welcome again to um the first of our summer webinar series with micro101 and environmental monitoring my name is thomas jones i'm the senior director of analytical services here at safe food alliance in our main laboratory in kingsburg it's a real pleasure to welcome you folks today we'll start right off with food microbiology 101 so why are we here today really what are microorganisms they're basically microscopic living creatures they're not visible to the naked eye so by definition we can't see them if we just are looking around in the world around us and this is why they're incredibly tiny thirteen thousand e coli cells placed end to end would equal about an inch so that's you know that's not very much um it's like looking down at the earth from outer space and hoping to see humans on the surface right we just can't see them with our eyes that's why we have to use other tools and techniques to make sure that we keep those guys in control and know what's going on what are some of the microbes of importance in foods i've split these out into really five basic categories right the bacteria the yeasts the molds viruses and the protozoa the bacteria yeasts and molds are really important because they can grow in foods and so those are the organisms that we'll spend a lot of time talking about today because we can modify conditions or impose practices that can keep those organisms from growing in areas where we don't want them to and causing issues viruses and protozoa can also cause illness in foods but we control those mostly through good agricultural practices and preventing contamination of the crop a little bit about the bacteria the bacteria really grow as single cells as individual cells each cell is an organism so our bodies are made of millions of cells but the bacteria each cell is an individual now sometimes they can produce what are called heat resistance endospores those are very difficult to kill the vegetative cells the actual cells of the bacteria are reasonably easy to kill but these endospores are are literally like seeds they're survival pods as it were for these organisms and they can be really resistant to heat chemicals etc in fact when you test a steam sterilizer like with ones we use in the laboratory or in the hospital setting they actually use bacteria to produce these spores to test the effectiveness of that sterilizer so um if it basically can survive 250 degrees fahrenheit for 15 minutes at 15 pounds per square inch steam i mean that's that's pretty tough anything slightly below that and these endospores can survive so those are organisms that are a little more challenging to control um most of these organisms are going to form the shiny smooth colonies on a petri dish so if you're looking at a bacterial dish in a laboratory if you come to our lab and take a tour you'll see these little smooth bacterial colonies they tend to prefer more moderate conditions of moisture and ph or acidity than the molds do they like a lot of the same conditions that humans do that's why they can be a real challenge in terms of food safety and spoilage many of the same nutrients that we use to sustain ourselves proteins carbohydrates and fats are the ones that the bacteria use as well so they're probably one of the the main organisms that we have to be concerned about in terms of control we'll talk a little bit about some key pathogens or key organisms of concern salmonella is a big key pathogen there's over 2500 different serotypes or strains of salmonella in the natural environment humans and animals um serve as reservoirs for salmonella both wild and domestic animals and um it was long thought that that was the principal way in which foods would become contaminated is through human or animal activity fecal material that sort of thing um and historically we look at animal-based foods like milk or meats as or poultry as the primary source of illness related to this organism we have though become aware in recent years that fresh fruits foods can be linked to some melosus outbreaks as well and we mentioned some here oranges tomatoes peppers uh orange juice and cantaloupes so there's a number of different produce-based foods that can also become contaminated we also know now through more modern research that these organisms can survive well in the environment and tolerate some extreme conditions especially heat and dryness and so the historic thought that they were just only associated with animal contamination is kind of changed much of that um understanding came from some major uh outbreaks and recalls related to some low moisture foods like peanuts tree nuts spices dried coconut um in which we did research and found that some of these organisms could survive for example in an almond orchard for five to ten years in the soil it is still true that the main reservoir for these organisms is humans and animals but once out in the environment they can hang on and survive long enough to recontaminate food and ultimately cause illness another key bacterial pathogen we talk about is toxogenic e coli so the regular e colis or normal inhabitants of the intestines of humans and animals if you take antibiotics for too long uh if you're ill for example you can actually kind of throw your normal digestive flora out of whack and actually wind up with some digestive issues and that's one of the organisms that you have to be concerned about is e coli it actually manufactures the uh vitamin k12 and so we can't make that