibility test devices —
Part 1:
Broth micro-dilution reference method for testing the
in vitro activity of antimicrobial agents against rapidly
growing aerobic bacteria involved in infectious diseases
Sensibilité in vitro des agents infectieux et évaluation des performances des dispositifs pour
antibiogrammes —
Partie 1: Méthode de référence pour la détermination de la sensibilité in vitro aux agents antimicrobiens
des bactéries aérobies à croissance rapide impliquées dans les maladies infectieuses
ICS: 11.100.20
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 20776-1:2018(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2018

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oSIST prEN ISO 20776-1:2018
ISO/DIS 20776-1:2018(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
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oSIST prEN ISO 20776-1:2018
ISO/DIS 20776-1:2018(E)
ISO CD 20776-1
13 Contents
14 Foreword . iv
15 Introduction. v
16 1 Scope . 1
17 2 Normative references . 1
18 3 Terms and definitions . 1
19 4 Test procedures . 3
20 4.1 General . 3
21 4.2 Medium . 3
22 4.3 Antimicrobial agents . 3
23 4.4 Preparation of inoculum . 5
24 4.5 Inoculation of micro-dilution trays . 5
25 4.6 Incubation of micro-dilution trays . 6
26 4.7 Reading results . 6
27 4.8 Special test situations where the MIC result might give unreliable results . 7
28 5 Quality control . 7
29 Annex A (normative) Requirements for Mueller-Hinton broth . 8
30 Annex B (normative) Solvents and diluents for making stock solutions of selected
31 antimicrobial agents . 11
32 Annex C (normative) Preparation of working dilutions of antimicrobial agents for use in
33 broth dilution susceptibility tests . 16
34 Annex D (Informative) Special test situations . 17
35 Bibliography . 18
36
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37 Foreword
38 ISO (the International Organization for Standardization) is a worldwide federation of national standards
39 bodies (ISO member bodies). The work of preparing International Standards is normally carried out
40 through ISO technical committees. Each member body interested in a subject for which a technical
41 committee has been established has the right to be represented on that committee. International
42 organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO
43 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
44 electrotechnical standardization.
45 The procedures used to develop this document and those intended for its further maintenance are
46 described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
47 different types of ISO documents should be noted. This document was drafted in accordance with the
48 editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
49 Attention is drawn to the possibility that some of the elements of this document may be the subject of
50 patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
51 patent rights identified during the development of the document will be in the Introduction and/or on
52 the ISO list of patent declarations received (see www.iso.org/patents).
53 Any trade name used in this document is information given for the convenience of users and does not
54 constitute an endorsement.
55 For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
56 expressions related to conformity assessment, as well as information about ISO's adherence to the World
57 Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL:
58 www.iso.org/iso/foreword.html.
59 This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in vitro
60 diagnostic test systems.
61 This second edition cancels and replaces the first edition (ISO 20776-1:2006), which has been technically
62 revised.
63 The main changes compared to the previous edition are as follows:
64 — Revised to a broth micro-dilution only performance document
65 — Removal of S,I, R breakpoint definitions and information
66 — Moved embedded Tables to Annexes
67 — Removed quality control range Table.
68 — Updated Table (now Annex B) on solvents and diluents for antimicrobial agents used globally.
69 — Updated information on special culture media and method performance for specific currently used
70 antimicrobial agents.
71 A list of all parts in the ISO 20776 series can be found on the ISO website.
72
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73 Introduction
74 In vitro antimicrobial susceptibility tests are performed on micro-organisms suspected of causing
75 disease, particularly if the organism is thought to belong to a species that may exhibit resistance to
76 frequently used antimicrobial agents. The tests are also important in resistance surveillance,
77 epidemiological studies of susceptibility and in comparisons of new and existing agents.
78 Dilution procedures are used to determine the minimum inhibitory concentrations (MICs) of
79 antimicrobial agents for antimicrobial susceptibility testing. MIC methods are used in resistance
80 surveillance, defining identifying wild type phenotypes, comparative testing of new agents, to establish
81 the susceptibility of organisms that give equivocal results in routine tests, for tests on organisms where
82 routine tests may be unreliable and when a quantitative result is required for clinical management. In
83 dilution tests, micro-organisms are tested for their ability to produce visible growth in broth (broth
84 dilution) containing serial dilutions of the antimicrobial agent or on a series of agar plates (agar dilution).
85 The lowest concentration of an antimicrobial agent (in mg/l) that, under defined in vitro conditions,
86 prevents the appearance of visible growth of a micro-organism within a defined period of time is known
87 as the MIC. The MIC is a guide for the clinician to the susceptibility of the organism to the antimicrobial
88 agent and aids treatment decisions. Careful control and standardizationis required for intra- and inter-
89 laboratory reproducibility of broth MIC tests. The MICs generally span two to three doubling dilutions
90 with a dominant central value.
