Development of ISO 11731:2017. Simon in t Veld Manager Microbiology Vitens Laboratorium Convenor ISO TC7/SC4/WG10 Legionella
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1 Development of ISO 11731:217 Simon in t Veld Manager Microbiology Vitens Laboratorium Convenor ISO TC7/SC4/WG1 Legionella
2 Vitens
3 Vitens Laboratory
4 Content Standards ISO 11731:1998 ISO :24 ISO 11731:217 Membrane filters Culture media Sampling Procedure Sample pre-treatment Culture/Incubation/Examination Performance data Decision Matrix
5 Standards ISO 11731: 1998 ISO : 24 T9-431:214 (France) AZ/NZS 3896: 28 (Australia/New Zealand) Standard Methods (USA) NEN 6265:27 (Netherlands) WAC/V/A/5:214 (Belgium)
6 ISO 11731: 1998
7 ISO 11731: 1998 Scope All kinds of environmental samples including potable, industrial and natural waters and associated materials such as sediments, deposits and slime. Membrane filters Nylon of polycarbonate Sampling Transport (6-18) C within two days
8 ISO 11731: 1998 Procedure Direct plating (>15 cfu/l Legionella) Membrane filtration (wash method) Centrifugation Dilution (high concentration non Legionella)
9 ISO 11731: 1998 Treatment Untreated Heat treatment (5 C for 3 minutes) Acid treatment (ph 2,2 for 5 minutes) Culture media GVPC BCYE and BCYE-cys for confirmation ph culture media (6,9 ±,4)
10 ISO 11731: 1998 Examination Three occasions at intervals of two days to four days during the 1 days incubation.
11 ISO : 24
12 ISO : 24 Scope Water intended for human use (e.g. hot and cold water, water used for washing), for human consumption and for treated bathing waters (e.g. swimming pools). It is especially suitable for waters expected to contain low numbers of Legionella. As the growth of Legionella may be inhibited by overgrowth of other bacterial colonies on the membrane, the method is only suitable for waters containing low bacterial counts.
13 ISO : 24 Sampling Same working day, transport at ambient temperature Otherwise, cool samples (5 ± 3) C and start analyses within two days
14 ISO : 24 Membrane filtration black nitrocellulose membrane filter Treatment Acid treatment (ph 2,2 for 5 minutes) Culture media GVPC BCYE and BCYE-cys for confirmation ph culture media (6,8 ±,2)
15 ISO : 24 Examination At least two occasions starting at day three or four during the 1 day incubation time.
16 ISO 11731:217
17 ISO 11731:217 1 Scope These methods are applicable to all kinds of water samples including potable, industrial, waste and natural waters. These methods can be used for water related matrices, e.g. biofilms, sediments, etc.
18 5.5 Membrane filter (1) Membrane filter for concentration and elution Polycarbonate or polyethersulfone membrane filters, diameter 47 mm to 142 mm with rated pore sizes of,2 μm; see Reference [4]. These types of membrane filters are used for concentration followed by a washing procedure.
19 5.5 Membrane filter (1) Membrane filter for concentration and elution Source: LORI SMITH, KAREN CARROLL AND SUSAN MOT[ICE: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1993, p
20 5.5 Membrane filter (1) Membrane filter for concentration and elution Source: LORI SMITH, KAREN CARROLL AND SUSAN MOT[ICE: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1993, p
21 5.5 Membrane filter (1) Membrane filter for concentration and elution Source: LORI SMITH, KAREN CARROLL AND SUSAN MOT[ICE: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1993, p
22 5.5 Membrane filter (1) Membrane filter for concentration and elution Source: LORI SMITH, KAREN CARROLL AND SUSAN MOT[ICE: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1993, p
23 5.5 Membrane filter (2) Membrane filter for direct placing on culture media Membrane filters from cellulose nitrate or mixed cellulose esters, diameter 47 mm to 5 mm with rated pore sizes of,2 μm or,45 μm. These types of membrane filters are used for direct placing onto the culture media after filtration. Filters shall be evaluated prior to use in accordance with ISO 774. NOTE Black membrane filters contrast better with the white Legionella colonies than light-coloured membrane filters.
