HomeMicrobiological AnalysisEN ISO 11133 Microbiological Culture Media

EN ISO 11133 Microbiological Culture Media

Riche E (PhD)1, Corpinot M (PhD)1, Plouhinec J2, Flint T1, Fischer S (PhD)3, Gerten B3, Hemy JC1, Airaud A1

1Lab Water Solutions, Merck, Guyancourt, France, 2Assystem Care, Courbevoie, France, 3LS-OII-Q Quality Specialties and BioMonitoring, {hcompany], Darmstadt, Germany

Centrally purified water vs. an autonomous water purification system

EN ISO 11133 and the quality control of microbiological culture media

Microbiology testing is critical to guarantee the safety and quality of food. Laboratories performing these tests are required to deliver accurate and reliable results and meet regulatory requirements while under time pressure. Many tests and procedures depend upon culture media being capable of providing consistent and reproducible results.

Microbiological culture media is used to test for contamination of food and water

The EN ISO 11133 standard establishes the context that ensures the quality of culture media and specifies the requirements for media preparation when used for the microbiological analysis of food, animal feed and water.1 In order to be compliant with the standard, each element of the process must meet specific requirements.

Water can potentially affect culture media performance since it is the largest component of microbiological media by volume. The purpose of the present study was to prepare culture media and to test their performance according to EN ISO 11133 using water from two alternative sources.

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.

Water sources used to prepare culture media

Each medium was prepared in duplicate by dissolving dehydrated medium in water from two different sources:

  • Purified water readily available in the QC laboratory: The purified water was centrally produced by a combination of reverse osmosis (RO), ion exchange and a bactericidal UV lamp, and delivered via a distribution loop. The water conductivity remained below 2 µS/cm throughout the study and the microbial level was < 102 cfu/mL, as required by the EN ISO 11133 standard (Table 1).
  • Water from a Milli-Q® IX 7003 water purification system: This system was installed in the lab and directly connected to tap water. It combined several water purification technologies to deliver pure water: RO, Elix® electrodeionization and bactericidal UVC LED lamps. Freshly purified water was collected from the system’s E-POD® dispenser, which was fitted with a Millipak® 0.22 µm filter to ensure low microbial levels. This system is validated to deliver purified water with a conductivity < 0.2 µS/cm, typically 0.1 µS/cm at 25 °C (equivalent to a resistivity > 5 MΩ.cm, typically 10-15 MΩ.cm), and microbial level < 102 cfu/mL, and fits the requirements of the ISO 11133 standard (Table 1).
Table 1. Compliance of the two types of water tested with EN ISO 11133 requirements.1

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.

Table 2. Application of culture media used in this study and the standards specifying them.
Table 3. List of control strains for performance testing of culture media in the present study in accordance with the List of control strains for performance testing of culture media and reagents from published standards from ISO/TC 34/SC 9 & SC 5 (food microbiology)3 and ISO/TC 147/SC 4 (water microbiology)4.

bStrain to be used as minimum; cSome national restrictions and directions may require the use of a different serovar. Refer to national requirements relating to the choice of Salmonella serovars; dStrain free of choice; one of the positive strains has to be used as a minimum; gStrain optional. (Only the required footnotes are reported here).

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.

Table 4. Physical parameters and specifications of the seven culture media used in the study, as well as results obtained with either centrally purified water or water from a Milli-Q® IX system.

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.

Table 5. Results of the performance tests of agar-based media prepared with either centrally purified water or water from a Milli-Q® IX system.

*Additional strains used in the experiments of the study. Strains tested for malt extract agar were chosen by the QC laboratory.
Table 6. Results of the performance tests of broth prepared with either centrally purified water or water from a Milli-Q® IX system.

*Additional strains used in the experiments of the study. Strains tested for mTSB were chosen by the QC laboratory.

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.

Suitability of the Milli-Q® IX water purification system to prepare and performance test microbiological culture media

This study demonstrates that culture media prepared with water from a Milli-Q® IX system has equivalent characteristics and performance as media prepared with the centrally purified water routinely used in the accredited QC lab. This indicates that water from this system can be used with confidence when preparing media according to the EN ISO 11133 standard. The Milli-Q® system can be used to prepare media manually or in combination with innovative automated instruments, such as the ReadyStream® system which simplifies the process by preparing and dispensing preheated culture media.

