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Microbiological testing: what food businesses need to know

A safefood Knowledge Network webinar in partnership with the Food Safety Authority of Ireland


00:00:00 Lisa O’Connor – Why should you do microbiological testing?  Transcript

00:33:44 Mary Lenahan – How to test and what the results mean. Transcript

1:27:30 Una McCormack – What food businesses need to know. Transcript

01:51:45 Q&A Transcript

Lisa O’Connor – why should you do microbiological testing? 

Thank you for that introduction Mairead. 

For the next 30 minutes or so I’m going to cover why we do microbiological testing of food and help you understand how to find out the microbiological parameters that are appropriate for testing your food. Between myself and my colleague Mary Lenahan we’ll take you through four easy steps to understand testing of food. 

So I will cover knowing your product and knowing what microbiological criteria could apply, and then Mary will go through how to take samples and get them tested and know how to assess the results. But before we do any of that I would like to remind you of a few things.  

Microbiological testing does not guarantee the safety of a batch of food and I suppose Mary and I have come across, on a number of occasions, people putting too much emphasis on the results and what they mean. And because the reality is when pathogens or harmful microorganisms is not detected in food it does not mean the food is safe. For example, when a test is done Salmonella if it’s not detected in food, that doesn't mean that the batch of food is safe. It could be that you tested a part of the food that wasn't contaminated. So please bear that in mind during this presentation and we can go through it in more detail. And if you have any questions at the end. 

But at the end of the day food safety is managed to the effect effective design and implementation of food safety management systems based on the principles of HACCP. Microbiological testing is a useful tool but it's only one of the tools that businesses can use to validate and verify the correct functioning of your food safety management system. And for those of you very familiar with the legislation and in EU law food safety management systems are based on our HACCP-based procedures so they’re based on the principles of HACCP, hazard analysis and critical control point. 

So the other reason to test is simply you absolutely legally have to. So in the microbiological criteria legislation there are some foods which have criteria set and actual sampling frequencies and we’ll say a little bit more about that later. So then another reason you might be testing is to validate your shelf life. In terms of food safety shelf-life validation. I suppose Listeria monocytogenes is the most common example but broadly when we come across shelf-life studies mostly what businesses have done are studies that that help them establish at what point their product spoils and therefore and what shelf-life they should put on it. When it comes to safety, you know at the end of the day the safety of the product is really controlled by your food safety management system. 

Okay, so then from time to time businesses come across the challenge of having contamination either in their raw materials or they discover it in their final product and so the level of testing will clearly increase when they're investigating the cause, they're trying to identify the root cause analysis, and then something that has sort of no legal bearing from our point of view or no legal requirement, but clearly when your customers buy from you so for example retailers or caterers who might buy from people and from businesses producing food, they are likely to set customer specifications and often times those specifications go over and above the minimum legal requirements. And so that's just between you and your customer. 

So the burning question of what tests should be carried out, well it really depends on the type of food and the microbiological hazards typically associated with that food. And I suppose that really boils down to the intrinsic characteristics of the food which I’ll go into in more detail later, and included as part of that is how you produce the food, how it will be used. Will it be cooked, for example? And also considering the vulnerability of your end consumer: are you supplying hospitals? Do you have to pay particular care to concerns around Listeria monocytogenes in ready to eat foods if you're supplying hospitals, where vulnerable groups are consuming foods.  

In terms of what tests to do, it also may depend on your reason for testing. So are you doing routine monitoring? Are you verifying your HACCP procedures? Are you investigating an ongoing incident with spoilage or a pathogen? Are you validating your shelf life? Or again is it the customer specifications? And really it will depend on whether there's legal criteria set and so this is the key piece of legislation Commission Regulation 2073 of 2005.  

But for those of you familiar with that legislation you'll be aware that it has a limited number of food categories and microbiological parameter combinations to test for. So for example it doesn't have Salmonella in all ready-to-eat foods; it has them in certain ready-to-eat foods. It does have Listeria monocytogenes in all ready to eat foods, but for those of you who are producing ready-to-eat foods for which there isn't a Salmonella criteria you still must make sure that Salmonella isn't in your food if it's possible that it could be in your raw materials. So you may well need to test depending on the assessment you do.  

So in FSAI Guidance Note 3 which provides guidance on ready-to-eat foods sampled on the market, and we give some of those parameters, that aren't covered in the regulation. And for those who need to sort of dig a bit deeper into what's relevant to their particular food and they're not finding it in best practice or industry guidance, really the ICMSF, the International Commission for the Microbiological Specification for Food, has a series of books on sampling and identifying appropriate microbiological criteria and sampling plans and that really is the go to for us when we get tricky queries. And also the Institute of Food Science and Technology in the UK has some quite useful guidance as well. So the links are there at the bottom here for you to have that as a resource. 

Okay so moving into the main part of the presentation: the testing in four easy steps.  

So the first thing is you have to know your product, and what that really means is describe it in detail. So think about all the ingredients you're using and the specifications you may need to have with your supplier for those ingredients and the processing parameters. Is there a heating step? Is there a step in your process that will destroy pathogens? Will it destroy the vegetative cells or will it destroy, if you're concerned if you're producing canned food and you need to be sure that you're destroying Clostridium botulinum, and that's a much higher cooking temperature because that organism produces spores. And I’ll give you references for where you can understand that information in more detail.  

Packaging details, are you packaging in modified atmosphere packaging? So some organisms like a lot of oxygen when they're growing. Others like a minimum amount of oxygen or are very sensitive to it, so is your product vacuum packed where most of the air is removed, or have you added gases? And all of those will influence what microorganisms can grow.  

And then of course, are you producing a ready to eat food or not? So for example, are you providing cooking instructions to the consumer on your labels? Have you validated those instructions to ensure they would destroy any pathogens that could be present in terms of shelf life declaration?  

Mary will go through that in a bit more detail, but it may be that you need to do testing to determine whether or not you need a use buy or a best before date.  

And then the storage conditions. Is your product chilled, frozen, or ambient? And I suppose one thing to remind you about the chilled and frozen, so for example there are some foods that grow at refrigeration temperatures like Listeria monocytogenes. It will grow slowly, so ready to eat foods with long shelf lives that are stored in the fridge become unsafe over time, if there was a risk that Listeria might be there in the first place.  

And then with frozen food something to just remind you, while freezing can damage some microorganisms, actually unfortunately with the majority of them it preserves them, as it preserves foods and the nutrients in food. So freezing isn't the friend of microbiologists in terms of destroying pathogens.  

And then any other relevant details so something just to remind an Irish audience. Private wells if badly managed or badly sourced can be a very problematic, so you may need to be doing extra testing, or unfortunately maybe in a part of a country where the public system is having problems. 

Okay I’ve mentioned Listeria monocytogenes a number of times and certainly for it and also for some of the Salmonella criteria in the legislation you will need to be very clear whether you're producing a ready-to-eat food or not. And while it might seem obvious we found it necessary to produce a decision tree to help businesses be really clear on whether or not their food is ready to eat. So for example, just at a very high level, baked beans: a can of baked beans, you could eat those without reheating them. The reheating step that you're doing isn't going to destroy the most heat resistant microorganisms which would be Listeria monocytogenes, in terms of the vegetative cells and those not enough and not in spore form. 

So this decision tree - I won't go through it in detail in this talk - but the FSAI Guidance Note 27 goes through this in detail and is a very useful reference for understanding that better. 

So getting down to more detail now and how you actually understand your food. I would say that we typically talk about two types of characteristics: the intrinsic characteristics and the extrinsic. In terms of the intrinsic, the background microbiological flora that might already be in your raw materials. Will they be there in the end product, will they help control pathogens? Or will they create an environment that allow pathogens to grow, or will pathogens be a part of that microbial background? And so the microbial quality of your ingredients are critically important. And the final pH of your product, but actually also the behaviour, the acidity of your product over its shelf life.  

So for example a food like cheese has a criteria in the legislation to test for Staphylococcus aureus during the cheese making process where it is most likely to be detected, because it does tend to decrease in number. But the problem there is you will find that it can produce, which is high enough number, it can produce a toxin that can survive in the food. So Listeria monocytogenes is a big concern in some [mould ripened?] cheeses and soft cheeses where the pH of the surface can change and become a more favourable environment for Listeria to grow, maybe sometimes in comparison to the core of the cheese.  

So one of the things just to reiterate, pH can change over the shelf life of the product and actually through your product matrix. And water activity similarly can change, so the water activity of a salami for example would be quite low. And for those people who produce salamis that are not heat treated, the water activity will be one of the really critical steps and making sure you get that quite low or as low as possible and or as low as you identify as being critical for the safety of your product. Some products are preserved using additives such as nitrite, nitrite or nitrates, so if you have a preservative in your food you need to know you can take that into consideration as to whether or not you need to test for a certain type of microorganism. 

Then moving on to the extrinsic characteristics. At the end of the day I’m just reiterating that you're managing having good hygienic practices, good manufacturing practices, and a robust well implemented food safety management system, and should control safety of your product the temperature during production, storage, distribution. But I think I’ve covered that well enough already, is that ambient, chilled or frozen and the gas I’ve mentioned as well. What kind of atmosphere is your product stored in, the type of packaging might have an influence. 

And then you definitely need to consider consumer practices. So that outbreak of frozen corn that happened across Europe a few years ago, the manufacturers of those frozen corn and the other mixed vegetables didn't intend for those products to be consumed raw, they were intended to be cooked. So those businesses weren't adhering to the Listeria monocytogenes requirements that are ready to eat and the manufacture must adhere to. So consumers also often change their practices. For example, we see people using a lot more raw foods or sort of wanting to eat in a healthier way, and that’s potentially one of the things that was behind that outbreak. People not reading labels, that’s another problem for manufacturers.  

You do go to the effort of doing your due diligence, identifying how your product should be handled, but consumers don't necessarily always do what you expect them to do. So you do need to anticipate some of that within reason. 

