Fast food

Cheryl Mooney explains how the new AFNOR validated Precis rapid culture methods from Oxoid offer speed and convenience in the isolation and confirmation of Salmonella and Listeria in food, animal feed and environmental samples

It is extremely important for food and animal feed manufacturers to quickly and reliably confirm that their products are free from foodborne pathogens, such as Salmonella and Listeria monocytogenes, in order to ensure the safety of consumers. In fact, most national and international food safety standards have zero tolerance for these pathogens, requiring manufacturers to demonstrate their absence in ready to eat foods and the processing environment using validated methodology.

Traditional methods for the identification of Salmonella and L. monocytogenes require the microorganisms to be grown using bacteriological culture media, involving pre-enrichment, enrichment and selective isolation steps. These methods have been well refined over the years and offer high sensitivity, specificity and reliability. Such performance is extremely important in the search for potentially life threatening pathogens, however traditional culture methods can take up to 5 days to perform (up to 7 days for Listeria), which can be unacceptable for ready-to-eat products with short shelf lives. More and more manufacturers are looking for faster methods which allow positive release of products, protecting both the health of consumers and the reputation of the company.

Many rapid methods for Salmonella, L. monocytogenes and other pathogens have emerged in recent years. The desire, in the development of these techniques, is to match the sensitivity and specificity of reference culture methods, whilst offering a faster turnaround of results. A variety of rapid biochemical, immunological and molecular methods are now available, but many of the assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis1 due to the very low numbers of target organisms in samples and the presence of inhibitory materials in foods. Furthermore, many rapid methods require positive results to be confirmed which usually requires isolated colonies to be obtained by culture.

Culture methods, on the other hand, dilute the effects of inhibitors in the enrichment step; allows differentiation between viable and non-viable cells (unlike molecular methods, for example); and allows the repair and recovery of stressed and injured cells resulting from food processing. In addition, the development of new rapid culture methods which offer confirmed results within just two days has also helped to overcome the issue of speed.

Oxoid has developed the Oxoid Precis rapid culture methods for the isolation, detection and confirmed identification of Salmonella and L. moncytogenes in foods in just 2 days and without the need for specialised equipment (Figures 1 and 2).

These methods involve a single enrichment step, which combines resuscitation and selective growth of target organisms in an overnight incubation, followed by a single 24-hour plating step, which yields isolated presumptive positive colonies that can be confirmed, directly from the plate, in less than 10 minutes.

Salmonella Precis Method
Figure 1: Protocol for the Oxoid Salmonella Precis Method
The first step in the Salmonella Precis Method is an overnight 18-hour enrichment in Oxoid ONE Broth-Salmonella. This is a highly nutritious medium that promotes the recovery and growth of Salmonella, even when present in very low numbers, whilst inhibiting competing organisms.

Following enrichment, a sample is plated on Oxoid Brilliance Salmonella Agar for 24 hours. This is the first in a new class of chromogenic media that incorporates Inhibigen technology, a selective agent that improves the recovery of Salmonella by reducing background flora. This highly sensitive and selective medium is capable of growing a wide range of salmonellae, including atypical strains such as the lactose fermenting and non-hydrogen sulphide producing strains, which are often missed on traditional media. The chromogenic agents in Brilliance Salmonella Agar produce brightly coloured, purple colonies, which are easily differentiated from the blue and colourless colonies of other Enterobacteriaceae which are able to grow. Presumptive positive colonies are confirmed in minutes using the Oxoid Salmonella Latex Test.

The Salmonella Precis method has been validated and approved by AFNOR according to ISO 16140, against the reference method ISO 6579:2002 standard for the detection of Salmonella in food, animal feed and environmental samples2. For flexibility, confirmation was validated using both Oxoid Salmonella Latex Test and the tests outlined in ISO 6579:2002. Alternatively, biochemical panels such as Microbact GNB 24E or RapID ONE Panel, may be used.

Listeria Precis Method
Figure 2: Protocol for the Oxoid Listeria Precis Method
The enrichment step for the Listeria Precis method is performed using Oxoid ONE Broth-Listeria. This novel enrichment broth incorporates a carefully balanced mixture of peptones, carbohydrates and salts to give optimal resuscitation, recovery and growth of even low numbers of Listeria species from food samples in just 24 hours. At the same time, selective agents within the medium inhibit a wide range of competing organisms. Listeria recovery levels at 24 hours are at least equivalent to the ISO enrichment method for Listeria and in some cases are improved3,4.

