Search: Microbiology

In EU member states, Council Directive 80/777/EEC on the approximation of the laws of the Member States relating to the exploitation and marketing of natural mineral waters of 15 July 1980 became the basis for the implementation of existing laws at national level (current version dated 18 June 2009: Directive 2009/54/EC).

Article 5 of Directive 2009/54/EC regulates the requirements for mineral water both at the source and in the bottled water.

Paragraph 1 of Article 5 specifies the permitted total colony counts. At source, the total colony count (CFU) must not exceed 20 CFU/ml at 20 °C to 22 °C/72 h and 5 CFU/ml at 37 °C/24 h. These values are to be understood as GUIDE figures [3].

After bottling, LIMITS apply to both colony counts if the total bacterial counts were analysed within 12 hours of bottling. The maximum permitted concentrations are defined as 100 CFU/ml at 20 °C to 22 °C/72 h and 20 CFU/ml at 37 °C/24 h [3].

No limit values are set for the total colony count for the marketing stage in paragraph 3 of Article 5. This takes account of the fact that mineral water is not sterile and that, under appropriate conditions, there may be an increase in microorganisms (e. g . storage conditions in retail outlets or at the consumer's premises), but this may only originate from the natural microbial flora it had at source. In addition, the mineral water must be free from organoleptic defects [3].

Paragraph 2 of Article 5 defines the requirement that mineral water must be free from parasites and pathogenic microorganisms [3]. This is considered to be fulfilled if Escherichia coli, coliforms, faecal streptococci and Pseudomonas aeruginosa cannot be detected in a sample volume of at least 250 ml and sporulated sulphite-reducing anaerobes cannot be detected in any 50 ml sample examined ("indicator principle").

Annex I Section II Number 1.3. sets out the criteria for microbiological tests at the source (quantitative determinations of indicator bacteria, sample quantities, incubation temperatures, incubation times). In the author's view, these criteria also apply to the filling of mineral water, even if this is not explicitly stated.

The EU Directive does not specify (apart from quantitative determinations) which methods should be used for carrying out microbiological tests. This is therefore left to the discretion of each Member State within the framework of national implementation.

In Germany, the "Table Water Ordinance" of 12 November 1934 was replaced by the implementation of the EU Directive with the German Mineral and Table Water ordinance (MTVO) of 1 August 1984 (current version of 5 July 2017).

It should be noted that the German implementation deviates from the EU directive in some respects. The "quantitative determination" requirement is not fulfilled in the MTVO or in the corresponding chapter in the collection of official test methods (Section 64 of the German Food and Feed Code LFGB, L59.00) for "indicator organisms". Qualitative methods are listed here.

There is a further deviation in the incubation time for the total colony count of 20 °C to 22 °C. The EU directive specifies 72 hours [3], while the MTVO and § 64 LFGB specify 48 hours [1, 2].

There are also differences between the two German regulations with regard to the higher incubation temperature. The MTVO lists 37 °C ± 1 °C [1], whereas § 64 LFGB lists  36 °C ± 1 °C [2]. From the author's point of view, this deviation has no foreseeable significant relevance to the growth behaviour of the bacteria.

In the following explanations of the test methods, only one of the two temperatures is used, namely 37 °C ± 1 °C (however, 36 °C ± 1  C can also be used in daily laboratory practice).

Qualitative detection of faecal streptococci in mineral water, spring water, table water and other drinking water bottled for distribution to the consumer by means of enrichment culture in double-concentrated azide-glucose broth [1].

Membrane filtration (MF) and immersion of the filter in single-strength azide-dextrose broth (corresponds to the methods in Annex 2, point 2b of Section 4 (3) of the German Mineral and Table Water Ordinance [1] and ASU Section 64 LFGB L59.00-2) [2].

Membrane filtration (MF) and placement of the filter on tetrazolium sodium azide agar (Slanetz-Bartley agar) (corresponds to method Annex 2, point 2b of §4 para. 3 of the German Mineral and Table Water Ordinance) [1].

Qualitative detection of Pseudomonas aeruginosa in mineral water, spring water, table water and other drinking water bottled for distribution to the consumer by means of enrichment culture in double-concentrated malachite green peptone broth [1].

Qualitative detection of Pseudomonas aeruginosa in mineral water, spring water, table water, and other drinking water filled for delivery to the consumer, by means of membrane filtration (MF) and soaking the filter in single-strength malachite green peptone broth [1, 2].