91 Broth dilution is a technique in which containers holding identical volumes of broth with antimicrobial
92 agent solutions in incrementally (usually geometrically) increasing concentrations are inoculated with a
93 known number of micro-organisms.
94 Broth micro-dilution denotes the performance of the broth dilution test in micro-dilution trays.
95 The method described in this part of ISO 20776 is intended for the testing of pure cultures of aerobic
96 bacteria that are easily grown by overnight incubation on agar and grow well in standardised micro-
97 dilution trays containing standardised Mueller-Hinton broth (volume of ≤ 200 µl), which may need to be
98 modified depending on the antimicrobial agent being tested.
99 The broth micro-dilution method described in this part of ISO 20766 is essentially the same as those used
100 in many countries, and as the methods published by the Clinical and Laboratory Standards Institute
101 (CLSI)[1] and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)[2]. These
102 methods are based on those described by Ericsson and Sherris[3].
103
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104 Susceptibility testing of infectious agents and evaluation of
105 performance of antimicrobial susceptibility test devices — Part 1:
106 Broth micro-dilution reference method for testing the in vitro
107 activity of antimicrobial agents against rapidly growing aerobic
108 bacteria involved in infectious diseases
109 WARNING — The use of this part of ISO 20776 may involve hazardous materials, operations and
110 equipment. This part of ISO 20776 does not purport to address all of the safety problems associated
111 with its use. It is the responsibility of the user of this part of ISO 20776 to establish appropriate safety
112 and health practices and determine the applicability of regulatory limitations prior to use.
113 1 Scope
114 This part of ISO 20776 describes one reference method, broth micro-dilution, for determination of MICs.
115 The MIC may be a guide for the clinician, and reflects the activity of the drug under the described test
116 conditions, by taking into account other factors, such as drug pharmacology, pharmacokinetics, or
117 bacterial resistance mechanisms. This allows categorisation of bacteria as “susceptible” (S),
118 “intermediate” (I), or “resistant” (R). In addition, MIC distributions can be used to define wild type or non-
119 wild type bacterial populations. Although clinical interpretation of the MIC value is beyond the scope of
120 this part of ISO 20776, modifications of the basic method are required for certain antimicrobial agent -
121 bacteria combinations to facilitate clinical interpretation. These modifications are included in a separate
122 Annex of this document. It is necessary to compare other susceptibility testing methods (e.g. disc diffusion
123 or diagnostic test devices) with this reference method for validation, in order to ensure comparable and
124 reliable results.
125 2 Normative references
126 There are no normative references in this document.
127 3 Terms and definitions
128 For the purposes of this document, the following terms and definitions apply.
129 ISO and IEC maintain terminological databases for use in standardization at the following addresses:
130 — IEC Electropedia: available at http://www.electropedia.org/
131 — ISO Online browsing platform: available at https://www.iso.org/obp
132 3.1
133 antimicrobial agent
134 substance of biological, semi-synthetic or synthetic origin that inhibits the growth of or kills bacteria, and
135 is thus of potential use in the treatment of infections
136 Note to entry: Disinfectants, antiseptics and preservatives are not included in this definition.
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137 3.2 Antimicrobial agents — properties
138 3.2.1
139 potency
140 potency is a measure of drug activity expressed in terms of the amount required to produce an effect of
141 given intensity.
142 Note to entry: The potency is expressed as mass fraction in milligrams per gram (mg/g), or as activity
143 content in International Units (IU) per gram, or as a volume fraction or mass fraction in percent, or as an
144 amount-of-substance concentration (molar fraction) in mole per litre of ingredients in the test substance.
145 3.2.2
146 concentration
147 amount of an antimicrobial agent in a defined volume of liquid
148 Note 1 to entry: The concentration is expressed as mg/l.
149 Note 2 to entry: mg/l is the designated ISO unit.
150 3.3
151 stock solution
152 initial solution used for further dilutions
153 3.4
154 minimum inhibitory concentration
155 MIC
156 lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within a
157 defined period of time
158 Note to entry: The MIC is expressed in mg/l.
159 3.5
160 breakpoint
161 BP
162 specific values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical
163 categories “susceptible”, “intermediate” and “resistant”
164 Note to entry: For current interpretive breakpoints, reference can be made to the latest publications of
165 organisations employing this reference method (e.g. CLSI and EUCAST).
