ISO 14461-2:2005

Milk and milk products — Quality control in microbiological laboratories — Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps

ISO 14461-2|IDF 169-2:2005 describes a routine procedure for the evaluation of results of the enumeration of microorganisms using colony-count methods with subsequent 10-fold dilution steps and one plate or two parallel plates within each dilution step. This routine procedure is applied regularly in each laboratory performing colony counts. It provides criteria for the acceptability of differences between the results from parallel plates and subsequent dilution steps, as follows. The results (colony counts) obtained from parallel plates are compared with tabulated limits for given colony counts. If these limits are exceeded, a technical problem when performing the parallel determinations may be indicated. The results (sums of colony counts) of two parallel plates of two subsequent 10-fold dilution steps are compared with tabulated limits for given sums of colony counts. If these limits are exceeded, a technical problem when performing the dilutions may be indicated. If the limits mentioned above are exceeded in more cases than expected, this indicates that the test procedure lacks reliability.

Lait et produits laitiers — Contrôle de qualité en laboratoire microbiologique — Partie 2: Détermination de la fiabilité des comptages de colonies en boîtes parallèles et des dilutions décimales suivantes

General Information

• Status: Published
• Publication Date: 11-Apr-2005
• ICS: 07.100.30 - Food microbiology
• Technical Committee: ISO/TC 34/SC 5 - Milk and milk products
• Drafting Committee: ISO/TC 34/SC 5 - Milk and milk products
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 23-Sep-2020

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 14461-2
IDF
169-2
First edition
2005-04-01

Milk and milk products — Quality control
in microbiological laboratories —
Part 2:
Determination of the reliability of colony
counts of parallel plates and subsequent
dilution steps
Lait et produits laitiers — Contrôle de qualité en laboratoire
microbiologique —
Partie 2: Détermination de la fiabilité des comptages de colonies en
boîtes parallèles et des dilutions décimales suivantes




Reference numbers
ISO 14461-2:2005(E)
IDF 169-2:2005(E)
©
ISO and IDF 2005

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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
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ii © ISO and IDF 2005 – All rights reserved

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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle . 2
5 Procedure. 2
5.1 General. 2
5.2 Counting results of two parallel plates. 2
5.3 Sum of counting results of subsequent dilution steps. 2
6 Evaluation . 3
6.1 Tables of results. 3
6.2 Examples of testing parallel plates . 7
6.3 Examples of testing sums of two subsequent dilution steps . 7
7 Calculation formulae and examples. 15
7.1 In Table 1. 15
7.2 In Table 2. 16
7.3 Examples. 16

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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organizations, governmental
and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 14461-2IDF 169-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC
International. It is being published jointly by ISO and IDF and separately by AOAC International.
ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products — Quality
control in microbiological laboratories:
 Part 1: Analyst performance assessment for colony counts
 Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 14461-2IDF 169-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee
SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC
International. It is being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team, Statistics of analytical data, of the Standing
Committee on Quality assurance, statistics of analytical data and sampling, under the aegis of its project
leaders, Dr. H. Glaeser (EU) and Prof. Dr. H. Weiss (DE).
This edition of ISO 14461-2IDF 169-2, together with ISO 14461-1IDF 169-1, cancels and replaces
IDF 169:1994, which has been technically revised.
ISO 14461IDF 169 consists of the following parts, under the general title Milk and milk products — Quality
control in microbiological laboratories:
 Part 1: Analyst performance assessment for colony counts
 Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Introduction
Every microbiological method consists of several steps that are followed in a specific sequence (sub-sampling,
diluting, plating and counting). The final result has a margin of uncertainty that is determined by the variability
of all the steps involved.
In order to obtain results with a margin of uncertainty not much larger than what can be expected from the
correct application of the method, it is necessary to follow the rules of Good Laboratory Practice (GLP).
The three most important factors in obtaining a correct plate count are
 the homogeneity of the sample material,
 the exactness with which the dilutions are performed, and
 the technique of inoculation and/or counting of the plates.
By homogenizing a sample material very well, making multiple dilution series, and inoculating several plates
from the same dilution, it is possible to assess how well a laboratory can perform the colony-count technique,
taking into account the expected variability of the method.
Too large a variability indicates that at least one of the steps in the performance of the method is out of control.
The identification of those steps is carried out by comparison of the replicate inoculations, the different dilution
levels and the dilution series. When the steps with excessive variability have been identified, necessary
measures should be taken to bring these steps under control.
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ISO 14461-2:2005(E)
INTERNATIONAL STANDARD
IDF 169-2:2005(E)

