🦠 Salmonella spp. in water — detection with pre-enrichment in BPW, selective enrichment (RVS, MKTTn) and plating on XLD
In short
The water sample (up to 10 ml directly, larger volumes — even 1000–5000 ml — through a 0. The test is performed according to PN-EN ISO 19250:2013-07 (ISO 19250:2010); Volume of the sample: < 10 ml directly into 50 ml of BPW; > 10 ml through a 0.45 µm filter into 50 ml of BPW; drinking water 1000–5000 ml. The procedure comprises 7 steps; it is used for: Drinking water and groundwater — large volumes of 1000–5000 ml concentrated by membrane filtration, Polluted surface waters and wastewater (smaller volumes; for wastewater a shorter pre-enrichment or direct plating), Epidemiological and outbreak investigations (with additional media; note S. Typhi and S. Paratyphi).
At a glance
- Standard: PN-EN ISO 19250:2013-07 (ISO 19250:2010)
- Category: Microbiology
- Procedure steps: 7
- Volume of the sample: < 10 ml directly into 50 ml of BPW; > 10 ml through a 0.45 µm filter into 50 ml of BPW; drinking water 1000–5000 ml
- Pre-enrichment: BPW, (36 ± 2) °C, (18 ± 2) h
- Selective enrichment: RVS 0.1 ml/10 ml, (41.5 ± 1.0) °C, (24 ± 3) h [max. 42.5 °C], optionally up to (48 ± 4) h; MKTTn 1 ml/10 ml, (36 ± 2) °C, (24 ± 3) h
Overview
ISO 19250:2010 specifies a method for the detection of Salmonella spp. (presumptive or confirmed) in water samples; for epidemiological purposes or in outbreak investigations additional media may be needed. The standard warns that the method may not recover all strains of Salmonella ser. Typhi and ser. Paratyphi. A semi-quantitative approach is possible by MPN tests with suitable volumes of sample — the volume of the buffered peptone water is then adjusted. The detection requires four successive stages: pre-enrichment (which makes it possible to detect small numbers or damaged cells of Salmonella; some strains require a longer incubation), selective enrichment (Salmonella occurs in small numbers in the company of far more numerous other Enterobacteriaceae), plating onto solid media, and recognition and confirmation. For wastewater a shorter pre-enrichment or direct inoculation into the selective medium gives better results.
The typical volumes for drinking water are 1000–5000 ml, concentrated by membrane filtration; for polluted surface waters and wastewater they are smaller. Samples kept at ambient temperature are examined within 12 h of sampling; exceptionally (5 ± 3) °C for up to 24 h is allowed. In Poland it is in force as PN-EN ISO 19250:2013-07 (Polish version, published on 20-09-2022). The national suppliers of media offer a complete set for this standard: buffered peptone water, RVS broth, MKTTn broth, XLD agar and sera for the serological confirmations (somatic O and Vi antigens).
Method principle
Pre-enrichment: the buffered peptone water (BPW) is inoculated at ambient temperature with a known volume of the sample or of its dilutions and incubated at (36 ± 2) °C for (18 ± 2) h — volumes below 10 ml are added to 50 ml of BPW; volumes above 10 ml are filtered through a sterile membrane filter with pores of 0.45 µm, which is immersed in 50 ml of BPW (for turbid waters a filter aid is used, typically 15 ml on the support and 15 ml mixed with the sample; the final volume of BPW is 100 ml); the culture can also be distributed into an MPN series. Selective enrichment: 0.1 ml of the culture is transferred into 10 ml of Rappaport-Vassiliadis soya broth (RVS) incubated at (41.5 ± 1.0) °C for (24 ± 3) h (42.5 °C must not be exceeded) and, where slow-growing strains have to be detected, for a further (24 ± 3) h up to (48 ± 4) h in total; in parallel 1 ml is transferred into 10 ml of Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn), incubated at (36 ± 2) °C for (24 ± 3) h (MKTTn recovers most strains, including some S. Paratyphi, but not S. Paratyphi C; it is not used where S. Typhi is suspected after selenite cystine broth). Plating: from each selective culture xylose lysine deoxycholate agar (XLD) is inoculated, incubated inverted at (36 ± 2) °C and examined after (24 ± 3) h for presumptive Salmonella colonies, together with a second solid selective medium complementary to XLD, suitable as necessary for lactose-positive strains, S. Typhi and S. Paratyphi (e.g. brilliant green agar, bismuth sulfite agar), incubated according to the manufacturer's instructions. Confirmation: the presumptive colonies are subcultured and confirmed by suitable biochemical and serological tests.