Okay so we've talked about the parameters, roughly described parameters that influence the survival and growth of specific organisms and foods and this reference FSAI Guidance number 18, table 6 goes through minimum and optimum and maximum temperatures and pH, water activity, salt concentration, the gas atmosphere and so on, and gives some although there are limited number, of examples of typical foods. And so that's an extremely useful reference if you want to identify what might be relevant and to test for. 

The other thing, just before I leave, is to remind you that a combination of factors in your food could result in multiple hurdles that ultimately ensure that your food is safe from pathogen growth.  

So an example might be a pH and a water activity combination, and those of you familiar with the legislation will know that those footnotes associated with Listeria monocytogenes explain either the minimum pH or the minimum water activity or then the combined minimum pH and water activity which are higher.  

So then, how do you go about identifying what you should be testing your food for? So really it's a bit of legwork using your laptop, starting from Google leading you to scientific literature, looking obviously at websites like our own for guidance. So we have a section micrological criteria which Mary will take you through. And then research institutes and professional food industry parties may be able to help you and international organizations. 

Okay so moving on to identifying or trying to decide what criteria would apply. And I’m going to give a brief recap. I’ve mentioned this already, but you know the principal criteria that we talk about are the legal criteria and the principal piece of legislation is this commission Regulation and 2073 of 2005, although there are some other pieces of legislation. For example, there’s criteria associated with raw milk in the hygiene legislation Regulation 853 and the bottled water legislation where they have specific criteria.  

Okay so then the guideline criteria, I’ve mentioned FSAI’s Guidance Note number 3 and the consumer specifications, so I’m not going to go through the text here in detail. This information is here for your reference.  

I am just going to go through food legislation in a very quick whistle-stop tour. Specifically, I’m going to look at the general food law, the hygiene legislation and then how the microbiological criteria legislation stems out of that.  

So in terms of the general food law, this is Regulation 178 of 2002, and I will say it's incredibly important in terms of understanding definitions and it's your go-to place to understand basic definitions that underpin food law. That legislation obliges you, the food business operator, not to place unsafe food on the market, and while it's obvious it is stated in article 14. Unsafe food is defined as both food that would be injurious to health or also unfit for human consumption, and when assessing if something is injurious to health or unfit, you and the competent authorities need to take into consideration, looking at normal conditions of use by the consumer, the stage of production, processing and distribution, including that information provided to the consumer, so all of which I’ve mentioned already. 

So then the hygiene legislation itself. There's two key pieces of legislation: there’s Regulation 852 which applies to all food businesses, and then 853, which applies to businesses producing foods of animal origin. And then with 852 there's general requirements at farm and at business level. These general requirements and you might hear us refer to as prerequisite requirements or PRP’s for short, and then specific requirements. So in the specific requirements it is expected that maintenance of the cold chain will be observed by all businesses along the chain. There's reference in 852 to microbiological criteria, and then the specific legislation where these criteria are aligned and it is Regulation in 2073. But as I said with bottled water, there's other legislation and then sampling and analysis so these specific well they're called specific hygiene requirements. I will say they're kind of general, they're a general catch-all and especially when it comes to the microbiological criteria, you then need to go to other pieces of legislation to actually find the details. 

Okay then, ju st to give you a picture of where the micrological criteria apply. I’m not talking about animal feed in this talk and these criteria, this Regulation, applies to food stocks, not to not to feed, but Regulation 1708 applies, it's the green line here, applies to all stages before consumption. So the microbiological criteria and the legal criteria in 2073 apply from primary production through to retail and catering. So whichever stage of the food chain you're at, you do need to double check whether there's a criterion in that legislation that applies to your business.  

And then, here, the first thing to say is, in a lot of our guidance, you'll see us abbreviating food business operators, which is effectively the legal owner of business or the person legally responsible in a business (FBO). And so, what the legislation says is the FBO must take measures as part of their procedures based on HACCP and GHP or the PRPs to ensure microbiological criteria are met, and that's Article 3 of that legislation. And then, FBOs shall test as appropriate, so this is the tricky one for you, and we get asked a lot, "What is appropriate?" We have some tools for you to assess when it's appropriate to test. When you're designing your food safety management system, you may really want to validate using microbiological testing. And when you're operating your food safety management system and producing food periodically, you may want to verify that the procedures are working correctly. And this legislation all the time goes back to your food safety management system. 

And I just pause here just to remind people about the differences between these two: validation is obtaining evidence that the food safety management system is effective, whereas verification is checking that the validated system has actually been implemented correctly.  

Okay, so this slide is just showing you a snapshot from the legislation, and there's just a couple of things I want to cover here. The first is the reference to the microbiological criteria, so they're all contained in the annex to the legislation, and it refers to process hygiene criteria and food safety criteria. And again, Guidance Note 27 will explain and guide you through this legislation in great detail. 

I also want to just take a tangent here to help you in understanding legislation. When legislation is amended, you should check for a consolidated version, which collates or puts together all the previous amendments. Now this particular snapshot here from the FSAI website only includes amendments since 2017. It highlights that a consolidated version of the legislation was produced in 2020. And I know for a fact that no further amendment was made since this consolidated version, but I would always caution that if you do find a consolidated version, it is wise to double-check that there isn't a more recent one. You can contact the FSAI if you have trouble figuring that out. And for those of you who are more proficient with legislation, Eur-Lexwebsite is where legislation will be published first, and you can check there. 

So going back to the criteria, Annex I lays out in Chapter 1 the food safety criteria, and the majority of those are for ready-to-eat foods, although not all. There are quite a few there for raw meat.  

Chapter 2 is about process hygiene criteria, and Chapter 3 then are the set rules for sampling and preparation of test samples. So this is a snapshot of Chapter 1, so the first five criteria there, you can see the first three are for Listeria monocytogenes in ready-to-eat foods. It then moves on to minced meat and meat preparations intended to be eaten raw, which is common practice in some parts of the continent. And minced meat and meat preps made from poultry meat intended to be eaten cooked. So here, this is recognizing that poultry meat has a high risk for Salmonella, and even if it's going to be cooked, minced meat and meat preps are products known in the past to have outbreaks associated with them, made from poultry meat, and that's why there's a criterion there for testing for a pathogen in a raw meat that is going to be cooked. 

Looking at Annex I, when you go to Annex I of the legislation, this is a snapshot or this is what you will see. Moving on, so we've covered Chapter One with safety criteria, and as I said, I just showed you the first five. I think there's 28 criteria at this stage. Chapter Two on the process hygiene criteria, covers the following categories of foods: meat and meat products, milk and dairy products, egg products, fishery products, and vegetable fruits and products made from them. And then it also has in Chapter 3, then you have rules for sampling and preparation of test samples. Okay, so in Annex, and yeah, so that was Annex One. 

So moving on to Annex II, this is really the shelf life and studies. So it says, as necessary, FBOs are responsible for the manufacture of the product shall conduct studies to investigate compliance with the criteria throughout the shelf life of the food. And really, as necessary, it may not be, when you go through your intrinsic parameters, for example, you may identify and sorry, when you go through both your intrinsic and extrinsic parameters, you may identify that your project will be stable throughout its shelf life.  

Okay, so testing against the criteria, I'm not going to repeat what's up here, but the purpose of this slide is really to remind you, those of you who might be more familiar with the older regime of end product testing, is that the principles now are to be proactive to identify the hazards in your food, to identify ways of controlling them, and then periodically to test. So really, end product testing isn't a fail-safe way of ensuring the safety of your food, and we're now in this more proactive approach with this legislation. 

To describe in a bit more detail, microbiological criteria, typically they define the acceptability of a batch of food, and that would be a food safety criteria or a production process. And the criteria are usually based on the absence, presence, or the number of microorganisms or the quantity of their toxins and metabolites. So a toxin, for example,   Staphylococcus aureus produces the toxin, or pathogenic E. coli, and some of them produce toxins and metabolites. The example here is histamine, which is produced by some spoilage organisms. 

Okay, so getting down to the detail of what a microbiological criterion contains. Obviously the food, the microorganism, it then includes a sampling plan. So n is the number of samples, and c is the number of samples that can be between the lower limit and the larger limit. The lower limit is the little m, and the larger limit is referred to as big M. It also includes the analytical reference method and the stage where the criterion applies. So taking this example of cheese, butter and cream made from raw milk, one that has undergone a lower heat treatment and pasteurization, our food category is here. The organism is Salmonella, five samples, five units from the batch must be taken. None of those samples can have Salmonella detected, so in this case, the little m and the big M are the same; they're just the limit and is not detected in 25 grams. The analytical reference method is here, and if an alternative method is used, there's detailed requirements in the legislation about how you demonstrate that the alternative is appropriate and is as good as the reference method. And then the stage where the criterion applies, so products placed on the market during the shelf life, is this one. 

So some definitions that are important to remember: the end of the manufacturing process means when a product is still with the manufacturer but is ready for sale. Products placed on the market during their shelf life, means products placed for sale within their specified shelf life, the issued use by or best before date. And then the term placing on the market, there's a definition for this in Regulation 178. It means the holding of food or feed for the purpose of sale, including offering for sale or any other form of transfer, whether free of charge or not, and the sale and distribution and other forms of transfer themselves.  

So this first part, the holding of food or feed for sale, for the purpose of sale, so that does mean that when you finish, at the end of your manufacturing process, and you produce the food and you're storing it in your premises prior to dispatch, that it is effectively considered placed on the market from the point of view of the food safety criteria in Regulation 2073 where the stage of sampling is placed on the market. 

Okay, so a few slides on sample and attributes and to explain that the different sampling plans that you see in the legislation. Basically, we can think of them as a pass-fail or a conforming non-conforming, and the food safety attributes are typically pass-fail and the conforming and non-conforming are typically process hygiene criteria. So a little bit more detail in this slide explaining what n, little m, big M, and c mean, and we've already seen a two-class sampling plan with the Salmonella criterion cheese and where m, little m, was equal to big M, and here you see an example of both at the bottom here. And you can see in this case, five samples need to be taken for E. coli and meat preparations, and two of those samples can be between 500 and 5000, and this upper limit cannot be exceeded, so no sample can exceed the upper limit. 