Milk and milk products — Quality control in microbiological
laboratories —
Part 2:
Determination of the reliability of colony counts of parallel
plates and subsequent dilution steps
1 Scope
This part of ISO 14461IDF 169 describes a routine procedure for the evaluation of results of the enumeration
of microorganisms using colony-count methods with subsequent 10-fold dilution steps and one plate or two
parallel plates within each dilution step.
This routine procedure is applied regularly in each laboratory performing colony counts. It provides criteria for
the acceptability of differences between the results from parallel plates and subsequent dilution steps, as
follows.
a) The results (colony counts) obtained from parallel plates are compared with tabulated limits for given
colony counts. If these limits are exceeded, a technical problem when performing the parallel
determinations may be indicated.
b) The results (sums of colony counts) of two parallel plates of two subsequent 10-fold dilution steps are
compared with tabulated limits for given sums of colony counts. If these limits are exceeded, a technical
problem when performing the dilutions may be indicated.
c) If the limits mentioned above are exceeded in more cases than expected, this indicates that the test
procedure lacks reliability.
NOTE The formulae for calculating the values in Table 1 and 2 are given and explained in Clause 7.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 14461-1IDF 169-1, Milk and milk products — Quality control in microbiological laboratories — Part 1:
Analyst performance assessment for colony counts
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply
3.1
colony count
number of microorganisms found, as determined by the method specified in ISO 14461-1|IDF 169-1
NOTE The number of microorganisms is expressed per gram or per millilitre of test sample.
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
4 Principle
The counting results obtained are compared with tabulated limits for given colony counts. Decisions are based
on the way the limits are exceeded. The tabled values are calculated and explained.
5 Procedure
5.1 General
The procedure shall be applied routinely in laboratories carrying out colony counts. A standardized method for
performing colony counts must be applied in any case.
If the applied method is not in accordance with an International Standard or another accepted standard, a
detailed description of the method shall be available and followed precisely.
In the case that a method is followed with only one plate per dilution step, the procedure described in 5.2 shall
be carried out with a certain minimum frequency (e.g. once per hundred sample units tested).
5.2 Counting results of two parallel plates
Compare the results (colony counts) of two parallel plates with the limits tabulated in Table 1.
Compare the upper and lower colony counts of an observed pair of results with the corresponding colony
counts given in Table 1. Use the upper colony count given in Table 1 as basis for the comparison. Then
compare the lower colony count given in Table 1 with the observed lower count.
A lower observed count below the lower colony count of Table 1 indicates that the difference between the
colony counts obtained with the two parallel plates is unacceptably high. (See the results of the first dilution
step in Examples 1 and 2 in Clause 7.)
A lower observed count, which is at least equal to the lower colony count, indicates that the difference is
acceptable. (See the results of the second dilution step in Examples 1 and 2 in Clause 7.)
5.3 Sum of counting results of subsequent dilution steps
5.3.1 Use for the following test the colony counts from the two sets of parallel plates that passed the test in
5.2. Compare the sums of colony counts from parallel plates over two 10-fold dilution steps with the tabulated
limits in Table 2.
–x –(x+1)
For an observed sum with dilution step 10 , compare the sum obtained with dilution step 10 with the
–(x+1)
tabulated lower limit for the sum. Observed sums for the dilution step 10 within the ranges given in
Table 2 are acceptable.
Observed sums outside these limits indicate that the ratio of the colony counts obtained over two 10-fold
dilution steps deviates significantly from the expected ratio.
5.3.2 Use for the following test the colony counts from the parallel plates that did not pass the test in 5.2 or
those colony counts from one plate per dilution step. Compare the colony counts from plates over two
subsequent 10-fold dilution steps with the limits tabulated in Table 2.
–x
For an observed colony count with dilution step 10 , compare the colony count obtained with dilution step
–(x+1) –(x+1)
10 with the tabulated lower limit for the count. Observed colony counts for dilution step 10 within the
ranges given in Table 2 are acceptable.
Observed colony counts outside these limits indicate that the ratio of the colony counts obtained over two
10-fold dilution steps deviates significantly from the expected ratio. (Two comparisons of results are given in
Examples 1 and 2 in Clause 7).
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
See Figure 1 for a flowchart of the procedure.

