Ask the Expert: How to Choose the Right Method for Microbial Testing in Infant Formula?

By: Dr. Andreas Bubert, Senior Global Product Manager Culture Media F&B

Interview with our food microbiology expert on microbial infant formula testing

Andreas Bupert

Infant formula and their ingredients are very sensitive foods which must be carefully microbiologically analyzed before release to market. Recent recalls of infant formula products due to microbial contamination raise the question which methods QC labs should apply to correctly detect microorganisms, especially pathogens.

Dr. Andreas Bubert has a PhD in molecular microbiology and over 20 years’ experience in the food and beverage industry. He is a member of AOAC RI Advisory Council, German VAAM and has authored over 40 publications.

Which microorganisms do we need to test for in powdered infant formula (PIF) and their raw materials?

Salmonella bacteria

Figure 1. Salmonella bacteria

Most critical is the absence of Cronobacter spp. and Salmonella in PIF. While Salmonella is able to infect all humans, Cronobacter spp. (formerly Enterobacter sakazakii) is a pathogenic bacterium that can cause severe diseases, primarily in infants under 6 months and immunocompromised adults. This organism is able to survive in low moisture foods, such as PIF, for up to two years.

In the EU, Commission Regulation (EC) No 2073/2005 requires the absence of Cronobacter and Enterobacteriaceae in 10 g and of Salmonella in 25 g respectively in dried infant formulae and dried dietary foods for special medical purposes intended for infants below six months of age. This food must also be tested for presumptive Bacillus cereus.

In the US, the Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as "a food which purports to be or is represented for special dietary use solely as a food for infants by reason of its simulation of human milk or its suitability as a complete or partial substitute for human milk" (FFDCA 201(z)). FDA regulations define infants as persons not more than 12 months old. All finished dried product infant formula samples (domestic or import) designated for microbiological analysis need to be tested for Salmonella and Cronobacter. For Salmonella analysis, each sample should consist of 25 grams or larger, for Cronobacter analysis it is 10 grams or larger.

Which quality control methods can be used?

Most countries have standards for quality control (QC) of PIF and their ingredients. In ISO following countries, QC labs can choose to follow either culture media-based ISO standards or ISO 16140 validated rapid methods. In 2017, ISO published revised culture media-based standards for Salmonella (ISO 6579-1: 2017), Cronobacter spp. (ISO 22964:2017), and Enterobacteriaceae (ISO 21528:2017), which are all relevant for testing of PIF and its ingredients.

These standards should be carefully read. ISO 6579-1 gives guidance to reduce the risk of false-negative detection results for atypical Salmonella strains. A recently recalled infant formula contained the atypical, H2S-negative strain Salmonella agona. Such a strain is difficult to detect, particularly by less experienced users.  

For Cronobacter spp., two new culture media types are included in the revised ISO 22964:2017. This version was validated, not only with infant formula, but also with its ingredients, e.g. milk powder, soy milk powder, starch, and environmental samples. It does not describe the usage of molecular methods for detection or confirmation. However, it is stated that alternative methods can be used to confirm the isolate as Cronobacter spp. provided that the suitability of the alternative method has been verified. Updated guidance for method verification is intended to be published in 2019 in ISO 16140-part 3 named “Protocol for the verification of reference and validated alternative methods implemented in a single laboratory.”

Infant Formula

Figure 2. Infant formula

In the US, FDA-BAM standards need to be followed. The Bacteriological Analytical Manual Chapter 29 Cronobacter contains both a real-time PCR method for rapid screening and a cultural method for the detection/isolation of Cronobacter spp.. Chromogenic agars are used to isolate the culture for confirmation. A pre-enrichment step is used to grow the bacteria to a level (≥ 103 CFU/ mL) detectable by PCR and chromogenic agars. The culture media portion of this method is a complete detection/isolation method, so it can be used as a stand-alone method if PCR technology is unavailable. It is completely different from the ISO method. The PCR portion of the method is a screening method, whose positive results should always be confirmed with the culture method. The PCR method may be used to confirm pure cultures as Cronobacter spp.