So this is a slide to remind you, or give you a visual of the guidance that we most commonly refer to. So aside from the legal criteria, as I said, the ICMSF book series, book eight is probably the one that we most typically refer to. Here are examples of just snapshots of, here's Guidance Note 3, so here you can see the Bacillus cereus, for example, is here, and it's an example of an organism where there's a limit, an enumeration limit, whereas pathogens like Campylobacter and Salmonella and STEC and there's no limit. So there's a mixture when it comes to the pathogens, some you just can't have in ready to eat foods, and others you can tolerate a certain level.  

This is a Guidance on cooked chill systems, which is a very particular for the food service sector, but some of you might be familiar with it. And here is just a snapshot of two of the ICMSF books, here looking at an example of the category of cocoa, chocolate, and confectionery. So it goes through the hazards, here are the most significant hazards. And as we all know and with the recent outbreak associated with Kinder chocolate, Salmonella can be very problematic for chocolate and the control measures are listed here. 

 The initial level, what can reduce the level, what might subsequently increase the level, the kind of testing, and then considerations around spoilage. And book 8 here gives recommendations. So for example, and Enterobacteriaceae testing, it recommends testing five units. And here with Salmonella, you can see it recommends testing 10 units. So that's what you're likely to find when you when you go to those books.  

So in summary, you should document the research that you've done to come to the conclusion that you have, whether you're going to test or not test. And these are the resources: the legislation, obviously, national guidance, EU guidance, industry best practice, international guidance, for example, Codex Alimentarius, and obviously, published literature is probably one of the first things that will come back when you do a Google search, and especially when you use Google Scholar. 

Okay, we haven't covered why and what we test for and having gone through part one of testing in four easy steps, so it's over to Mary now. And just to finally say, if you have any questions and anything that I've covered that if we don't end up covering it in the Q&A session, always remember that the info at FSAI advice line or the info FSAI email address is there is a one-stop shop to getting an answer to your questions. Okay, so I'm going to stop sharing and hand you over to Mary. 

Mary Lenahan – How to test and what the results mean 

So thanks, Lisa, for that very nice presentation. So I'm just going to continue on for me, so it's the second part of the presentation, and I'm going to cover how to test and what those results mean.  

So before I get into the second part of testing for easy steps, I'm just going to mention some points you might need to take into consideration for shelf life testing and environmental monitoring, mostly related to ready to eat food. And then I'm going to go into the second part of knowing how to take your samples and get them tested, and then how to assess those results and any follow-up actions you may need to take. And I'm going to finish off the FSAI series of presentations just giving a summary of some guidance and resources you may find helpful. 

And so I'm just going to mention a few points around shelf life of food. I know that this is one of the reasons that you may be required to test the food that you're producing. So shelf life is the length of time food maintains its quality and its safety over reasonably foreseeable conditions of distribution, storage, and use. This piece of legislation called the Food Information to the Consumer (FIC) lays down the requirements you need for setting a use by or best before date on food and how you label that on food.  

Use by dates relate usually to foods that you need to take into consideration for food safety. These are usually foods that are highly perishable and they're likely to constitute an immediate risk to human health once that use by date has passed. So it is illegal to sell food once it's gone past the use-by date, and because that food is considered unsafe under article 14 of Regulation 178. So these are foods that are more likely to need microbiological testing. A lot of them are falling into the food categories that Lisa mentioned in Regulation 2073. So you're looking at things like raw meat and fish, a lot of chilled ready-to-eat foods, and things like pre-cut fruit and vegetables and sprouted seeds. 

For the other types of food where they're mostly stored at ambient temperatures, they're the types of foods that you're really concerned about food quality more than food safety. So it's a best before date or referred to in the legislation as a date of minimum durability. And these are foods where you're concerned with their sensory properties being retained more so than concerned about food safety. So you want to, you're probably focusing more on the spoilage organisms, they're less likely to need microbiological testing from a food safety point of view, but you might want to carry out some testing because of, say, yeast and moulds, for example. And so you're looking at things at the appearance of the food, you know, the odor, the texture, the flavor. So a lot of these are stored at ambient temperatures, and we're talking about dried foods, canned foods, jar foods, and some bakery products like bread and cereals. Frozen food has a long shelf life, and these would be your best before. And this legislation to say that raw shell eggs or hen eggs they are a best before date rather than use by date. And other things with high sugar content, you know, like jams, jellies, and chutneys, for example. 

So anybody who packages or sells a food that requires a shelf life declaration is responsible for validating and setting shelf life on food. And there are many different factors that will affect the safety of the food. As Lisa described in her presentation, you know, it's around whether the food is a ready-to-eat food or not, what are the intrinsic characteristics, you know, does it have a low pH a lower water activity  

And then the extrinsic characteristics, how is that food packaged? Is there, you know, is it packaged with a modified atmosphere, a gas that prolongs the shelf life of the food? And all of these things will lead to a variation in the shelf life, so it's very much on a case-by-case basis. 

I would say if you're carrying out microbiological testing to validate a shelf life on a food, it would be best to use the worst-case scenario for your shelf life study. So in terms of storage temperatures, we know that while the national recommended best practice temperatures for storage of chilled food is less than five degrees Celsius, there's potential for consumers' fridges not to be operating at that optimal temperature. So you may need to take into account some form of temperature abuse, so this is accounted for in the legislation under taking into account reasonably foreseeable conditions of distribution, storage, and use. For pH or water activity, if you have some information on the pH of different batches of your food, is there variability within the pH of those batches that you make? And if there is, use the worst-case scenarios of the highest pH for that food when you're carrying out your tests. 

I'm not going to go into shelf life in detail today, but we do have our FSAI Guidance Note 18 which gives you a very logical step-by-step way to determine and validate the shelf life of food. And it's not unlike the process that we've described today. I will be giving a breakfast bite for the Food Safety Authority of Ireland on the 20th of September, so you can sign up to our events and you'll be notified when that's going to happen, and I'll go through shelf life in more detail on that day. 

I would like to mention, and Lisa has mentioned it already, in Commission Regulation 2073, Annex 2 does say that "as necessary, the food business operator responsible for the manufacture of the product shall conduct studies to investigate compliance with the microbiological criteria throughout the shelf life of the food." So in particular, this applies to ready-to-eat and chilled foods that are supporting the growth of Listeria Monocytogenes. These are laid down, usually foods that come under food category 1.2 in Regulation. So Lisa has shown a snapshot of the first three categories in the food safety criteria in Regulation 2073, and these are all concerned with Listeria

 This slide is just going to show you how to figure out where your food, if it's ready-to-eat food, falls in under the Listeria Monocytogenes categories. So if you're manufacturing a food that's for infants or for special medical purposes, there's food category 1.1. I see an answer to a question in the Q&A box there, in that one there's actually, it asks you to take 10 samples because it's a more stringent criteria, whereas the other ones are five samples. Then there's, Lisa had mentioned, you know, in Regulation 2073, a lot of the detail is in the footnotes. And when you're looking at that criteria and if there's any food categories that are relevant to the foods that you are producing, make sure you take into account any footnotes that might, you might have to comply with as part of that legislation.  

So footnote 8 lays down certain criteria for knowing whether your food is automatically a category 1.3, so these are ready-to-eat foods that cannot support the growth of Listeria Monocytogenes. And so this could be a shelf life a ready-to-eat food with a shelf-life of four days or less, written in the legislation as the less than sign, five days. If your food has a pH of less than or equal to 4.4, if it has a water activity of less than or equal to 0.92, or as Lisa mentioned if there is a hurdle and you have a combination of a pH of less than five, less than or equal to five, combined with the water activity of less than or equal to 2.94. 

And there could be other foods that fall into that category under subjective scientific justification. So an example is a frozen food. So Listeria monocytogenes will not grow, but it will survive in a frozen food. And so the food, while it remains frozen, would fall under category 1.3. If you had instructions on that food, so say for example it was a frozen cheesecake, and you had instructions on it that you can defrost the food and consume within five days, then that defrosted food would potentially fall into category 1.2 because the shelf life is five days.  

Any ready-to-eat food that does not fall in under the different stipulations for footnote 8, or if it's not for infants or special medical purposes, by default falls under category 1.2. And this is ready-to-eat foods that are able to support the growth of Listeria monocytogenes. Or if you have information where you've done your homework and you found that you're producing a cheese, and it's well known to support the growth of Listeria monocytogenes, so soft cheese, and this will fall into category 1.2. 

So again, Guidance Note 27 is our bible really for how you comply with Regulation 2073, and I'd highly recommend you take a look at that guidance after our webinar.  

So a food category 1.2 is a bit unusual in that it within that microbiological criterion, it splits into two parts. And the first part is a limit of 100 CFU (colony-forming units) per gram. And footnote 5 is related to that limit, and it says as a food business operator, if you can demonstrate that that limit of 100 CFU per gram will not be exceeded in that ready-to-eat food throughout the shelf life of the product, you will be allowed to have that less stringent limit that you can comply with throughout the shelf life of your food. But if you're unable to provide sufficient evidence that a competent third party deems appropriate to show that that limit of 100 CFU per gram will not be exceeded throughout the shelf life of the products, a more stringent limit of not detected in 25 grams applies. So the difference here is the two different test methods that are carried out for the detection method. It's a more sensitive method, and the sample is put into what's called an enrichment broth, and it helps any sub-injured cells grow. And it's a more sensitive method. The enumeration method is where you get a count of Listeria, so that sample is directly placed onto the agar, and it's a less sensitive method. 

So how you gather that evidence? There is a European Union Reference Lab (EU-RL) for Listeria monocytogenes, and they've published this technical guidance document. And it details how you carry out challenge tests and durability studies for assessing the shelf life of ready-to-eat foods related to Listeria Monocytogenes. And this is the guidance that would need to be followed to show that that limit of 100 CFU per gram would not be reached. 

So just a few words on environmental monitoring. So Article 5 of Regulation really kind of sets environmental monitoring as a legal requirement for two different types of foods. So the first is if you're manufacturing a ready-to-eat food which supports the growth of Listeria monocytogenes, so those category 1.2 foods that we just talked about. Or if you're a manufacturer of dried infant formula or dried foods for special medical purposes intended for infants below six months, which pose a Cronobacter species risk, then let's do environmental monitoring for Enterobacteriaceae.  