Water purified by the Milli-Q® IX water purification system, fitted with a 0.22 µm final filter, is validated to deliver water meeting or exceeding the water quality requirements of the EN ISO 11133 standard (Table 1). Other standards and methods, such as the American Public Health Association (APHA) Standard Methods,5 make specific recommendations regarding water quality and purification technologies, to which the Milli-Q® IX system abides by its design and performance. As recommended, it includes a combination of technologies to produce pure water, resistivity monitoring, as well as a storage tank equipped with UV irradiation. The Milli-Q® IX system delivers water with a conductivity below 0.2 µS/cm, microbial levels below 102 cfu/mL, and free from traces of contaminants likely to inhibit the growth of microorganisms, such as chlorine, ammonia and metals. Since the system delivers water that suits EN ISO 11133 water quality requirements, it will help to ensure a lab’s regulatory compliance. However, it is important to also ensure that this water is well suited for the preparation of microbiological culture media.

Design of the Milli-Q® IX system ensures constant quality pure water

Using water of consistent quality to prepare culture media is an important factor in ensuring accurate and reproducible test results. The Milli-Q® IX system is designed to produce constant quality pure water and to safeguard stored water purity via several key features:

  • Water purified by the system is stored in a high-quality polyethylene tank, which does not release extractables into water
  • The tank is protected against airborne contaminants by a vent filter.
  • Reliably low microbial levels are ensured by automatic recirculation of the pure water over a UVC LED bactericidal lamp, as well as the 0.22 µm screen filter placed at the system’s point-of-delivery.
  • The Elix® electrodeionization module included in the system delivers constant water quality, especially regarding conductivity6

The low level of extractables from the storage tank, the low microbial level in the water, as well as consistently low water conductivity are important water quality parameters highlighted in the EN ISO 11133 standard. Having a reliable and independent source of freshly purified water prevents any risk of unexpected changes in water quality or punctual unavailability of water from a central loop due to manufacturing constraints or maintenance.

Benefits of the Milli-Q® IX water purification system to prepare and performance test microbiological culture media

Using a Milli-Q® IX water purification system offers many benefits to labs performing microbiology testing.

  • According to the EN ISO 11133 standard, water conductivity (or its reciprocal, resistivity) should be checked before use. The Milli-Q® IX water system lets users accurately measure water resistivity on-line and displays it on the interface of the E-POD® dispenser, allowing a convenient monitoring solution. Checking water quality parameters is easy to perform via the intuitive touchscreen compatible with gloves.
  • The E-POD® dispenser allows time gain and precision as the flow rate can easily be adapted to the needs of the experiment, and the screen lets the user program a specific volume to be dispensed. Moreover, the dispenser brings flexibility as it can be used over the sink or rested on its arm to fill larger containers.
  • A foot pedal can be added to facilitate serial dispensing while freeing the user’s hands.
  • Regular system maintenance is easily done by any member of the lab, or can be delegated to a service engineer to ensure the system operates optimally.
  • The system stores all data related to water quality parameters, as well as information such as consumable changes and other service activities, thus providing data traceability and paperless data management. Easy-to-retrieve data can simplify the lab’s audit preparation. An online service is available to store all documentation and enable online service contract management.
  • For additional efficiency, the system can be used as a direct water source for the ReadyStream® system, to conveniently prepare large amounts (up to 100 L) of preheated media right where it is needed in only 15 minutes. The purified water delivered by the system can also be used for other tasks, such as glassware rinsing, and to feed autoclaves or glassware washers. This makes the Milli-Q® IX system a comprehensive solution for microbiology laboratories.
E-POD<sup>®</sup> dispenser of the Milli-Q<sup>®</sup> IX pure water system

The results of this study revealed that culture media prepared with water from the Milli-Q® IX water purification system performed similarly as the culture media prepared with the lab’s routinely used centrally purified water. This Milli-Q® water purification system enabled the lab to prepare media performing as required by the standard, while bringing additional benefits, such as ease of use, convenience, data traceability and autonomy. Combined, these features allow for efficient water system management, confidence in water purity and improved lab efficiency, which can keep scientists working at maximum productivity.


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