our ourselves we actually rely on the e coli in our systems to do that for us when we talk about the toxogenic e coli it's a different group toxigenic strains of e coli cause serious foodborne illnesses okay so the symptoms can include vomiting diarrhea fever and kidney failure so there are a number of very very serious um outcomes for people that become ill with this toxic strain of e coli and a number of foods have been linked to this this bad e coli as we call it meats fruit juices milk nuts flowers one of the earliest outbreaks associated with this was the infamous jack-in-the-box recall or outbreak back in the early 1990s but there have been fruit juice related illnesses um flour was one of the more recent ones they actually had an outbreak of this organism over this illness related to cookie dough consumption raw cookie dough consumption and they ultimately determined that that was the most likely pathway for the contamination was in the flour probably the biggest food that has been impacted by this is lettuce and leafy greens um there have been like 22 outbreaks between 1995 and 2006 that were related to this organism in lettuce and leafy greens it ultimately led to the leafy greens marketing agreement here in the state as well as in i think arizona and other nearby states that produce a lot of leafy greens to try and control uh some of the conditions in the field and reduce the contamination and these organisms also are good survivors in the environment they're ph resistant that's why they've caused such an issue in some of these foods like leafy greens another key bacterial pathogen if we're looking at kind of the big three of these pathogens or disease-causing organisms is listeria monocytogenes this is a very common uh soil bacterium there are a number of species historically there were six species of listeria genus listeria but now through the miracle of modern genetics we found many many more species of this organism interestingly though it still appears that one species listeria monocytogenes is really a human pathogen or causes disease in humans so that's the primary one that we are worried about now when we talk for example about salmonella or the toxic toxic e coli that we were talking about earlier there are multiple strains multiple species that can cause illness listeria it's this one species we know that it can grow well under refrigeration even down to freezing which is a challenge so if it contaminates fresh produce sometimes it can slowly continue to grow in refrigerated conditions it can also tolerate very high salt foods up to ten percent sodium chlorate so those are some of the challenges that folks in the food industry can face when they're doing a risk evaluation for um listeriosis or listeria monocytogenes contamination is looking at the way those foods are produced or stored and trying to understand if it can still grow there if there is contamination foods linked to listeriosis the illness caused by the el mono include melons cabbage deli meats soft style cheeses and milk products these are some of the most common outbreaks that we've seen in recent years and the outbreaks can often be traced to lack of pasteurization for example in in cheeses or milk products and or post-process contamination and i show a really nice picture here i'm hovering my pointer over it on this really nasty french drain and that's something that you really have to look out for with listeria is controlling water on the floors your drains and drain sanitation because that's an ideal harborage spot for listeria mono this is a little chart that i put together seven years ago i think that's really useful i kind of cobbled together data from the centers for disease control as well as the leatherhead food research institute which is in the uk and you basically are looking at all three of these primary main pathogens or disease-causing bacteria that we've talked about their incubation times infectious doses cases per year in america and the death rate in america the incubation time is really the time it takes from when we ingest contaminated food to actually seeing those symptoms and the infectious dose is the number of cells or the number of microbes in that infected food serving or that contaminated food serving that we actually have to ingest to get ill so if we first look at that we can see listeria monocytogenes is very challenging because it can take anywhere from three to seventy days for the symptoms of the illness to actually show up uh that can be a real challenge for investigators because who the heck can remember what they ate uh two months ago let alone yesterday right so it's a very challenging investigation with el mono the infectious dose is relatively high they think it's greater than a thousand cells and there are some countries for example in the eu they actually have a tolerance for el mono in foods ready to eat foods um the united states does not have a tolerance for it it's a zero tolerance food but that's part of the reason why because they think you need to have a fairly high level of these cells to cause illness we move over into e coli 157 which is one of those toxogenic e coli it's the most common strain that has been looked for uh the incubation time is short one to six days it doesn't take very long after ingesting that contaminated food to become ill and the infectious dose is less than 100 cells so this is a very pathogenic organism it is going to infect uh at a fairly low level