The FDA-BAM Chapter 5 (Salmonella 8th ed.) method for detection, isolation and confirmation of Salmonella was revised in 2018. It is mostly culture media-based but differs completely from ISO 6579-1:2017 in the usage and/or composition of the media, e.g. use of XLD agar. It now also contains a real-time quantitative PCR method for confirmation of pure cultural isolates as Salmonella spp.  

Countries which do not describe ISO or FDA-BAM based standards have their own methods which may be very different. For example, in China, GB standards need to be followed: GB 4789.40-2016 is the national food safety standard for food examination of Cronobacter while GB 4789.4-2016 is for examination of Salmonella. Both standards were also issued in 2016 by the China Food and Drug Administration. For Salmonella, the method is different from both ISO and FDA-BAM in the usage culture media. For Cronobacter spp., the current GB method is very similar to the previous ISO TS 22964: 2006. Both GB standards do not describe a rapid method for the testing of these pathogens.

It is important for the export of powdered infant formula products that the QC procedures of the receiving country are always followed, e.g. a PIF exported from Europe for sale in China must be tested according to the Chinese GB standards.   

What to consider when choosing the right method?

For such sensitive food, QC labs of food companies exporting PIF need to be aware of the food testing regulations in the countries of destination. Culture media-based methods are described in all countries, but a careful check of the compliance of culture media products used in quality control is important. The culture media should be of high quality and should be accompanied by a clear documentation about the compliance to the given standards. This compliance information should be visible in both the product and batch documentation provided by the media supplier. Such documentation also supports QC managers in audit situations and lab inspections.

As described above, a rapid method can only be applied if permissive in the country of destination. These methods are only acceptable if validated according to ISO 16140 or AOAC International standards.

Figure 3. Cronobacter sakazakii WDCM 00124 on ChromoCult® CCI agar acc.

Molecular methods for detection of Cronobacter and Salmonella are preferred as they are able to detect these organisms in low numbers. There are several systems on the market available. To choose the right supplier, users should have a close look at the performance data of the individual system with PIF as food matrix. A sample preparation procedure that efficiently extracts the target nucleic acid is important, e.g. by magnetic separation, which leads to higher test reliability. Users should carefully read the approval validation protocols before to decide for a system.   

Our solutions for infant formula testing

We offer a broad range of culture media solutions, from traditional powdered and granulated media to ready-to-use media. Our culture media solutions meet the highest industry performance standards and our compliance documentation to international regulations is trend-setting in the food industry. In addition, we also offer a comprehensive portfolio of Microbiological Certified Reference Materials (CRMs) which can be used for media performance testing and quality control. Our Assurance® GDS molecular detection systems include very efficient DNA extraction using immunomagnetic separation and provide faster results with increased accuracy and high sensitivity required to meet today’s food safety testing needs.

Chromucult® granulated mediaGranulated Media

Figure 4. ChromoCult® granulated media

Check our complete portfolio for microbiology food safety testing here: Food Microbiology Testing. Contact your specialist to find the microbial testing solution matching your needs.