166 3.6
167 wild type
168 absence of known resistance mechanisms to the antimicrobial agent for a given strain
169 3.7
170 reference strain
171 catalogued, characterized bacteria with stable, defined antimicrobial susceptibility phenotypes and/or
172 genotypes
173 Note to entry: Reference strains are kept as stock cultures, from which working cultures are derived.
174 They are obtainable from culture collections and used for quality control.
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175 3.8 Susceptibility testing method
176 3.8.1
177 broth dilution
178 technique in which containers are filled with appropriate volumes of an antimicrobial solution,
179 employing incrementally (usually two-fold) increasing concentrations of the antimicrobial agent and
180 appropriate volumes of broth with a defined inoculum
181 Note to entry: The aim of this method is the determination of the MIC.
182 3.8.2
183 micro-dilution
184 performance of broth dilution in micro-dilution trays with a final test volume of ≤ 200 µl per well
185 3.9
186 broth
187 fluid medium used for the in vitro growth of bacteria
188 Note to entry: For the broth reference method the medium is standardised Mueller-Hinton broth (see
189 Annex A)
190 3.10
191 inoculum
192 number of bacteria in a suspension, calculated with respect to the final volume
193 Note to entry: The inoculum is expressed as colony-forming units per millilitre (CFU/ml).
194 3.11
195 inoculum effect
196 change in MIC related to change in inoculum concentration
197 4 Test procedures
198 4.1 General
199 The tests are performed in polystyrene micro-dilution trays. The method is based on the preparation of
200 antimicrobial agent working solutions, either in 50 µl volumes per well (with the addition of an inoculum
201 also in a volume of 50 µl), or in a volume of 100 µl per well (with the addition of a maximum of 10 µl
202 inoculum volume).
203 4.2 Medium
204 Mueller-Hinton broth shall be used (see Annex A for details and Annex D for special test situations).
205 4.3 Antimicrobial agents
206 4.3.1 General
207 Antimicrobial agents shall be obtained directly from the manufacturer or from reliable commercial
208 sources; pharmaceutical preparations for clinical use are not acceptable. The antimicrobial agents shall
209 be supplied as powders with a lot number, potency, an expiry date and details of recommended storage
210 conditions. Substances shall be stored in tightly closed containers in the dark, with a desiccant at the
211 recommended temperature of the supplier. Hygroscopic agents should be dispensed into aliquots, one of
212 which is used on each test occasion.
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213 To avoid condensation, allow containers to warm to room temperature before opening.
214 4.3.2 Preparation of stock solutions
215 The use of a calibrated analytical balance is required to weigh antimicrobial agents. Allowance for the
216 potency of the powder shall be made by use of the following formula to obtain the amount of antimicrobial
217 agent substance or the volume of diluent needed for a standard solution:
V
m
P
218 (1)
mP
V 

219 (2)
220 where
221  is the concentration of the stock solution, in mg/l;
222 m is mass of the antimicrobial agent (powder), in g;
223 P is the potency of the antimicrobial agent (powder), in mg/g;
224 V is the volume of diluent, in l.
225 Concentrations of stock solutions should be 1000 mg/l or greater, although the solubility of some agents
226 is a limiting factor. The actual concentrations of stock solutions depend on the method of preparing
227 working solutions (serial dilutions). Agents should be dissolved and diluted in sterile distilled water
228 unless the manufacturer states otherwise. Some agents require alternative solvents (see Annex B).
229 Note: For newer antimicrobial agents not identified in Annex B of this current document, consult
230 manufacturer information on the most appropriate solvent and diluent for the specific agent. Sterilisation
231 of solutions is not usually necessary. If required, sterilisation should be done by membrane filtration, and
232 samples before and after sterilisation should be compared by assay to ensure that adsorption has not
233 occurred.
234 Unless information is available on stability of stock solutions under specified storage conditions, they
235 should be prepared fresh for each test batch.
236 4.3.3 Preparation of working solutions
237 The range of concentrations selected for testing depends on the micro-organisms and antimicrobial
238 agent. The chosen range shall allow full endpoint MIC determination for appropriate reference strains. A
239 two-fold dilution series based on 1 mg/l is prepared in Mueller-Hinton broth. Dilutions should not be
240 prepared by serial dilution steps, but according to the procedure outlined in Annex C. Working solutions
241 shall be used the same day unless information is available on stability of the solutions under specified
242 storage conditions.
243 4.3.4 Preparation of micro-dilution trays
244 Working solutions are dispensed into micro-dilution trays at 50 µl per well with double the desired final
245 concentrations of antimicrobial agent, or at 100 µl per well in the desired final concentrations.
246 At least one well, containing 50 µl or 100 µl of antimicrobial agent-free medium, should be included as a
247 growth control for each strain tested. Likewise, a well containing 100 µl of antimicrobial agent-free
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248 medium should be included as an un-inoculated negative control well for each micro-organism type
249 tested.