So, I would imagine most of the people who are participating in the webinar today would fall in under the first requirements. So if you are manufacturing ready-to-eat food which supports the growth of Listeria monocytogenes, you must do minor environmental monitoring for Listeria monocytogenes. So we would recommend that you follow again the EU-RL, the European Union Reference Lab, and Listeria monocytogenes guidelines on sampling the food processing area and equipment with the detection of Listeria monocytogenes. That details how you take your samples and all the different equipment you might need, for example, and where you should take them from. If you do find Listeria monocytogenes on a food contact surface or a non-food contact surface, and this FSAI publication, the control and management of Listeria monocytogenes, includes and will give you information on what corrective actions you need to take out or carry out.  

I know that some of you may also carry out environmental monitoring to check the efficacy of your cleaning and disinfection processes in your processing environment. I'm not going to mention that today, but that might be another type of environmental monitoring that you may want to take and carry out. I think Una might be touching on that in her presentation today. 

And so, I'm going to move on to the second part of the testing in four easy steps. 

So Lisa covered knowing your product and what criteria might apply. Now that you've gathered all this information, you're at the halfway point, and it's time to stop and think. And on the basis of everything that you have gathered, really think about is microbiological testing required for your product or products. And this will be based on all of the things that Lisa describes, the type of food, particularly if it's ready-to-eat food, and does it have any intrinsic or extrinsic characteristics that will prevent or limit the growth of bacteria? Is there any biological hazards in particular that you need to pay attention and control? Are there any legal criteria you need to comply with, or is there any guideline criteria? Are you testing because you want to validate the shelf life of your food, and that could be for food safety reasons or potentially for food spoilage reasons? Do you have to carry out environmental monitoring? Or have you agreed something outside of these points in your customer specification that goes above and beyond what you need to do legally and that you need to comply with as part of your agreement with the customers you're supplying? 

I'm going to give two examples here. So, in the first example, we have Martin, and he is a small food business owner, and he produces kimchi and sauerkraut, so fermented plant-based products. He sells about 20 jars of these each week at his local farmers’ market, and they are ready-to-eat plant-based foods. He's done some research, and he knows that there can be some biological hazards associated with these foods. Clostridium botulinum because they are jarred food and there's an anaerobic environment, and Listeria monocytogenes because they're ready-to-eat food. And he knows there has been some issues with Salmonella and shiga toxin-producing E. coli or STEC in contamination of the fresh produce that he uses as ingredients, but potentially that could be a problem. But from his research, he's also found out that if the foods are fermented properly under good hygienic conditions, they have a good history of microbiological safety, and this is really down to during the fermentation process the pH of those products lowers to below 4.2, usually for a lot of them it’s around 3.5 up to 4, and this prevents pathogens growing if they're present. So, on this basis, he has determined that he does not need to carry out microbiological testing on his products, and instead he's going to monitor the pH of every batch of the products that he makes as a critical control point, and he has set the critical limit of less than or equal to 4.2. We recently published Guidance Note 37, and we recognized that there's been a big increase in the production of these kinds of fermented plant-based products, and this guidance goes through the HACCP-based procedures that you know you need to bear in mind for the different types of products. And everything that Martin does, he makes sure he documents and keeps it as part of his food safety management system. 

In our second example, we have Sheila, and she already owns a big food manufacturing business that manufactures a lot of different types of products, and they've recently decided to go into a new manufacturing line, and this is for salami. In her product specification, she has recognized that this is a raw ready-to-eat fermented meat product, and they're producing a lot of it and they're distributing internationally. From her research, she knows that Listeria monocytogenes, Salmonella, and STEC could be potential biological hazards, and she also knows there's been some occasional outbreaks linked to fermented meat. And she knows that there are some legal criteria, microbiological criteria that apply, and also some guideline criteria she can use.  

So, she has determined that yes, she needs to carry out microbiological testing, and she has identified in Regulation 2073 Listeria Monocytogenes. So, if she has pH or water activity information, and that Aw is the symbol for water activity, if she has no information, it'll fall into category 1.2 by default. If she has pH and water activity information and they comply with the conditions in footnote 8, it could potentially fall into category 1.3, so there's a less stringent limit there over 100 CFU per gram. She's also identified as a raw fermented meat product, Salmonella needs to be tested for under category 1.8. And in the Guidance Note 3, she's identified criteria for STEC under the pathogens in Table 1 and Enterobacteriaceae as a hygiene criteria in Table 4. So, we also have Guidance Note 33, and this is good manufacturing practices for the production of ready-to-eat raw fermented meat products. So you're going to follow that, and everything that she does again is documented and kept as part of her food safety management system.  

So now that Sheila has decided that yes, she has to take samples for her products and get them tested, she needs to know how to take those samples and how to get them tested properly. So, as a really important take-home message, I want you to remember this slide and to remember that the distribution of bacteria in food is not uniform. So, you can see here, we have an example of a soft cheese, and you can see that there's some bacteria there, but it's in no way distributed in a uniform way. And this could be for lots of different reasons, it could be that the contamination is on the surface of the cheese and not in the middle of the cheese. It could be that the composition of the food, so you know the bacteria cannot easily move throughout the cheese and become distributed and evenly. If the cheese is left on the table for a number of hours and you have some increased temperature, a bit of temperature abuse, if there is a small bit of bacteria on the surface, there's kind of hot spots on the outside of the cheese could get warmer before the inside of the cheese, so you get a little bit of growth on the outside and not in the middle. And bacteria as well tend to like to clump together.  

So, this all means when you take your sample, it's really a lottery as to whether you will take the portion of the sample that has the contamination in it. So, if you take just a single sample, you know your chances of finding the bacteria in that food if it's contaminated are much lower than if you take a number of samples. So, the more frequently you test and the more samples you take from a batch, the better your chance of finding that bacterial contamination if it is present. 

So, as Lisa mentioned, at the start of her presentation, it's a really important take-home message that the absence of bacteria in a sample that you test is not a guarantee of safety. And that microbiological testing is a really useful tool to verify that the critical control points that you have implemented, critical limits that you implemented as part of your HACCP-based procedures, and are working properly and are implemented as they should be. But you can only make conclusions on the sample portion that you've tested as to whether that batch is contaminated or not. So, if you in this example, if you've only taken the first sample of that piece of cheese, you may have thought, you know the rest of the cheese was contaminated, but you just didn't pick it up in sample number one. Of course, you cannot test 100% of the batch that you're manufacturing, so it's really a compromise between food safety and what's commercially viable. And so, the more samples that you test from the same batch, the higher your chance of the higher probability that the contamination will be identified, and a lot of the microbiological criteria have worked out for you already how many samples to take and that will give you the best chance of finding the bacteria in food should it be present. 

So, when you're taking your samples from a batch of food, you need to take them in a way that's representative of the batch. A batch of food is a quantity of food that's manufactured under the same kind of characteristics, and it's really up to the food business operator to determine what constitutes a batch of their food, and Guidance Note 10 gives some guidance on that and how to determine about your food and some information about product recall and traceability. 

And so, for some food business operators, you might if you make a lot of products, it might be, you know, one particular product, and you have an hour's worth production of that product, and that's a batch. For some other food business operators, they don't make so much food, it might be one day's worth of production. So, it's really up to the business operator to determine what makes the best sense for them in terms of a batch.  

When you're taking samples, you need to take them from a single batch, so that you can come to a conclusion about the contamination in that batch. Take them at random and try to spread taking the samples throughout the batch, so you might take some from the beginning, some from the middle, some from the end. And you may also have a sampling process, so you try to cover every possible combination of factors that could potentially produce and contribute towards contaminating your food products so that you have the best chance of identifying the contamination if it's there. So, make sure that you cover different days of production, and if you've different food products, you need to test all the different products that you make. And if you make them on different production lines, in case there's a problem with production line three and you always take your samples off production line one, you'll never find the problem with production line three. Equally, and staff changeovers, it's good to try and cover that, you know where maybe some staff are better trained than others. And so just try and know your process and try and cover everything that could potentially contribute towards contaminating your product.  

As I mentioned, the microbiological criteria indicate how many samples that you should test. So, in Regulation 2073, for most of the criteria, you're required to test five samples per batch. Some of them might require more if it's a more stringent criteria, like category 1.1 asks for 10 samples to be tested. They sometimes refer to taking five samples as a batch sample. In Guidance Note 3, this is for single samples, and that's because Guidance number 3 was really written mostly for the monitoring or surveillance of ready-to-eat food already placed on the market. And it's used by our official control inspectors for when they take samples of food placed on the market as a random check to see if there's any contamination. And when they're taking samples placed on the market, it's very hard for them to get five samples from the same batch. So, suppose it's a compromise in terms of what's practical for them. But if you're using Guidance note 3, you may choose to take more than one sample, knowing that the more samples you take from a batch, the more likely you are to find contamination. Regulation 2073 does permit reduced sampling numbers for the criteria, but this must be agreed prior to this with the competent authority, and it must be based on the evidence of your HACCP-based procedures being effective and your historical data. And in FSAI, note 27, we would recommend that if you are looking at reducing sampling, that you look to reduce sampling frequency rather than sampling numbers, because the more samples that you take from a batch, the higher your chance of finding contamination if it's there.  

As Lisa mentioned, there are some set sampling frequencies for some food categories, and these are set down in chapter 3 of the Regulation, and they're mostly for meat-related products, so the carcasses of cattle, pigs, sheep, goats, and horses and some meat preparations, poultry, and for sprouts. If there's no set frequency for the relevant food category for the food that you're producing, as a food business operator, you're supposed to determine the frequency of testing based on risk assessment. So this will be where you're taking into account the biological hazards of the food, is there a vulnerable end consumer, is it ready-to-eat food, what type of food business have you got – is it a large business, a small business - and do you have to carry environmental monitoring, do you have a good food safety management system in place? So again, Guidance number 27 will give you a lot of guidance on that.  