Figure 1: Protocol for the Oxoid Salmonella Precis Method

The enriched sample is then plated on Brilliance Listeria Agar. This chromogenic medium detects the presence of ß-glucosidase activity, which is common to all Listeria species, resulting in distinctive blue/green colonies. Other organisms that possess this enzyme, such as enterococci, are inhibited by selective agents within the medium, whilst amphotericin inhibits the growth of yeasts and moulds. L. monocytogenes and pathogenic Listeria ivanovii are then further differentiated by their ability to produce the phospholipase enzyme, lecithinase. This enzyme hydrolyses the lecithin in the medium, producing an opaque white halo around the colony.

L. monocytogenes is confirmed in just 10 minutes using the rapid biochemical confirmation test, O.B.I.S. mono.
The Listeria Precis method has been validated and approved by AFNOR according to ISO 16140, against the reference methods ISO 11290 Part 1:1997 and Part 2:1997 incorporating Amendment 1:2004 for the detection and enumeration of L. monocytogenes in food, animal feed and environmental samples5,6. For flexibility, confirmation was validated using either the O.B.I.S. mono test or tests outlined in ISO 11290. Alternatively, biochemical panels, such as Microbact 12L or RapID CB Plus Panel, may be used.

Figure 2: Protocol for the Oxoid Listeria Precis Method

The Oxoid Precis methods offer time savings compared to standard culture methods. Fewer steps and fewer incubations save on laboratory space and, in particular, incubator space, reducing the volume of waste to be disposed of and allowing laboratories to increase their capacity.
It is also significant that Precis presumptive positive results yield isolated colonies that are immediately available for confirmation tests, unlike some other rapid methods, such as PCR, lateral flow or ELISA.

One laboratory that has experienced the cost and time saving benefits of the Salmonella and Listeria Precis methods, is the Earlybird Farm laboratory based in Standerton, South Africa.

Earlybird Farm Earlybird Farm is part of the Astral Foods group and is one of the largest poultry producers in South Africa, providing frozen, fresh and value-added chicken products to both the retail and food services sectors. The two processing plants in Standerton and Olifantsfontein process, on average, 800 tonnes of chicken every day. Processing 400-450 samples every month, the simpler protocol and faster results were significant factors to consider. “We want to have results back before products leave the premises,” explains Laboratory Manager, Louise Zwarts. “Using the Precis methods we get results several days earlier than before, allowing us to get products out to the market much sooner.” “We also save time in setting up and performing the methods. We have much less media preparation and there are fewer steps and less subculturing compared to the ISO methods.” “The fact that the Precis methods were already AFNOR validated made it easier for us to adopt the method,” Louise continues. “It gave us the confidence to try them and reduced the amount of in-house validation that we had to perform.” “For our own validation we performed the Precis methods alongside the ISO methods on routine samples and on spiked control samples. We found that the Precis methods gave us equivalent results and were much easier to perform. They also resulted in fewer false positives, which gives us greater confidence in the results.” “The chromogenic media made the differentiation of Salmonella and Listeria colonies much easier. They were much more distinctive and this has resulted in less confirmation work, which also saves time and money.” The time saved in the Earlybird Farm laboratory has given them capacity to expand their in-house test repertoire and to spend more time on method development. Furthermore, the faster turnaround of results has allowed them to clear products more quickly and to save on expensive storage space, benefiting the business as a whole. Further information about the Salmonella and Listeria Precis methods can be obtained at www.oxoid.com.

References: 1. FDA Centre for Food Safety and Applied Nutrition (2001) Bacteriological Ananlytical Manual Online Appendix 1 Rapid Methods for Detecting Foodborne Pathogens 2. AFNOR validation certificate UNI 03/06 – 12/07, www.afnor-validation.com. 3. Oxoid Poster - Folio No. 1033, July 2004. 4. Data on file at Oxoid. 5. AFNOR validation certificate UNI 03/04 - 04/05 (Detection), www.afnor-validation.com. 6. AFNOR validation certificate UNI 03/05 - 09/06 (Enumeration), www.afnor-validation.com.