it's a good survivor as it goes through our digestive tracts and it readily attacks the intestine and starts to to infect and invade the body so again that's a challenge for control we really don't have a tolerance for those cells in any kind of food we want to get those numbers basically to zero salmonella again is is a relative of e coli uh they're in the same kind of family of bacteria as it were infectious um doses are 10 to 100 cells again um you know fairly low levels it doesn't take much to survive that's another challenge in controlling salmonella you really don't need much contamination to potentially make someone ill the incubation time is also quite a bit longer i think from the standpoint of e coli one five seven generally is a little longer 12 to 72 hours again it's it's a lot shorter than listeria monocytogenes but typically within a few days you start to feel ill now when we look at the cases per year and the death rates it's really interesting with el mono you don't have that many cases of illness per year but the death rate is relatively high at 30 percent so you can have fairly few cases but the folks that do get ill there's a fairly high mortality rate and many of these are very tragic outcomes um it is a risk factor for um expectant mothers for example and has been known to kill both the mother and the unborn child also for the elderly or the very young um looking at e coli 157 again a few more cases 73 000 per year the death rate is is 5 to 10 again fairly high lower than el mono and many of the folks that do get deal with this organism have those complications we talked about earlier kidney damage and other issues salmonella is the winner in terms of the number of cases per year 4.8 million cases per year probably most of us on this this webinar may have had a case of salmonellosis for some from some food that we ate in the past the death rate fortunately is relatively low even though it is the most common of the three in terms of illnesses and outbreaks uh the death rate is relatively low most people um recover from this without needing medical attention there are some cases where people do become seriously ill with it though so it's just an interesting comparison again these are some numbers or some factors that you can use in in your risk evaluations for these pathogens in your food products let's keep going and looking at some of these other microbes we talked about yeasts for example grow as single cells like bacteria and they're going to form sort of a shiny colony on a petri dish sort of like a bacterium would they are much bigger than bacteria remember there were 13 000 e coli cells per inch there's 3 200 cells of yeast per inch still too too small for us to see but they are bigger compared to the uh to the bacteria um they're often fairly acid tolerant and they grow well in high sugar foods so if your food is one of those foods that has a more acidic ph and a fairly high sugar content this is one of those organisms that you have to think about particularly with regards to spoilage of course they can also be very beneficial right because some of the food products that we really love beer and wine and bread are produced by yeast cells producing you know the necessary changes in that food and growing in that food to create the flavors that we really like however of course if those yeasts are growing in a food that we don't want uh to be converted into something different it could be a spoilage issue and even within like beer wine or bread if you get the wrong type of yeast in there it can cause spoilage and it will often manifest itself in terms of you know gas production if it's a liquid product a sour or off flavor molds are related to the yeasts and these basically are growing as filaments which we call hyphae again very very narrow the individual height here like one five thousandth of an inch uh they produce a lot of different types of spores we talked about bacterial endospores before as being very resistant these are also fairly resistant they are not as resistant as the endospores but they can also cause some real challenges in terms of trying to control uh mold growth or mold spoilage on a plate the colonies often appear fuzzy or cottony and really what's going on is you have literally thousands and thousands of these little structures with the spores coming off of them they're very very tolerant of extreme conditions of acidity low moisture they can grow in conditions that you wouldn't really think anything could grow and some of them have what are called osmo tolerant capabilities they're able to grow an extremely low uh water activity or high sugar high salt foods molds are a really major source of food spoilage um they're one of the biggest pains in the neck for the food industry and for people trying to ship foods uh to get them to the consumer um they spoil the appearance of the purdue food and they produce a musty off flavor so we see the spoiled stone fruit to the to the left and to the right we see some walnuts that are actually a photo from a sample that i took in which the the nuts had been shipped the container that they were in had gotten wet during sea transport and this is this was the outcome so they can really uh wreck that food and make it unusable also they have the ability to produce some chemicals that are very toxic to animals to humans there are a wide range of them perhaps one of the most common ones we talk about is aflatoxin which is there uh