The legislation also lays down a reference method on the next one that you should use to test these products, but you may use alternative methods, and the conditions for that are laid down in article 5.5. So usually, if the alternative methods, a lot of them are rapid methods if they're validated and certified, so you may be familiar with AFNOR, and this validates and certifies methods according to ISO 16140 part two, and there's other groups as AOAC, and you can use those methods if it's your competent party beforehand.  

We have this guidance, it's just a fact sheet really on how to assess if a lab is suitable to test the foods that you need to get tested, so we would always recommend that they're accredited to the Irish national accreditation board INOW accreditation 17025. And if you go through that fact sheet, it'll give you some important things you need to take into consideration when you're getting your samples tested. 

Just to mention, sometimes you might see different bacterial names used on a lab report, so a typical one is ACC's or TVC or TBC, so these are all the same thing: aerobic colony count, total viable count, or total bacterial count. So the generic test that counts the total number of bacteria in a food, but it doesn't differentiate between the different types of bacteria. It can be a useful indicator in terms of microbiological quality, so you know if high counts are present, it might be indicating quality issues or poor temperature control. And because you can't just distinguish what the difference, what that type of bacteria is, it doesn’t go beyond that. Just to know if it's not suitable for any kind of fermented foods as we might have purposely added a starter culture because of course you've added bacteria to those. So Guidance Note 3 gives you some ACC limits for ready-to-eat foods when they're placed on the market. 

Another one you might see is Enterobacteriaceae. And just to kind of give you a visual, there's a few different bacteria and types that all fall in under that Enterobacteriaceae family. And so, they’re a broad group and they include pathogens such as Salmonella or spoilage organisms such as Citrobacter. And they indicate maybe inadequate cooking or post-process contamination if the food is fully cooked, and then afterwards you find a high level of Enterobacteriaceae, it would indicate maybe cross-contamination after it was cooked. And there's a high, typically a high number of Enterobacteriaceae naturally on fresh food, fruit, vegetables, and salads. And so, we wouldn't recommend that you use Enterobacteriaceae as a quality indicator or hygiene indicator on those products. 

Another one you might see are coliforms and faecal coliforms. So these are a group of bacteria that are kind of grouped together based on their characteristics. So they ferment lactose with both gas and acid production at 35 degrees Celsius, faecal coliforms incubate at a higher temperature of about 42 degrees. So they're typically found in the gut, soil, and water and they can be used to indicate quality. Total coliforms indicate drinking water quality and faecal coliforms are a subset, and they're considered a better indicator of faecal contamination specifically. Again, E. coli these are a subset again of this sort of Enterobacteriaceae family. And they’re used to assess the hygiene of food and they can indicate the presence of faecal contamination. And so, they can indicate raw material or cross-contamination, and so could be, for example, if you find E. coli on a carcass, it could be a contamination feature, contamination from the animal during slaughter, and could be an indication of inadequate heat processing, or a problem with cleaning.  

So drinking water should have no E. coli. And there are some process hygiene criteria for E. coli in Regulation 2073. And then we have pathogenic E. coli, so it's a very small subset of the E. coli, and these are a very particular type of E. coli that produce toxins called verotoxin or also known as Shiga toxins. So you might see VTEC (verocytoxigenic E.coli) or STEC (Shiga toxin-producing E. coli) are same thing. And these have a particular gene that allows them to produce this toxin and a very small number of these bacteria will cause a very serious illness, mostly where it affects the kidneys.  

You might find these is one of the reasons why we tell consumers to fully cook their burgers and also raw milk and cheese have contributed to some outbreaks. Increasingly fresh produce can be contaminated with VTEC or STEC, and it’s usually from faecal contamination, maybe from water that was contaminated, or maybe there's animals nearby. There was a very large outbreak related to pathogenic E. coli in sprouts in 2011 …. And there is a regulation, there is a category for sprouts in Regulation 2073.  

So just moving on to how to read a microbiological lab report. So when you send off your samples, they get tested and then you get a lab report back. There's generally five different names or types that you may see on a report. And so, detected or not detected in a certain quantity of food, so in grams or milliliters. And secondly, a specific test result, so it could be 100 CFU, a colony-forming units per gram or millilitre. A less than result, so a less than 10 CFU program results, and a log result or it might be written in scientific notation or a greater than result.  

And just to mention that, a decimal dilution, so sometimes you may see, you know, in the very high amount of results, if the lab expects the number of colony-forming units in a particular sample to be high, they will need to dilute the samples so that they can get a result on their agar plate. So they do this, it's called a decimal dilution, so it's usually maybe one ml or one gram of the food into nine mls or have a daily one, two, three some kind of a buffer. So when they carry out the dilution, then they have to, if they get a count on a neighbor place, they have to multiply back up by that dilution factor. So if the lab carried out a 1 in 10 dilution, they have to multiply the results by 10, or if they had to dilute it by a factor of 100, they multiply the final result back by 100 or a thousand, so on.  

So just to explain these different types of results and what they mean, so for the first one, the not detected as I explained, it's the most sensitive test method. So that particular sample is enriched to give any sub-injured cells the best opportunity to grow, and the result really means either the bacteria was found or was not found in the food portion that was tested. So generally it's 25 grams, sometimes you might see 10 grams, depends on the microbiological criteria.  

For the second one, it's really a specific number of colony-forming units have been found on the petri dish or the agar plate per gram or ml of food tested.  

And the third one is where you have a less than result, so this indicates that there are no colonies growing on the petri dish at the decimal dilution places. So if you remember, sometimes labs have to dilute the sample to get a result, they may not find any colonies growing on the agar plate, and in this case, it's usually due to the limit of detection for the method. So at less than 10 CFU per gram, that's the limit of detection, and so that means that there are no bacteria growing on that plate at 10 CFU, 10 colony-forming units or above, but there could be bacteria present below that, so it could be nine CFU, eight CFU, you know, up to one CFU, but it just, what they weren't able to detect it at that limit.  

So if you were, if you were constantly finding your lab were finding no bacteria growing at say less than a hundred or less than a thousand CFU, you might ask them to place a lower decimal dilution to this to see if you can find it at a lower dilution factor.  

For the fourth result, so you have a log result or scientific notation, this is really where a high dilution, a decimal dilution was used, and that high result had to be condensed. So each log is worth a multiple of 10 or they may use scientific notation, and so that's the number of tens multiplied by the superscript number. 

And so just to give you an example there, so for five log ten CFU per grams, that's five, that's 10 by 10 by 10 by 10 by 10, so you're five tens multiplied by each other that gives you a hundred thousand CFU per gram. 

In the second example, if you had scientific notation, so if you had one, a number, so in this case, it's one by ten to the five CFU programs, or you might just have ten to the five CFU program. So that's the number 10 and multiplied by 10 five times. So the number from this example is number one multiplied by 10 by 10 by 10 by 10 by 10, so it's a hundred thousand CFU per gram.  

You can work out these numbers on Microsoft Excel, so if you want to work out what a log number is and CFU per gram, into the cell, you will put equals 10 and that little sort of inverse of V and by the numbers, in this example, it's five. Or if you want to work out what the log of the CFU per gram number is, you would choose the log 10 from the Excel drop-down and present the number, and it can give you what the log is.  

And then the last result, a greater than result, so say greater than 10 to the five and CFU per gram. So this is where there's too many bacteria growing on a petri dish or an agar plate to count at that decimal dilution placed. So, that sample would need to be diluted further to get a colony count that could be considered accurate. 

So, I would caution if your test results say it’s over 10 to the five CFU per gram, it doesn't tell you how much over the limit the result is, just that it's over the limit. So, in terms of determining the risk associated with that, it's hard to know because it could be a million or it could be, you know, 10 million of that your actual result is, but your reported result just is greater than 10 to the 5.  

So just to mention, Guidance Note 3 has a lot of information on all the different bacteria types, and I’d recommend you look at that, it’s in section 7 of that Guidance Note and in the appendix it also lists all the different types of foods that there is legislation for.  

So then we're coming to the last part, so now you've done all your testing and you've got your lab report, how do you assess the results?  

So for Regulation 2073, it actually explains how you interpret test results. So, I’m just giving an example of an extract from the legislation there. So if we go through this food safety criteria to give you an example of how you interpret those results. So, this is the one Lisa has also mentioned earlier for cheese, butter, and cream made from raw milk and milk that has undergone a lower heat treatment from pasteurization, and it's for Salmonella, and you need to take five samples and out of those, you're allowed to have none that have Salmonella detected at 25 grams.  

So the master food then is designated as satisfactory when all of the five sample units meet the criteria limit of Salmonella species not detected in 25 grams. The batch is designated as unsatisfactory if you have any of those five sample units that had Salmonella species detected in 25 grams. So the whole batch then is deemed unsatisfactory, and if you have an unsatisfactory batch, you have to take actions as laid out in article seven of the legislation and together with other corrective actions defined in your HACCP-based procedures or other actions necessary to protect the health of consumers.  

And for process hygiene criteria in Regulation 2073, as we mentioned, it's usually a three-class plan. So, for this example, we have the same cheese made from milk or whey that has undergone heat treatment, and it's for E. coli, and as this example, you need to take five samples from that batch and test them, and you are allowed to have a maximum of two samples that are between the limits on a small m 100 CFU per gram up to a big m 1000 CFU program. So all of the factors designations are satisfactory if all of those five samples that you've taken have a test result that is below the limit of 100 CFU per gram. The batch is designated as acceptable if you have a number of sample units between the lower limit, so 100 CF per gram, and the upper limit 1000 CFU program does not exceed the number c, so two in this case in the annex. So, if you had two samples that were 110 CFU, 120 CFU, and the remaining three samples were 80 CFU your sample would be designated as acceptable. If you have any test results that are above the upper limit of a thousand CFU, so big m in this case, the batch of food is considered unsatisfactory. So, if you had any of those five test results that were at 2000 CFU per gram, that whole batch would be considered unsatisfactory or if you had taken five samples and three of those were between 100 and a thousand CFU per gram because the number c, number two, has been exceeded that batch will be considered unsatisfactory. 

