Temi ambientali
Servizio Sanitario Regionale
Arpae Emilia-Romagna
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Methods of analysis

Bathing water monitoring is done every season through the collection of at least 5 samples (the exact number is set every year by Emilia-Romagna Region). The first one is considered supplementary and shall be taken shortly before the beginning of the bathing season. Analysis is done on two microbiological parameters (Escherichia coli and Intestinal enterococci) that determine if water is compliant with the requirements for bathing. If only one of the parameters is over the limits, the entire bathing area shall be closed and bathing temporarily prohibited. The area can be opened again after a sample showing that values are below the limits of law.

Sampling procedures

Sampling procedures are defined in the Annex D of D.M. 30/03/2010. Samples shall be taken in the sea strip usually used by bathers, around 30 cm under the surface of the water, at a distance from the shoreline so that the seafloor is from 80 to 120 cm deep, with the exception of areas with sheer cliffs or steeply descending seafloor: in these cases, samples shall be taken near the cliffs or the shoreline. In Emilia-Romagna the right depth is to be found a few tens of metres from the shoreline. The variability depends on tide levels.Samples shall be taken from 9 to 16 o´clock and registered on specific reports.

The following weather and marine parameters shall be collected and reported in the monitoring report:

• air temperature (°C)

• water temperature (°C)

• wind: direction and speed

• state of the sea, direction of waves, visual estimation of wave height

• surface current: intensity and direction

• weather conditions: rain, cloud cover


Sample containers are to be sterile and transparent, with a capacity of at least 500 ml. They shall be stored at a temperature of around +4°C until they are taken to the laboratory. Analysis shall take place if possible on the same working day, and in any case within no more than 24 hours.
Microbiological analysis are done in the "Bathing water" Lab of Oceanographic Structure "Daphne" of Arpae in Cesenatico.

 

METHODS OF ANALYSIS

Methods of analysis comply with the legislation:

 

Escherichia coli (EN ISO 9308-2:2012) 

Intestinal enterococci (Enterolert®-E/Quanti-Tray®)


ESCHERICHIA COLI - The EN ISO 9308-2: 2012 method is based on the growth of target organisms in a liquid medium and on the calculation of the "most likely number" (MPN), using the multi-well Defined Substrate Technology (DST). It foresees an incubation of the specimen, suitably diluted, at 36 ± 2 ° C for 18-22 h. The nutrient substrates used, distributed in series of wells (Quanti-Tray®) in which the sample to be tested is inoculated, are added with chromophores and fluorigens compounds which hydrolysed by specific enzymes (β-D-galactosidase and β-D-glucuronidase) produced by the sought-after microorganisms, make it possible to highlight their growth through the yellow coloring of the wells and confirm the appearance of blue fluorescence under ultraviolet light 365 nm (Figures 1 and 2).

 

Figure 1: Colilert after incubation:
yellow wells, presumably positive
Figure 2: Colilert after incubation exposed to ultraviolet light for confirmation: fluorescent wells for the presence of Escherichia coli.

 

INTESTINAL ENTEROCOCCI - The method used to detect Intestinal Enterococci in marine waters is Enterolert®-E / Quanti-Tray® based on the multi-wells Defined Substrate Technology (DST). The sample, suitably diluted, is incubated at 41 ± 0.5 ° C for 24 h. Enterococci use their β-glucosidase enzyme when present in the sample to metabolize the Enterolert® 4-methylumbutylated β-D glucoside nutrition indicator by releasing the 4-methylumbelliferone compound which, if it is observed under ultraviolet light at 365 nm, emits a signal fluorescent blue (Figure 3).

 

 Figure 3: Enterolert after incubation exposed to ultraviolet light.

 

Both methods require, when they are used with marine waters, a dilution of 1:10 in sterile water. Concentration counts for both wanted microorganisms are obtained by the Quanti-Tray® or Quanti-Tray® / 2000 method (single-dose trays are composed by 51 or 97 wells) that allows semi automatic quantification based on the most probable number (MPN). The Oceanographic Structure Laboratory Daphne uses the Quanti-Tray® / 2000 enumeration method at 97 wells, which makes it possible for marine waters samples to quantify both the microorganisms sought in the range of <10 to 24190 MPN per 100 ml.

 

The following table briefly describes the methods of analysis.