EN ISO 11133 and Water for the Preparation and Performance Testing of Microbiological Culture Media

Centrally purified water vs. water from a Milli-Q^® IX system and culture media performance

This study investigated whether a modern water purification system, the Milli-Q^® IX pure water system, installed in a microbiology laboratory could advantageously replace centrally produced purified water obtained from a distribution loop when preparing and testing microbiological media. Solid and liquid culture media for important pathogens and hygiene indicators were included, such as Listeria, Salmonella, Escherichia coli serogroup O157 and coliforms, as well as culture media for yeast, molds and lactic acid bacteria.

Culture media preparation

Seven different dehydrated culture media were selected (Table 2) to obtain representative results. All media are compliant with the corresponding EN ISO/FDA Bacteriological Analytical Manual (BAM)/USDA-FSIS/APHA and other standards and methods were involved in performance testing. The standards specifying the media and the ones specifying their relative performance testing are reported in Table 3, as well as the control stains, and the WDCM and ATCC^® (American Type Culture Collection) numbers.

The media were prepared following manufacturers’ instructions. Specifically, the required mass of the dehydrated media was added to 500 mL of centrally purified water or Milli-Q^® IX water. All media were dissolved, heated and, if required, autoclaved as needed according to manufacturer’s instructions.

The study was performed by the Life Science QC laboratory for microbiological products at Merck, Darmstadt, Germany. The lab is accredited by the German accreditation authority DAkkS as registered test laboratory D-PL-15185-01-00 according to DIN EN ISO/IEC 17025² for the performance testing of media for microbiology according to DIN EN ISO 11133.

Culture media evaluation: physical and chemical quality control

The seven culture media studied were prepared with water from the two alternative sources. Each prepared culture medium was assessed by visual inspection to ensure that it conformed to stated recommendations, e.g. appearance, color, homogeneity, gel consistency and pH.

Table 4 describes the physical parameters and the expected specifications, as well as the results obtained in both conditions. In each case, the media prepared with water from the Milli-Q^® IX system matched the required specifications and gave similar outcomes as media prepared with the centrally produced purified water of the QC laboratory.

Performance testing of prepared microbiological culture media

Growth and inhibition were assessed by either quantitative or qualitative methods as described in the EN ISO 11133 standard.

• Productivity is the level of recovery of a target microorganism from the culture medium under defined conditions.
• Selectivity is the degree of inhibition of a non-target microorganism on or in a selective culture medium under defined conditions.
• Specificity is the demonstration, under defined conditions, that non-target organisms, if a grow on the medium, do not show the same visual characteristics as target microorganisms.

1. Bacteria, yeasts and mold preparation and inoculation

Control strains were obtained from ATCC^® and were selected according to the requirements of the EN ISO 11133 standard and/or other specific standards. They were diluted to the desired number of organisms for inoculation for testing on productivity, specificity and selectivity. Performance evaluation and interpretation of the results were obtained following the specifications given by EN ISO 11133.

2. Performance testing

• Quantitative methods: Quantitative productivity for solid culture media was measured by quantitative methods as described in EN ISO 11133.
• Qualitative methods: Productivity, specificity and selectivity were determined qualitatively using the methods as described by EN ISO 11133.

All the culture media prepared and tested met the required criteria for productivity, selectivity and specificity, when applicable. Quantitative productivity tests met the required criteria: productivity ratio (PR) ≥ 50% for selective media and PR ≥ 70% for non-selective media. Table 5 and Table 6 report the results obtained in the study. Qualitative and quantitative tests of the culture media led to results in accordance with the standard requirements with purified water used in the QC laboratory as well as with water delivered by the Milli-Q^® IX system.

Selecting a water solution for media preparation

When preparing culture media, it is important to select the correct water quality since water may contain impurities that can affect media productivity or cause abnormalities, such as incorrect pH, wrong color or precipitation, as indicated in Annex H of the EN ISO 11133 standard. The water quality requirements of the standard are shown in Table 1. An additional note in the standard indicates that water which has been passed through an ion exchange resin (demineralized or deionized water) can have a very high microorganism content. It is therefore advised not to use water from these sources without first checking microbial content. The standard warns that highly contaminated demineralized water, even if sterilized by filtration, can still contain substances that are inhibitory for the growth of certain microorganisms.