So, for the process hygiene criteria in Regulation 2073, at the end of that table, it gives you the action in case of unsatisfactory results, so it's usually improvements in process hygiene and selection of raw materials.  

For any unsatisfactory results for food safety criteria, article 7 lays down what you need to do. Guidance Note 27 gives some information, so usually to notify the competent parties, and then if the batch of food has reached the consumer, you may need to carry out a recall. So that's when the food has gone as far as getting on the shelves and it's reached consumers. Or if it hasn't yet reached retail level, you may be able to carry out withdrawals. That's just where you notify your customers that haven't actually gone on to the public, that you need to carry out corrective actions. So these should be defined in your HACCP-based procedures and any other actions necessary to protect the health of consumers. And you need to carry out a root cause analysis to try and find out why were these results unsatisfactory. And you need to kind of assess what, you know, when you find out why did this happen, is there some weakness maybe in your HACCP-based procedures that allowed this to happen, and is there some controls that you can put in place to prevent it happening in the future? And so, Guidance Note 27 will give you information on these actions.  

Article 9 of the Regulation also requires that you trend your test results, and I suppose this is to get the best value out of all of the testing that you might be carrying out. You can see in this example, it's for the cheddar cheese, so the food safety criteria and the process hygiene criteria, that I just went through. So the process hygiene criteria, they've plotted the results of the e coli test, and so they're taking five samples each time, and on two occasions, they've noticed that some of those samples are approaching the upper limit. And it's at this stage, so it's not unsatisfactory, but it's approaching unsatisfactory. So the food business operator sees this trend and tries and finds out why is this happening, it takes measures to address it.  

So Guidance Note 3 also gives you a number of tables that detail the actions you need to take if what's called a borderline result, which is the same as an acceptable result in Regulation 2073, so it's not unsatisfactory, but it's not satisfactory, it's in the middle. Or if your pathogen or your hygiene indicator is unsatisfactory. We briefly mentioned customer specifications, so you may have an agreement with the customers you're supplying about test batches that you test on, tests for how often you test for them, and what the limits are. And customers may require a more stringent limit for certain bacteria, maybe to what's in the legislation, and they can do that if they wish and as long as you agree to it. They may require you to supply them with some analysis to prove that you've been testing, and you may have to agree with them actions that you take if those limits are reached. 

So, just to summarize, I suppose, myself and Lisa's presentation. Lisa went through knowing your food and the potential hazards associated with this. So, if it's, you know, ready to eat food or not, and what's the pH, what's the water activity, how do you package it, is there anything in that food that will limit the growth of any hazards, biological hazards? And then, use all of that information to find out what microbiological criteria apply in particular, take account of anything that legally you need to comply with.  

Know how to take your samples according to the micro criteria. So, how many samples do you have to take? Take them representatively from the batch, make sure you're taking them all from a single batch. And then, when you get your lab reports, make sure you read the lab report, understand it, and you know, review whether you have any action to take. So, assess the test results against the relevant microbiological criteria that you've identified for your food and see if any of those are reaching the limit. If the limit is reached, take any corrective actions necessary under your food safety management system for unsatisfactory or borderline results. And remember to trend your results, and keep an eye on that, and then if any of them are approaching unsatisfactory, you know, take any actions necessary to prevent them becoming unsatisfactory and bring them back down to satisfactory.  

And so, just to finish off, and I'll just touch on some resources and guidance. So, the FSAI website has a huge amount of information and guidance. And if you click on the food business section on our website, you'll be brought to a page like this. And in particular, on the left-hand side, there's margins and buttons for microbiological criteria and shelf life, and that links out to a lot of the guidance that we mentioned today. In particular, I would mention, you know, Guidance Note 27 is how to comply with Regulation 2073. We mentioned that a lot today, and also Guidance Note 3. Guidance Note 18 has everything to do with shelf life. And then we have that best practice for testing foods factsheet, how to choose a lab on how to make sure you're using the right method.  

We have two e-learnings on the microbiological criteria, and they really go through everything that myself and Lisa have talked about today. They each take about an hour to do, and they're free. I mentioned that FSAI carry out events as well, so we often do breakfast bites. So, we had one yesterday on what to expect from your inspector. I will be giving one on shelf life on the 20th of September. So, if you go to our website, you can sign up for our events or any other alerts you might be interested in, such as e-alerts.  

And just to mention then, I suppose, lastly, some other useful non-FSAI resources. And so, we talked about the ICMSF, and they have, you know, a lot of books that are very useful. But they also have these free YouTube videos that go through testing and food safety management, and I really recommend those. They're short YouTube tips. If anybody has more interest in the background to statistical setting of microbiological criteria related to foods, and this WHO FAO document is really interesting and gives a lot of good information. Don't be put off by the title, there's lots of plain language in it, and it's a really useful resource for food businesses.  

You mentioned the professional food industry bodies, so Chilled Food Association and Campden BRI, in particular, have a lot of very useful resources and books. Some of those are free, some of those are available to members only or maybe at a higher price to non-members. We'd like to thank safefood for inviting us to take part in the Knowledge Network today and to acknowledge that, you know, safefood has a huge amount of resources and other webinars on their website that are very useful sources of information. And then lastly, there's Teagasc and UCC. They have food industry training, and they provide some free or at-cost courses, and they have a lot of very useful information and courses.  

So that's it for me. So, I'd like to say thanks to safefood for inviting us to take part in the webinar today. I hope you found the information useful. And as Lisa said, if after going through all of this process, you find you really can't find an answer to your query, you can email us at the advice line, [email protected], and we will endeavor to get back to your query within five working days. So, I'm going to hand over now to Una, and thank you, everybody, for joining us today.  

Una McCormack – What food businesses need to know 

Okay, firstly, I'd like to thank safefood for inviting me on this webinar, and I'd also like to thank Mary and Lisa for the presentations because that leads gently into what I want to talk about. Because we've heard about the Regulations, we've heard about the legislation, we've heard about criteria, about categories of food, and what you need to get tested. And now, I will continue on from a commercial laboratory perspective on what a food business needs to know to get this testing done.  

So, the first thing you need to do is to find your laboratory, and you need to build a good relationship with the laboratory that you have chosen. Now, to find a laboratory, you'll get a list of accredited labs available on the INAB website under the directory of laboratories or, indeed, you may get a recommendation, word of mouth, etc.  

Once selected, then contact details will be available on the laboratory website with a number and/or email for the FBO to get information on services provided. Communication between the food business operator and the laboratory is key to building a good relationship. You need direct contact with staff with experience and knowledge of the Irish food industry, to understand the customer requirements and familiarity with relevant legislation, as spoken about by Lisa and Mary there. A timely turnaround of results and communication on the progression of results, flexibility to act quickly to new customer requirements, the interpretation of results in conjunction with technical feedback, also important, and then to tailor and personalize the service offering complete confidentiality and develop familiarization and relationship with individual sites might have individual sites doing different products for the one company, etc.  

So then, we spoke briefly earlier about laboratories fit for purpose, and basically, this comes down to accreditation. It means that the laboratory is approved to test specific types of samples using approved methods. It does not apply to the laboratory in overall general terms, and you need to check with the lab if your product is within the scope of their accreditation status. And what's meant by that is if you decide to produce maybe a very unique product, it's not first to the market; you need to check that the lab is accredited to test that product under the scope of their accreditation. If not, if they're not accredited, the test can still go ahead, but your test will be marked as with an asterisk, perhaps something like that, that it doesn't come under the scope of their accreditation status.  

You may have heard of lab proficiency testing. These are testing schemes which are all parts of the requirements of the accreditation status, and it proves that the lab and staff are competent to carry out this analysis. So basically it involves the lab joining a PT testing scheme. The lab receives a spiked sample that has a designated quantity of the test organism, or it may not; indeed, it may not have it. And the lab staff, in their turn, test this as a regular sample and carries out normal procedure, and they return the results to the PT company, and those results are evaluated, and you get a PT score then for that test or whatever item. Under ISO 17025 this has a series of clauses in this standard, which must be part of the quality system of the laboratory, and the lab will be audited to ensure that the lab is fully compliant with these clauses in the standard. 

I spoke earlier about rapid methods and rapid methods versus traditional the ISO method. So, and I know this, as questions are sometimes asked of food business operators by inspectors as to what method has been used for testing their product. Your method reference will be stated on the certificate, and rapid accredited methods used by labs are perfectly acceptable and part of achieving its use is validating it against a standard method. So, you'd check your standard method, or you check your rapid method against the standard method; are you getting similar results? And the benefit of the rapid method is actually the turnaround time on test reporting; they are, in their name, faster.  

So, I’ll just take you through a step-by-step approach now. So, you've decided on a laboratory, what do you do next? So, you contact the lab through the contact details. Customer service will have a discussion with you, and they will issue a quotation to the client with details of test price, accreditation status of the testing, and this the quotation will contain turnaround time and standard terms and conditions, etc.  

So, once the client has reviewed laboratory quotation, has agreed with the lab to commence testing and the suite of tests to be used, to perform, the next step is the accounts set up. This is very easily done, the client completes a new customer form where you have contact details, telephone numbers, email addresses, a contract of the suite of tests to be used, is issued to the client detailing this suite of testing views, and again the cost. A detailed service level agreement or more basic service contract can be drafted. It depends on the size of the client, sometimes they want a very detailed one, sometimes the very basic one will be will suffice.  

So, then you arrange transport of the samples to the lab. They're all arranged, agreed and samples to be labeled clearly, description and batch number for traceability. So, the next step then is to complete the analysis request form. This is where you enter the description of the sample and you'll get your test code from your contract and the date of the analysis to be done. The description entered on the ARF is what will appear in your certificate of analysis, and this is to be sent electronically to the lab on the day or prior to dispatch. And we just go to just a side note here, is that for commercial labs that have a high throughput of sample each day, it's imperative that the ARF is completed fully and instructions of “do the usual tests” or “whatever was done last time” unfortunately cannot be accommodated in busy testing environments. On the receipt at the laboratory, samples are linked onto the LIMS system, and this you may be you may have heard of this before, the LIMS is the laboratory information management system used within the laboratory, and it assigns a unique sample ID label that's linked to the test to be carried out. This ID is entered onto the ARF for purposes of traceability with sample receipt department, and this label is actually on your product, so it goes through from receipt right through through sampling, right through to the number on the Cert for traceability.  