250 4.3.5 Storage of micro-dilution trays
251 Filled trays may be used immediately or may be stored for up to three months or until documented
252 quality control or other evidence indicates degradation of the antimicrobial agent. For storage the filled
253 trays should be sealed in plastic bags and immediately placed in a freezer at ≤  60 °C unless the
254 antimicrobial agents are known to be stable at higher temperatures.
255 Trays shall not be stored in a self-defrosting freezer, and thawed antimicrobial solutions shall not be re-
256 frozen, as repeated freeze-thaw cycles accelerate the degradation of some antimicrobial agents,
257 particularly -lactams.
258 Allow frozen plates to thaw for up to 2 hours and inoculate by 4 hours of removal from the freezer.
259 4.4 Preparation of inoculum
260 4.4.1 General
261 Standardization of the inoculum is essential for accurate and reproducible broth micro-dilution
262 susceptibility tests. Therefore, purity checks and viable colony counts shall be performed on every isolate
263 tested with this reference procedure
264 4.4.2 Broth culture method
265 The inoculum may be prepared by diluting a broth culture or by suspending several morphologically
266 similar colonies (when possible) from an overnight culture on non-selective agar medium in broth with
267 a sterile loop or a cotton swab. When suspending colonies, morphologically similar colonies should be
268 picked to avoid contamination of other species or atypical variants of the same species.
269 The broth used shall not be antagonistic to the antimicrobial agent tested. The broth is incubated at 35
270 ±1°C until the growth reaches a turbidity equal to or greater than that of a 0.5 McFarland standard[4]. If
271 needed, the culture is adjusted with saline or broth to give a turbidity equivalent to the 0.5 McFarland
272 standard. This can be done by means of a photometric device (using 625 nm wavelength and a 1 cm path
273 cuvette, the absorbance will be 0.08 – 0.13), or by employing a suitably calibrated nephelometer.
274 Alternatively, this can be achieved visually by comparing the appearance of black lines through the
275 inoculum and 0.5 McFarland standard suspensions (the inoculum and McFarland standard shall be in
276 tubes of the same size) or any other method that gives reproducible CFU/ml. The final inoculum shall be
5 5 5
277 5 x 10 CFU/ml (target range, 2 x 10 CFU/ml to 8 x 10 CFU/ml).
278 NOTE A 0.5 McFarland standard can be produced by adding a 0.5 ml aliquot of 0.048 mol/l barium
279 chloride (BaCl ) (11.72 g/l BaCl 2H O) to 99.5 ml of 0.18 mol/l sulphuric acid (H SO ), with constant
2 2 2 2 4
280 stirring to maintain a suspension.
281 4.4.3 Direct colony suspension method
282 Several morphologically similar colonies from a non-selective nutritive agar medium (incubated at 35
283 ±1°C for 18 h to 24 h, unless longer incubation is required) are touched with a sterile loop and the growth
284 transferred to sterile broth or saline. The suspension is adjusted to give a turbidity equivalent to that of
285 a 0.5 McFarland standard, as described in 4.4.2 for the broth culture method.
286 For all micro-organisms, the concentration of viable cells in the final inoculum depends on the growth
287 phase of the culture. This effect is most pronounced for fastidious organisms such as Streptococcus
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288 pneumoniae, where use of older cultures can significantly reduce the number of viable cells in the
289 suspension.
8
290 A correctly adjusted suspension prepared by either method contains approximately 1 - 2  10 CFU/ml
291 for the common relevant bacteria.
292 The adjusted inoculum prepared as above is diluted in broth to give a final cell number concentration of
5 5 5
293 5  10 CFU/ml (target range 2  10 CFU/ml to 8  10 CFU/ml). The dilution required depends upon the
294 bacterial species being tested and the method used for inoculum delivery. Transfer of 0,1 ml of
295 standardized micro-organism suspension to a tube containing 9.9 ml (1:100 dilution) of broth results in
6
296 a suspension of 1  10 CFU/ml which, when 50 µl is added to an equal volume (50 µl) of antimicrobial
5
297 agent solution, results in a final inoculum of 5  10 CFU/ml with many Gram-negative bacteria (e.g.
298 Escherichia coli). If the wells already contain 100 µl of antimicrobial agents in broth, an appropriate
299 dilution to give the required final inoculum should be prepared prior to addition of up to10 µl of the
300 diluted suspension to each well. Different dilutions of the 0.5 McFarland suspension may be necessary,
301 as determined by colony counts in preliminary tests[5]
302 4.5  Inoculation of micro-dilution trays
303 The trays shall be inoculated within 30 minutes of standardizing the inoculum suspension, in order to
304 maintain viable cell number concentration. To each well containing 50 µl of diluted antimicrobial agent
305 in broth (see 4.3), a volume of 50 µl of bacterial suspension (see 4.4) is added. For tray wells that contain
306 100 µl of dil
...