1. NEAGU LILIANA Microbiologist for Romanian National Institute of Public Health Dr.A. Leonte 1-3, Sector 5, Bucharest, Romania liliana.neagu@insp.gov.ro

2. European Drinking Water Directive 98/83 EC

3. E. Coli and coliformsEN ISO 9308-1 or EN ISO 9308-2 Intestinal enterococciEN ISO 7899-2 Total countEN ISO 6222 Pseudomonas aeruginosa bottled water only EN ISO 16266 Clostridium perfringens surface water only EN ISO 14189 Water quality – Sampling for microbiological analysis EN ISO 19458 General guide to the enumeration of microorganisms by culture EN ISO 8199

4. Limits for drinking water

5. Limits for bottled water

6. * According to this directive all tests (excluded the detection of total count) must be made by membrane filter method. * The membrane filter must have a pore size of o,45µm sufficient to retain the bacteria. * Sampling shall be carried out in accordance with ISO 19458.

7. Detection and enumeration of E.coli and coliform bacteria – according to EN ISO 7899-1

8. Membrane filtration unitDisinfect by flaming

9. Put sterile membrane filter on filtration unit

10. Filtrate 100 ml samplePut membrane filter on Chromogenic Coliform Agar (CCA) Incubation at (36±2)ºC for (21±3)h.

11. Escherichia coli Coliform bacteria - Count all colonies giving a positive β-D-galactosidase and β-D-glucuronidase reaction (dark –blue to violet) as E. coli - Count all colonies giving a positive β-D-galactosidase reaction (pink to red) as presumptive coliform bacteria that are not E.coli

12. Identification coliform bacteria  The confirm the presumptive coliform bacteria that are not E.coli.  Preparing subculture onto a non selective agar at (36±2)ºC for (21±3)h: - oxidase test (commercial tests can be used).

13. Detection and enumeration of E.coli and coliform bacteria – according to EN ISO 7899-1

16. Case after identification or confirmation After identification or confirmation, calculate for each of the plates the confirmed results as the proportion of presumptive colonies complying with the identification or confirmation criteria, usig Equation:

17. Where: x = is the estimated number of confirmed colonies per plate; k = is the number of colonies complying with identification or confirmation criteria among the inoculated colonies n; n = is the number of presumptive positive colonies inoculated from a plate for confirmation; z= is the total number of presumptive positive colonies counted on the plate

18. Detection and enumeration of intestinal enterococci – EN ISO 7899-2

20. CONFIRMATION AND ENUMERATION

21. Expression of results Calculate the results in accordance with ISO 8199.

22. Procedure – preparation and inoculation  Preparation of the sample  Place a volume of the test sample (or it dilution) not exceeding 2ml in the Petri dish.

23. Procedure – preparation and inoculation  Add 15 ml to 20 ml of the molten medium, allow to cool and maintain it at (45±1)ºC in the Petri dish.  Time between addition of the test sample (or it dilution) and addition of the molten medium shall not exceed 15min.  The inoculated doublets for incubation at each temperature.

24. Procedure – preparation and inoculation  Mix carefully by gentle rotation.  Leave the Petri dish on a flat surface aprox. 10 minutes until the gel completely solidifies.

25. Procedure – incubation and examination  Invert the plates and incubate one set at (36±2)ºC for (44±4)h;incubate the other set at (22±2)ºC for (68±4)h.  Examine the plates as soon as they are removed from the incubators.

26. Examination of the plates >300 colonies

27. Countig of colonies  Count all colonies (from the surface and interior of the culture medium).  A counter of colonies with a magnifying glass helps with counting small colonies grown in culture medium.  Calculate the results in accordance with ISO 8199.

28. ”Water is the essence of life”