So, once samples are logged by the laboratory office, a contract review then is generated and emailed to the client, and it's very important that this is reviewed by the client because it details all the samples have been received as sent, the tests are logged that are required and the test and the testing dates are correct.  

So, I just have an example here of a service level agreement. This is a quite detailed one, I won't go through all the points, but an SLA, you may have heard it called it that called that, it signifies a signed written agreement between the lab, in this case, ALT Merieux, and the client that documents the arrangements for the management and delivery of work, handling of samples, reporting of results and parties to the agreement are detailed in the SLA, both from the laboratory and the client. 

So, details in the sla may or may not contain some of the following points: sampling and consumables, how your sampling is done, what consumables are supplied for the samples, how the samples are transported, registered storage details (what I’ve spoken of earlier, logging them into the LIMS), the quality management system. ALT Merieux testing divisions carries out its activities in such a way to meet the requirements of the ISO standard technical requirements. Data integrity, results held on the LIMS system, out of specification reports can be provided, and I just speak more about that in the next slide. Communications are very important in the overall scheme, so ALT Merieux will provide contact names, numbers, and the responsibility of key advanced laboratory testing personnel, including those who are out of office hours, if the need arises.  

Expert support, invoicing, invoices are agreed and issued for the agreed work, contract review if it's going over a long period of time, maybe over a year, several a year or two, or even longer, you have a contract meeting to view that, to monitor requirements and develop and maintain the working relationship. Confidentiality, ALT Merieux and the client should keep confidential any information disclosed directly or indirectly. And you have agreement terms, notice period should you wish to terminate the work, standard terms and conditions, force majeure, and signatories.  

So, this is what I spoke about earlier, and this is just an example of a laboratory contract between the client and the lab itself. So, here we have a contract to set up for safefood. They have decided that they want to do some products for testing using the full suite. This your item code is what will be entered onto the ARF - it's only an abbreviation for the LIMS to recognize it. So, here we have Safe PDT for a product and six MT six micro tests. So, your six micro tests, in this case, are entero, E. coli, Listeria, Salmonella,  Staph, and TVC, and just an example price here. You also want to get a swab test done, and they're going to do that for Listeria, Listeria species, in this case, and they also want to get a hand swab done, and for, so they're getting one micro test on a hand swab and Staph aureus, and again just the price there.  

So, I spoke earlier about the ARF. This can also be called a requisition form. It can also be called a chain of custody form. All terms are interchangeable. So, again, we have our company name, our client, a contact name, some contact details. We have the sample type here. Is it raw, is it ready to eat, is it a swab, is it water? And the reason this is entered is that it meets the scope of the accreditation status. So, they can see are they accredited to test prepared dishes. This details the exact sample description to appear in the test certificate. This is what you enter on the ARF, and here we have safefood Homemade Lasagne batch 1, 2, 3, 4 for traceability used by the 9th of the 6th. It's not a shelf-life product. We want to test it on the 9th of the 6th and under 6 MT and so on and so forth there for the other items.  

I’ll just speak briefly here now, and it's been alluded to already, some of the lab terminology. So, ARF analysis request form can also be a COC chain of custody or requisition sheet, turnaround TIT, turnaround time for the various tests. We have OOS out of specification, accreditation is your certification. INAB is your Irish national accreditation board. ISO 17025 is the international standard existing laboratories. Your PT scheme, spoken about earlier, is some of the test terminology that you will come across. We have presumptive, you might get a presumptive Listeria that is suspect, and then you'll that will either be confirmed. We spoke heard of earlier about the CFU colony forming unit, it estimates the number of bacteria or fungal cells in a sample which are viable. Listeria or Salmonella spp. means Listeria, Salmonella species. ACC, TVC, all interchangeable terms, aerobic colony count, total viable count.  

Labs may use different terms but they all these are all interchangeable as I say. Enterobacteriaceae might be just shotened to Entero in a conversation. Listeria mono, monocytogenes.  Staphylococcus, to give it its full title, that might be just shortened to  Staph or  Staph aureus. Aseptic technique, this is how to handle samples without contaminating them, and I’m going to focus on that now in more detail.  

So, aseptic sampling, if samples are not in their final packaging, an aseptic technique must be used. Aseptic technique for sampling means using practices and procedures to prevent incidental microbial contamination. For the interpretation of your result, it is extremely important that the lab receives the sample which is true to the batch produced and not contaminated during sampling or damaged in transport. Samples should be submitted in original containers where possible. If not in original containers, then sample portions should be transferred to sterile or other suitable containers. Sometimes Ziplock bags are used, or ideally, the lab will supply you with sterile bags or containers.  

The area for your sample preparation should be sanitized with an appropriate surface sanitizing spray. Utensils, such as spoons, knives, all should be all washed thoroughly, dried and sanitized with alcohol wipes or alcohol spray, and you would be familiar with an example there Steri wipes. If sampling multiple products, ensure that you have enough utensils for each product. Wear disposable gloves or ensure your hands are washed and use sanitizer, and change gloves as your product type changes. And do not use the same utensil to portion out different products. You run the risk of cross contamination from one product to the next.  

This is a question that often comes up, then how much sample do I need to send you for testing? So just again briefly, where possible, decant directly from your bulk product into sterile containers and use alcohol wipes to sanitize the area where you will be opening up the product. And if using a scissors to cut an opening, sanitize the scissors also. So if it, for example, if it's a pouch and you want to cut a slit on top of the pouch, wipe the pouch with your alcohol spray, wipe your scissors with your alcohol spray, and decant directly and quickly into your container. 

So the amount of sample then required for testing, and I’m taking the example of my six MT, my six micro tests of entero E.coli, Listeria, Salmonella, staph, TVC, so typically, a hundred gram minimum is what was requested. And the reason for this is Listeria detection is per 25 grams, Salmonella is per 25 grams, and 10 grams can be used for the others. So there you have your 25, 25, that's 50, another 10 grams, 60. 

So the reason we asked for a little bit more is that the remainder of the sample is retained. In the event of a re-test might be required or else indeed you might decide, "Oh look, I actually need to get another test carried out on that same sample, can you add it onto it?" There's enough portion retained to carry out this request. If you're water sampling, the tap must be sanitized, and then again with your sanitizer spray allow it to run prior to sampling. The lab will supply you with sterile bottles specifically for this with defined volumes depending on the tests, but for the full suite of coliform, E. coli, and enterococci, a 500 ml sampling bottle is issued and full documented instructions on sampling can be obtained from any lab on request.  

Also mentioned earlier was environmental sampling. The effectiveness of cleaning practices is verified and monitored by environmental sampling, that is swabbing. Swabbing is carried out on both food contact surfaces such as equipment, utensils fillers, also on containers and non-food contact surfaces such as drains, cold room, maybe cold room fans, cold room walls, and protective clothing. Personnel hand swabs also form part of your environmental testing program as personnel can be a source of microbiological contamination both directly as in personal hygiene and indirectly through cross-contamination. Swabs can be taken on bare hands and gloved hands of staff handling food. Swabs are available as both a sponge which is generally used for Listeria and Salmonella testing and stick swabs which are used for hand swabs or surfaces where you're testing for the hygiene indicators such as entero or total viable counts. Instructions on how to use the swabs are freely available either on the packaging itself or separately on an instruction document from the laboratory or indeed on YouTube as well. There are several resources for that.  

For your transport of samples and storage, again to maintain the integrity of samples for testing, care must be taken in the transport and storage of samples. Containers should be leak-proof or well-sealed to avoid cross-contamination and labelled clearly. Samples should be refrigerated until agreed transport arrangements are in place. Cooler boxes with ice packs are acceptable for transfer if the lab has a depot or collection from clients. Generally, again by means of refrigeration vehicles, and these are monitored and recorded via temperature loggers. Chilled foods should not be frozen prior to testing. Precautions should be in place to prevent cross-contamination from raw products to ready-to-eat products. You can use separate cooler boxes or sturdy tear-proof bags and label these accordingly.  

This is a question that is often asked: when will I get my results? Electronic reporting of result is by email, i.e., that is your test certificate, which is in pdf format and secure. A final certificate is issued when all the tests are completed and approved for issue. Please note the turnaround time of the test on your quotation, as this determines the wait time for completion. If I can briefly just give you an example of this, so if you have a product in for your six, we'll say seven MT because you're adding Yeast and Mould to it now as well, your E. coli will complete in 24 hours, your TVC will complete in 48 or 72 hours, and Listeria might be up to three days.  

So you're not going to get your cert until your yeast and mould completes because that's the longest test that's five days. So your cert will be issued as soon as the longest test is complete.  

But we come down then to out of specification alerts, and this is an early alert system relating to a result based on product specification. It also applies to presumptive detective pathogens. So if you have this same product in for your seven MT and something suspect goes for Listeria, something suspect on the plate, it sets off at the out of alert specification. You will get an OOS email, and that will say presumptive Listeria, and that will await final confirmation, and that can take maybe two to three days extra to confirm that. Urgent results can be communicated to the client in agreement or via an interim report until the final Cert is issued. With the ALT Merieux online portal, this allows the client to log on using a unique password to access only the final results, and this gives you your option then for trending.  

So we now go on to advice and expertise available. Again, a statement I often hear again is, "I receive my search, but I don't understand what the numbers mean," and I’ll just go down through this briefly because I’ll start with the role of the laboratory is primarily to analyse and report the results, and a food business operator cannot be expected cannot be expected to be familiar with all the technical and scientific terms used on the certificate, and some of the legislation and guidelines may be daunting at first to read and understand, but there is help. Assistance can be given at the outset. Number one, on test selection appropriate to the product based on the legislation, guidelines, or client specifications.  