NOTE A minus sign (–) indicates an acceptable result within limits. A plus sign (+) indicates a result that is out of
limits, and is an indication of technical problems.
Figure 1 — Flowchart describing the testing of colony-counting procedure and its evaluation
6 Evaluation
6.1 Tables of results
For each test described in 5.3.1 and 5.3.2, the results outside the limits specified in Table 1 or Table 2 shall
not occur more often than once in a 100 cases. If results outside these limits occur more often, the
microbiological test procedure shall be scrutinized.
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Table 1 — Limits of agreement for colony counts of two parallel Petri dishes
(with a probability of 99 % per comparison)
Colony count Colony count Colony count
Upper Lower Sum Upper Lower Sum Upper Lower Sum
10 2 12 54 31 85 98 66 164
11 3 14 55 32 87 99 67 166
12 3 15 56 32 88 100 67 167
13 4 17 57 33 90 101 68 169
14 4 18 58 34 92 102 69 171
15 5 20 59 35 94 103 70 173
16 5 21 60 36 96 104 71 175
17 6 23 61 36 97 105 71 176
18 6 24 62 37 99 106 72 178
19 7 26 63 38 101 107 73 180
20 7 27 64 39 103 108 74 182
21 8 29 65 39 104 109 75 184
22 9 31 66 40 106 110 76 186
23 9 32 67 41 108 111 76 187
24 10 34 68 42 110 112 77 189
25 11 36 69 43 112 113 78 191
26 11 37 70 43 113 114 79 193
27 12 39 71 44 115 115 80 195
28 12 40 72 45 117 116 81 197
29 13 42 73 46 119 117 81 198
30 14 44 74 46 120 118 82 200
31 14 45 75 47 122 119 83 202
32 15 47 76 48 124 120 84 204
33 16 49 77 49 126 121 85 206
34 16 50 78 50 128 122 86 208
35 17 52 79 50 129 123 86 209
36 18 54 80 51 131 124 87 211
37 19 56 81 52 133 125 88 213
38 19 57 82 53 135 126 89 215
39 20 59 83 54 137 127 90 217
40 21 61 84 54 138 128 91 219
41 21 62 85 55 140 129 91 220
42 22 64 86 56 142 130 92 222
43 23 66 87 57 144 131 93 224
44 24 68 88 58 146 132 94 226
133 95
45 24 69 89 58 147 228
46 25 71 90 59 149 134 96 230
47 26 73 91 60 151 135 96 231
48 27 75 92 61 153 136 97 233
137 98
49 27 76 93 62 155 235
50 28 78 94 62 156 138 99 237
51 29 80 95 63 158 139 100 239
52 29 81 96 64 160 140 101 241
141 102
53 30 83 97 65 162 243
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ISO 14461-2:2005(E)
IDF 169-2:2005(E)
Colony count Colony count Colony count
Upper Lower Sum Upper Lower Sum Upper Lower Sum
142 102 244 188 142 330 234 182 416
143 103 246 189 143 332 235 183 418
144 104 248 190 144 334 236 184 420
145 105 191 144 237 185
250 335 422
146 106 252 192 145 337 238 186 424
147 107 254 193 146 339 239 186 425
148 107 255 194 147 341 240 187 427
149 108 195 148 241 188
257 343 429
150 109 259 196 149 345 242 189 431
151 110 261 197 150 347 243 190 433
152 111 263 198 151 349 244 191 435
153 112 199 151 245 192
265 350 437
154 113 267 200 152 352 246 193 439
155 113 268 201 153 354 247 194 441
156 114 270 202 154 356 248 194 442
157 115 203 155 249 195
272 358 444
158 116 274 204 156 360 250 196 446
159 117 276 205 157 362 251 197 448
160 118 278 206 158 364 252 198 450
161 119 207 158 253 199
280 365 452
162 119 281 208 159 367 254 200 454
163 120 283 209 160 369 255 201 456
164 121 285 210 161 371 256 202 458
165 122 211 162 257 202
287 373 459
166 123 289 212 163 375 258 203 461
167 124 291 213 164 377 259 204 463
168 125 293 214 165 379 260 205 465
169 125 215 165 261 206
294 380 467
170 126 296 216 166 382 262 207 469
171 127 298 217 167 384 263 208 471
172 128 300 218 168 386 264 209 473
173 129 219 169 265 210
302 388 475
174 130 304 22
...