Culture Media

Catalog No. Application Description Pack Size
1125350500 Sample Preparation GranuCult® Peptone salt solution (Maximum recovery diluent) acc. ISO 6887 and ISO 8199 500 g
1072280500 Pre-enrichment GranuCult® Buffered Peptone Water acc. ISO 6579, ISO 21528, ISO 22964, FDA-BAM and EP 500 g
1009080001 Pre-enrichment Readybag® Buffered Peptone Water acc. ISO 6579, ISO 21528, ISO 22964, FDA-BAM and EP, 29 g, irradiated 60 bags
1464040006 Pre-enrichment ReadyTube® 225 BPW ISO 6579, ISO 6887, ISO 21528, ISO 11290, FDA-BAM and EP 6 x 225 mL
1077000500 Selective enrichment Salmonella GranuCult® RVS (RAPPAPORT-VASSILIADIS-Soya) Broth (Base) acc. ISO 6579 500 g
1466940020 Selective enrichment Salmonella ReadyTube® 10 RVS Broth ISO 6579 20 x 10 mL
1058780500 Selective enrichment Salmonella GranuCult® MKTTn (MULLER KAUFFMANN Tetrathionate Novobiocin) Broth (Base) acc. ISO 6579 500 g
1052870500 Detection Salmonella GranuCult® XLD (Xylose Lysine Deoxycholate) agar acc. ISO 6579 500 g
1075000001 Detection Salmonella RAMBACH® Agar 4 x 250 mL
1467190020 Detection Salmonella Salmonella-Elektiv Agar (Rambach-Agar) 20 plates
1205970500 Selective enrichment Cronobacter GranuCult® CSB (Cronobacter Selective Broth) base acc. ISO 22964 500 g
1205950010 Selective enrichment Cronobacter CSB Selective supplement (Vancomycin) acc. ISO 22964 10 x 1 vial
1205960500 Detection Cronobacter Chromocult® CCI (Chromogenic Cronobacter Isolation) agar acc. ISO 22964 500 g
1054580500 Confirmation GranuCult® Tryptic Soy Agar EP, USP, JP, ISO and FDA-BAM 500 g
1464310020 Confirmation ReadyPlate™ Tryptic Soy Agar ISO, FDA-BAM, EP+USP 20 plates
1054500500 Confirmation GranuCult® Nutrient agar acc. ISO 6579, ISO 10273 and ISO 21528 500 g
1084920500 Confirmation Urea Agar (Base) acc. CHRISTIANSEN 500 g
1057120500 Confirmation MR-VP (Methyl Red-VOGES-PROSKAUER) 500 g
1109870500 Confirmation Phenol-red broth (base) 500 g
1050430250 Confirmation Potassium Iodide 250 g
1133000001 Confirmation Bactident® Oxidase 50 strips
1102750500 Detection Enterobacteriaceae GranuCult® VRBD (Violet Red Bile Dextrose) agar acc. EP, USP, JP and ISO 21528 500 g
1461270020 Detection Enterobacteriaceae ReadyPlate™ VRBG Agar ISO 21528 20 plates
1052670500 Detection presumptive Bacillus Cereus GranuCult® MYP (Mannitol Egg Yolk polymyxin) Agar (Base) acc. ISO 7932, ISO 21871 and FDA-BAM 500 g
1037840001 Detection presumptive Bacillus Cereus Egg-Yolk Emulsion sterile, 50 %, for microbiology 10 x 100 mL
1098750010 Detection presumptive Bacillus Cereus Bacillus Cereus Selective Supplement 10 x 1 vial
1461600020 Detection presumptive Bacillus Cereus ReadyPlate™ MYP ISO 7932, 21871 20 plates
1205890500 Detection presumptive Bacillus Cereus GranuCult® PEMBA (Polymyxin Pyruvate Egg yolk Mannitol Bromothymolblue Agar) (base) acc. ISO 21871 500 g
1467110020 Detection presumptive Bacillus Cereus ReadyPlate™ PEMBA ISO 21871 20 plates


Ready-to-Use Certified Reference Microorganisms for Performance Testing of Media

Catalog No. Strain CFU Range Pack Size
VT000097 Enterococcus faecalis WDCM 00009 50,000-150,000 10 Discs
VT000122 Escherichia coli WDCM 00012 15-80 10 Discs
VT000127 Escherichia coli WDCM 00012 50,000-150,000 10 Discs
VT000133 Escherichia coli WDCM 00013 80-130 10 Discs
VT000136 Escherichia coli WDCM 00013 1,000-10,000 10 Discs
VT000303 Salmonella Enteritidis WDCM 00030 80-130 10 Discs
VT000303 Salmonella Enteritidis WDCM 00030 80-130 10 Discs
VT000312 Salmonella Typhimurium WDCM 00031 15-80 10 Discs
VT000313 Salmonella Typhimurium WDCM 00031 80-130 10 Discs
VT000322 Staphylococcus aureus WDCM 00032 15-80 10 Discs
VT000877 Enterococcus faecalis WDCM 00087 50,000-150,000 10 Discs