24 5.5 Membrane filter (2) Influence membrane filter Cellulose Nitrate
25 5.5 Membrane filter (2) Influence membrane filter Mixed Cellulose Ester
26 5.5 Membrane filter (2) Influence membrane filter Mixed Cellulose Ester: Supplier X, Batch A
27 5.5 Membrane filter (2) Influence membrane filter Mixed Cellulose Ester: Supplier X, Batch B
28 6 Culture media and reagents Buffered charcoal yeast extract (BCYE) agar Buffered charcoal yeast extract agar without L-cysteine (BCYE-cys) Buffered charcoal yeast extract agar with selective supplements (BCYE+AB) Glycine vancomycin polymyxin B cycloheximide (GVPC) agar Modified Wadowsky Yee (MWY) agar.
29 Development culture media 1978 Mueller Hinton Agar + additives 1978 FG medium (L-cysteine and Iron (III)) 1979 CYE medium (active carbon and yeastextract) 1979 CYE+antibiotics (Polymixin en Vancomycin) 198 BCYE (ACES buffer) 1981 WY (CYE+antibiotics + glycin) 1981 BMPα (CYE + mix antibiotics+ α-ketoglutarate) 1982 MWY (WY+AB+dyes+α-ketoglutarate) 1984 GVPC (WY + antibiotics + α-ketoglutarate) 1984? BCYE+ab
30 Development culture media 1978 Mueller Hinton Agar + additives 1978 FG medium (L-cysteine and Iron (III)) 1979 CYE medium (active carbon and yeastextract) 1979 CYE+antibiotics (Polymixin en Vancomycin) 198 BCYE (ACES buffer) 1981 WY (CYE+antibiotics + glycin) 1981 BMPα (CYE + mix antibiotics+ α-ketoglutarate) 1982 MWY (WY+AB+dyes+α-ketoglutarate) 1984 GVPC (WY + antibiotics + α-ketoglutarate) 1984? BCYE+ab
31 Recovery Legionella pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. pneumophila 1 ± 6 17 ± 6 79 ± ± 5 serogroup 1 1 ± ± ± 3 69 ± 8 95 ± 4 8 ± 12 L. pneumophila 1 ± 25 9 ± ± 1 75 ± 8 86 ± 18 serogroup 3 1 ± 2 13 ± ± ± ± 18 L. pneumophila 1 ± 2 11 ± 4 92 ± 2 66 ± ± 3 serogroup 6 1 ± ± ± ± ± 23
32 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
33 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
34 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
35 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
36 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
37 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
38 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
39 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (=NEN) (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l L. dumoffii 1 ± 9 1 ± 9 9 ± 6 92 ± ± 7 98 ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 1 ± ± 1 91 ± 5 88 ± ± 6 1 ± ± ± ± ± 3 L. jordanis 1 ± 2 1 ± 16 1 ± 3 1 ± 7 1 ± 3 7± 3 66 ± 7 11 ± 8 87 ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± ± 7 88 ± 14 serogroup 1 1 ± ± 15 1 ± ± 7 serogroup 2 1 ± ± ± ± ± ± 5 L. bozemanii 2± ± 4 L. gormanii 78 ± 71 ± 35 ± 4 ± 77 ± L. micdadei 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± ± 9
40 Recovery Legionella non-pneumophila BCYE BCYE BMPα GVPC MWY non-selective +selective (Oxoid SR111) (Oxoid SR152 (Oxoid SR118) kve/l (=NEN) 1 ± 9 9 ± ± 7 1 ± 9 92 ± ± 9 6 ± 9 45 ± 8 L. longbeachae 1 ± 8 88 ± 6 71 ± 6 serogroup 1 1 ± ± 5 88 ± 1 52 ± 1 47 ± 8 52 ± 6 77 ± 2 L. longbeachae 1 ± ± ± 9 94 ± 7 95 ± 11 serogroup 2 1 ± 2 13 ± ± 1 79 ± ± 2 L. anisa 1 ± 5 91 ± 1 88 ± 9 91 ± ± 6 1 ± 9 1 ± 2 1 ± ± ± ± ± 1 81 ± 4 L. bozemanii 1 ± 8 99 ± 18 serogroup 1 1 ± ± 15 L. bozemanii 1 ± ± 7 serogroup 2 1 ± ± 28 2± 2 11 ± 8 1 ± 7 1 ± 3 7± 3 66 ± ± 4 1 ± ± 71 ± 35 ± 4 ± 77 ± L. dumoffii L. gormanii L. jordanis 1 ± 3 L. micdadei 96 ± 3 87 ± ± 14 7 ± 5 91 ± 11 9 ± 2 54 ± 8 49 ± 3 94 ± ± ± 7 88 ± ± ± ± ± 5 17 ± ± 9