Clients can also be made aware of the presence of independent food safety consultants who they can engage with for assistance and planning. Interpretation or comments on the results will not be documented on the ALT Merieux laboratory certificate of analysis; however, result interpretation and opinion can be expressed separately and communicated by dialogue through personnel authorized to do so. Result review reports can be requested from ALT Merieux as an option as part of the laboratory service, and this will be discussed at the initial stages for the client requirements quotation stage.  

The reason why the interpretation will not appear on the search is, and it's to do with the INAB accreditation in that ALT doesn't come under the scope of accreditation to comment on the results, and that is the reason on the basis for ALT Merieux. Food business operators that have dedicated quality teams may not need assistance but in the event of maybe a specific item or advice made can be made available. 

So just to conclude, I trust this presentation has given you the basics on how to select your testing laboratory, the steps needed to get registered on the laboratory system, an understanding of what is required by both client and laboratory for the testing of your product, the confidence to complete the required requirements on sample handling, transport, and the necessary paperwork to request the necessary tests, an understanding of the results issued, and the knowledge to the route for further assistance if needed through published guidelines from the FSAI and other parties. 

I’d like to thank you all for your attention and best wishes for future working relationships, and I wish you all well in your food business endeavors.  


Mairead Mccann: Thank you so much Una and to Lisa and Mary for taking the time out today to give us such informative presentations. Just as food businesses in their understanding of microbiological testing, you can see it's not straightforward and there's a lot of information food businesses need to be aware of in relation to micro testing. 

We have a number of questions in our Q&A box, I can see it at the minute. And if anyone has any questions that they'd like to ask, maybe pop them in there now before our speakers are finished. Okay, I’ll just check the box there and see what we have. 

So someone has asked, let me see, if a shelf life of a ready-to-eat food or ready-to-heat food is production plus two days, is micro testing required? That's quite a specific question, so I’ll leave it. 

Mary Lenehan: I’m happy, okay, yeah, so generally if it's just a two-day shelf life, we would say no testing is required because it's kind of similar to leftovers in your fridge. And again, I suppose, producing safe food would really be based on your food safety management system and good hygiene practice. 

Mairead Mccann: Perfect, Mary, thank you. And someone has asked in the Guidance Note for shelf life testing, it says some foods, such as coleslaw, will naturally be high in TVCs. If test for coleslaw doesn't reach shelf life because of high TVCs in results, how best to go about this?  

Mary Lenehan: I’d say, I’m not sure I understand the question exactly, but I think it's probably when you're setting yourself up for your coleslaw, there are probably a number of quality parameters you might want to take into account. So TVCs are just a general account that gives you a general idea of the quality of your product, and so raw vegetables would have naturally kind of a higher count, I suppose. But it is an indication of spoilage because as the product gets older, there are more bacteria, in general, that will grow, so a higher count towards the end will indicate that you're coming towards the end of the shelf life. And there is a count for, there is a criteria limit for coleslaw in Guidance Note 3, in that table on ACCs, and I can't remember what one it is, but it's foods mixed with dips, and coleslaw has been given as an example. 

Mairead Mccann: Okay, thank you, Mary. And someone wants to know why eggs are assigned a best before date and not a use-by date?  

Lisa O’Connor: Okay, I can take this one. It's not an easy one. In truth, it’s a quality issue, I suppose. You know, eggs do deteriorate over time. The issue here is that Salmonella is a concern, especially in certain parts of Europe. In Ireland, we have managed to control Salmonella in our egg-laying populations really well, so the risk is more associated with imported eggs in Ireland at this point. And so while a best before date is set, a limit of 28 days after laying is set to use those eggs, and it’s just a combination of the concern around Salmonella broadly across Europe. So, well, you might say, there's a set date, and it's relatively short, so therefore eggs are perishable, and it is actually a best before date. It's set at 28 days after laying. And there's another peculiarity around eggs, and some of you may have noticed that at retail level they're stored at ambient temperatures and not in the fridge, but when you bring those eggs home, you're recommended to store them in the fridge. The reason is that as eggs go in and out of chilled and ambient temperature, condensation can develop on the egg, and if the surface of the egg has pathogens on it, Salmonella the most likely one, it can go through, and the condensation can go through the shell. So while you might have had contamination the surface, you could then end up with contamination inside the egg, which is more problematic because a lot of eggs are eaten undercooked or even raw. So, yeah, I can put some guidance in the chat that we have on eggs, and I’ll just pop that in there now. 

Mairead Mccann: Okay, thanks very much, Lisa. Okay, we’ve another one on the shelf life of ready-to-eat foods. So if the shelf life of ready-to-eat food is not five days, but it is a ready-to-eat food, should you do testing for Listeria for that food, or just environmental testing required for that product?  

Mary Lenehan: Right, so if it's a ready-to-eat food with a shelf life of four days or less, it's falling into category 1.3. So that's ready-to-eat foods unable to support the growth of Listeria monocytogenes, and the limit for that is the less stringent limit of 100 CFU per gram, so you'd use the enumeration test for that. There's no set frequency for how often you test those samples, and it will depend on that risk assessment that you carry out around that product. So if you remember, you know what the type of product is, what the nature of the business is, who are you supplying, etc. So if you were producing ready-to-eat food with a short shelf life, such as chilled ready-to-eat food like a sandwich or a wrap or a salad, and you know that that's going to a hospital, for example, the end consumer is potentially more vulnerable to contracting listeriosis, so you may do more frequent testing on the basis of that. And so it's really, you know, you have to go through those steps of kind of knowing what the risk is associated with your food.  

Mairead Mccann: Okay, thank you, Mary. Okay, we have a question again about Listeria growth. So what evidence do the food business operators need to provide to demonstrate that Listeria monocytogenes will grow over the course of the shelf life so that the enumeration method of testing can be applied?  

Mary Lenehan: So if you remember that slide, it's that EU-RL, the European Union Reference Lab, and guidance on challenge tests and durability studies, and it's all in there, basically. And so they may also be able to use some combination of predictive microbiology to support their case, but you know, you'd want to caution that. You probably need the help of the trained microbiologist to interpret those models, but the EU-RL Guidance basically, there's two different types of tests you can do. So a challenge test is where an inoculum of Listeria monocytogenes is deliberately added to the food, and it's held under certain temperature conditions to see if the Listeria will grow, and durability study is where the food is naturally contaminated and it's held under different conditions to see if Listeria will grow. But it's all in that EU-RL Guidance basically.  

Mairead Mccann: Perfect, thank you Mary. Okay, we're getting a lot of questions coming in now. So will the amount of sampling you do relate to the size of the business? For example, what if a salami manufacturer was only making a few hundred kgs or if it was a butcher expanding its offering? So, I assume this related to what you're producing?  

Mary Lenehan: Yeah, exactly. So, again, that's part of that risk assessment piece where you would kind of take into all consideration all the different aspects of the food that you're producing. And sometimes, you know, producing a small amount of food is not necessarily safer, and it really depends on the type of food that you're producing, what the risks are associated with it. And for butcher shops and for some small throughput slaughterhouses, there are some derogations around sampling frequency for some of those products that there is a set sampling frequency on. So we have guidance on that in Guidance 35, and you can also find guidance on our website.  

Mairead Mccann: Perfect, thank you Mary. Someone has a question about taking environmental swabs and sending them to the laboratory. Is it okay to send them in the post or better to store them in chilled storage?  

Una McCormack: Yeah, I'll take that one. Yes, ideally, it's better to have them chilled or keep them chilled to get them to the laboratory as soon as possible, ideally within 24 hours through the post. You could run the risk, even though you can use cooler packs and all that, but things can go wrong with posts and couriers, as we all well know. So if you can get them to the lab asap, be chilled.  

Mairead Mccann: Perfect, thank you Una. And we have a question when conducting a shelf life for soft cheeses and the ACC result hits the maximum value so that's the first sample. Should we stop the test and start again?  

Mary Lenehan: So, I suppose the test result is related to that particular product, and what you really need to do is kind of step back and assess the situation. So an ACC is a process hygiene criteria, so it's really talking about improvements in your process and hygiene. So you review your raw materials, review your cleaning, all that kind of stuff, and to see can you find out where it is, why is there more ACCs growing on this particular batch of food maybe than you've seen before. I wouldn't recommend if you get a test result on a certain batch then go back and retest another sample from that same batch because it doesn't really change the overall outcome. And as I said, the distribution of bacteria is not uniform, you know, so you could take a second sample from that same batch and get the result that you want, but ultimately the first sample that had the higher result is really showing you that there is a contamination issue in your product, a spoilage issue maybe if it's ACCs in your product.  

Mairead Mccann: Okay, thank you Mary. So we have one more question. Someone's asking if they produce overnight oats with a vegan milk rather than dairy, would testing and shelf life be significantly different if you have any experience of that?  

Lisa O’Connor: Yeah, I guess I'll take this one. Well, the answer is I have no experience of it, and we would just start to google and just learn about the hazards associated with vegan milk relative to real milk.  

Mary Lenehan: Yeah, I think it all boils down to it's on a case-by-case basis, and for every product, you need to go through that process of figuring out what type of food It is, what are the hazards, and you know for that overnight oats, you know how long is the shelf life that you're intending to put on to it. And if there are sort of temperature control issues, spoilage issues, you just have to go through the whole process really for every different product and make sure you document it. 

Mairead McCann: Perfect, thank you. And thanks to you all for staying on so long to answer the questions for us.  

I suppose if any of our participants haven't joined our Knowledge Network, you can do so at, and that'll keep you updated with any of our events that are coming up on any of our new resources. And as Tracy said earlier, she will send out a link to an evaluation form that we would really appreciate if you take a few minutes just to fill out for us. Tracey, is there anything else I’m supposed to say? 

Tracey Thompson: But just a reminder to everyone that we have been recording, so the webinar will be available on the Knowledge Network website as well as the safefood main website. We will also send a link around to everyone, probably in about a week's time, once we've had it downloaded and all the rest. We'll send it on to anyone who has registered for the webinar today, and that's it. 

Mairead McCann: Okay, and thanks everyone for joining us and enjoy the rest of your day. 


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