Applications
- Drinking water and groundwater — large volumes of 1000–5000 ml concentrated by membrane filtration
- Polluted surface waters and wastewater (smaller volumes; for wastewater a shorter pre-enrichment or direct plating)
- Epidemiological and outbreak investigations (with additional media; note S. Typhi and S. Paratyphi)
- Semi-quantitative MPN determination of Salmonella with a series of sample volumes
- Control of the effectiveness of treatment and purification in waterworks and sanitary laboratories
Key parameters
| Parameter | Value |
|---|---|
| Volume of the sample | < 10 ml directly into 50 ml of BPW; > 10 ml through a 0.45 µm filter into 50 ml of BPW; drinking water 1000–5000 ml |
| Pre-enrichment | BPW, (36 ± 2) °C, (18 ± 2) h |
| Selective enrichment | RVS 0.1 ml/10 ml, (41.5 ± 1.0) °C, (24 ± 3) h [max. 42.5 °C], optionally up to (48 ± 4) h; MKTTn 1 ml/10 ml, (36 ± 2) °C, (24 ± 3) h |
| Plating | XLD agar, (36 ± 2) °C, (24 ± 3) h + a second selective medium (BGA, bismuth sulfite agar) according to the manufacturer |
| Confirmation | biochemical and serological tests (O, Vi antigens) |
| Storage of the samples | examination within 12 h at ambient temperature; exceptionally (5 ± 3) °C for up to 24 h |
Standard
- Standard number
- PN-EN ISO 19250:2013-07 (ISO 19250:2010)
- Title (PL)
- Jakość wody — Wykrywanie Salmonella spp.
- Title (EN)
- Water quality — Detection of Salmonella spp.
Step-by-step procedure
-
Sampling and storage
Take the sample and begin the examination within 12 h (ambient temperature); exceptionally store it at (5 ± 3) °C for up to 24 h. Choose the volume: drinking water 1000–5000 ml, polluted waters and wastewater — less.
-
Pre-enrichment — volumes < 10 ml
Inoculate 50 ml of BPW with the sample or with a dilution of it at ambient temperature and incubate at (36 ± 2) °C for (18 ± 2) h.
-
Pre-enrichment — volumes > 10 ml
Filter the sample through a sterile 0.45 µm membrane filter (for turbid waters: about 15 ml of filter aid on the support and 15 ml mixed with the sample), immerse the filter in 50 ml of BPW (100 ml of BPW in the end) and incubate at (36 ± 2) °C for (18 ± 2) h; for the MPN distribute it into a series of tubes.
-
Selective enrichment
Transfer 0.1 ml of the culture into 10 ml of RVS broth and incubate at (41.5 ± 1.0) °C for (24 ± 3) h (do not exceed 42.5 °C; extend it if necessary to (48 ± 4) h in total). In parallel transfer 1 ml into 10 ml of MKTTn, (36 ± 2) °C, (24 ± 3) h.
-
Plating onto solid media
From each selective culture plate XLD agar and the second selective medium; incubate the XLD inverted at (36 ± 2) °C and examine it after (24 ± 3) h, the second medium according to the manufacturer's instructions. Mark the presumptive Salmonella colonies.
-
Confirmation
Subculture the presumptive colonies (repeat the plating as in step 5; after (24 ± 3) h at (36 ± 2) °C) and confirm them by biochemical and serological tests (O, Vi antigens).