41 Influence of ph of culture media on the growth of Legionella species
42 7 Sampling Carry out sampling, transport and storage of the samples in accordance with ISO Take care not to expose the samples to adverse temperature conditions (e.g. freezing or overheating). NOTE The use of insulated containers is helpful in this regard.
43 8 Procedure 8.1 Samples and 8.2 Concentration of samples Direct plating (>14 cfu/l) Membrane filtration (filter on plate) Membrane filtration (washing procedure) Dilution (highly contaminated samples)
44 8 Procedure Centrifugation procedure moved to informative Annex Recovery centrifugation Percentage recovery Laboratory 6 7 8
45 8 Procedure 8.3 Sample pre-treatment Heat treatment (5 ± 1) C (3 ± 2) minutes Acid treatment (5 ±,5) minutes ph (2,2 ±,2)
46 8 Procedure 8.3 Sample pre-treatment Result ringtrial for validation standard ISO times concentrated acid solution; 1 part acid 9 parts sample
47 Acid treatment drinking water 8, 7, 6, 9 volume sample : 1 volume acid 5, 5 volume sample : 5 volume acid 4, 1 volume sample : 9 volume acid 3, 2, 1,,
48 Acid treatment cooling tower water 9, 8, 7, 9 volume sample : 1 volume acid 6, 5 volume sample : 5 volume acid 5, 4, 1 volume sample : 9 volume acid 3, 2, 1,,
49 Acid treatment drinking water 1 times concentrated acid 6, 5, 9 volume sample : 1 volume acid 4, 5 volume sample : 5 volume acid 3, 1 volume sample : 9 volume acid 2, 1,,
50 Acid treatment cooling tower water 1 times concentrated acid 8, 7, 6, 9 volume sample : 1 volume acid 5, 5 volume sample : 5 volume acid 4, 1 volume sample : 9 volume acid 3, 2, 1,,
51 8.4 Culture High concentration Legionella species Low concentration interfering organisms Direct plating Low concentration Legionella species Low concentration interfering organisms Membrane filtration: - Membrane filter on plate - Filtration with washing procedure
52 8.4 Culture High concentration interfering organisms Dilution Direct plating Concentrated (filtration with washing procedure) Extremely high concentration interfering organisms Direct plating Dilution
53 Incubation and examination 7 days to 1 days (36 ± 2) C First inspection after day, 2,3,4 or 5 followed by end inspection at the end of the incubation period.
54 Performance data
55 Performance data
56 Annex J: Decision Matrix The first step is to determine the origin and characteristics of the sample.
57 Annex J: Decision Matrix The second step is to determine the desired lower limit of detection level and to select one or more method(s).
58 Annex J: Decision Matrix
59 Annex J: Decision Matrix The third step is the selection of the required treatment(s). Besides the required treatment(s), optional treatment(s) can be carried out. The fourth step is the selection of the required culture media. Besides the required culture media, optional culture media can be used.
60 Annex J: Decision Matrix
61 Annex J: Decision Matrix
62 Annex J: Decision Matrix
63 Annex J: Decision Matrix
64
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