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Result
Give the presence of Salmonella spp. (presumptive or confirmed) or their absence in the volume of water examined; with an MPN series — the MPN.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Autoclave (121 ± 3) °C and (115 ± 3) °C | sterilisation of the media | — |
| Water baths or incubators at (36 ± 2) °C and (41.5 ± 1.0) °C | pre-enrichment, MKTTn, XLD / RVS broth | — |
| Water baths at (70 ± 1) °C and 50–55 °C | preparation of the media | — |
| Membrane filtration set with sterile 0.45 µm filters | concentration of volumes > 10 ml; a filter aid for turbid waters | — |
| pH meter with a calibration accuracy of ± 0.1 pH at 20–25 °C | control of the pH of the media | — |
| Petri dishes, tubes, sterile pipettes, loop | platings and subcultures | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Buffered peptone water (BPW) | — | non-selective pre-enrichment (resuscitation of damaged cells) |
| Rappaport-Vassiliadis soya broth (RVS) | — | selective enrichment at 41.5 °C |
| Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn) | — | second selective enrichment at 36 °C |
| Xylose lysine deoxycholate agar (XLD) | — | isolation plating — presumptive Salmonella colonies |
| Second selective medium (e.g. brilliant green agar, bismuth sulfite agar) | — | complementary to XLD, also for lactose-positive strains, S. Typhi, S. Paratyphi |
| Diagnostic sera (somatic O and Vi antigens) and biochemical tests | — | confirmation of the presumptive colonies |
Health and safety (OHS)
- Salmonella spp. are enteric pathogens (including S. Typhi) — work under the conditions of a microbiological laboratory, disinfect and autoclave all the cultures
- RVS broth at 41.5 °C and media after autoclaving — risk of burns
- Filtration of large volumes under vacuum — sound glassware, asepsis when transferring the filter
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 19250:2013-07 (ISO 19250:2010) — “Water quality — Detection of Salmonella spp.”.
How does this method work?
Pre-enrichment: the buffered peptone water (BPW) is inoculated at ambient temperature with a known volume of the sample or of its dilutions and incubated at (36 ± 2) °C for (18 ± 2) h — volumes below 10 ml are added to 50 ml of BPW; volumes above 10 ml are filtered through a sterile membrane filter with pores of 0.45 µm, which is immersed in 50 ml of BPW (for turbid waters a filter aid is used, typically 15 ml on the support and 15 ml mixed with the sample; the final volume of BPW is 100 ml); the culture can also be distributed into an MPN series.
What is the measuring range and accuracy?
Volume of the sample: < 10 ml directly into 50 ml of BPW; > 10 ml through a 0.45 µm filter into 50 ml of BPW; drinking water 1000–5000 ml; Pre-enrichment: BPW, (36 ± 2) °C, (18 ± 2) h; Selective enrichment: RVS 0.1 ml/10 ml, (41.5 ± 1.0) °C, (24 ± 3) h [max. 42.5 °C], optionally up to (48 ± 4) h; MKTTn 1 ml/10 ml, (36 ± 2) °C, (24 ± 3) h; Plating: XLD agar, (36 ± 2) °C, (24 ± 3) h + a second selective medium (BGA, bismuth sulfite agar) according to the manufacturer.
How long does the test take?
The procedure comprises 7 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Autoclave (121 ± 3) °C and (115 ± 3) °C, Water baths or incubators at (36 ± 2) °C and (41.5 ± 1.0) °C, Water baths at (70 ± 1) °C and 50–55 °C, Membrane filtration set with sterile 0.45 µm filters, pH meter with a calibration accuracy of ± 0.1 pH at 20–25 °C, Petri dishes, tubes, sterile pipettes, loop.
Where is this test used?
Drinking water and groundwater — large volumes of 1000–5000 ml concentrated by membrane filtration; Polluted surface waters and wastewater (smaller volumes; for wastewater a shorter pre-enrichment or direct plating); Epidemiological and outbreak investigations (with additional media; note S. Typhi and S. Paratyphi); Semi-quantitative MPN determination of Salmonella with a series of sample volumes; Control of the effectiveness of treatment and purification in waterworks and sanitary laboratories.
What safety precautions apply?
Salmonella spp. are enteric pathogens (including S. Typhi) — work under the conditions of a microbiological laboratory, disinfect and autoclave all the cultures; RVS broth at 41.5 °C and media after autoclaving — risk of burns; Filtration of large volumes under vacuum — sound glassware, asepsis when transferring the filter.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 19250.