in its molecular form on the lower right hand side of the the slide aspergillus flavius is the mold that typically produces that aspergillus parasiticus can also produce it um this toxin is um a carcinogen and is directly toxic to a number of different animals uh it was initially recognized in poultry because there was contaminated grain fed to turkeys and they died left and right and they called it turkey x disease and many years later we know a lot more about aflatoxin but it is a major uh major chemical contaminated or byproduct of these molds viruses again these are some of the organisms that don't grow in foods right so they're not growing in the food we're trying to control them through good agricultural practices and good handler practices viruses basically cause a variety of foodborne illnesses hepatitis a norwalk virus of course we all know about the covavirus that is a respiratory virus we've all been contending with in our lives viruses are very very small particles 850 000 virus particles per inch that's why it's so easy for someone to catch cold or covet or influenza from someone coughing or something being aspirated in the air because they're so tiny they can literally float on particles or droplets in the air and it doesn't take much uh in terms of viral contamination to cause disease 10 to 100 virus particles may cause a disease right so it really uh doesn't take much what happens the the virus particle will basically get into someone's body infection by taking over your host cells after you've ingested say contaminated food in this case if we're talking hepatitis or molar virus you eat that food the virus goes into your cells it says stop making copies of the cells and start making copies of me and it will make these copies of these viruses literally millions and millions of copies the cells erupt or explode or die releasing those viruses and continuing that cycle and of course in the meantime you can be shedding that virus and of course causing illness so in the food industry it really viruses are transmitted and cause disease through infected food handlers that's why food handling practices are so critical proper hand washing and disease control and that sort of thing the last one we talked about here in terms of groups of organisms are the protozoa these are also ones that are not going to grow in the food that can cause come from from contamination of that food these are basically parasites single-celled parasites or single-celled animals that cause foodborne disease again they're not growing in the food typically they come from fecal contamination either from humans or from animals i show the little watering can there because one of the vehicles for contamination is often irrigation water or spray water that was contaminated the spores or cysts are about the same size as a yeast cell and again these are sort of like those endospores we talked about are moles for is there a resistant structure that enables that organism to survive and get into the host one of the challenges is these cysts are often highly resistant to chlorine or other types of sanitizers and so there have actually been some interesting outbreaks in water systems uh city of milwaukee had an outbreak of cryptosporidium some years ago that caused a number of illnesses uh was traced to raccoons few animals uh basically getting into the water system and causing causing illness well it looks like we have a check going on here let me check this out all right we have a little question here when there's a spoilage we throw everything away or we can keep the fruit that's in good condition well that's a that's that's a good point that was one of the questions we had um it it kind of depends on what it is i mean certainly when you're dealing with um pathogens microbial pathogens uh if there's no good luck controls between the individual types of food products you really have to assume that that entire lot could be contaminated with the pathogen because you're at a really low risk or really high risk i should say zero tolerance uh type scenario i mean if you have a load of say stone fruit that comes in and there's some mold on that fruit some pieces are spoiled and some are not i don't think it's unreasonable to try and sort out the moldy product for example and try to salvage the sound product or clean product but of course you do have to be mindful of the fact that if some of that product is spoiled those spores could have been moving through the system and maybe cross-contaminated the other the other fruit of the other food products so you have to think about some way to possibly wash or decontaminate those or treat them with something that would reduce the chance of the growth of that mold so that's a good question spoilage it's you know it's more like getting rid of the spoiled product or what are those the contaminated um food items and getting those out of there you know trying to keep that mold spoilage or that you spoilage from moving through the product but with pathogens it's a whole different situation when you're doing your sampling for pathogens you really have to be aware that it takes so few cells to cause such serious illness in people that you really have to assume that whole load might be contaminated so good question okay so let's move right along here all right how do we control microorganisms in food there's a lot of key variety of variables or factors that we can use to influence the growth of bacteria yeasts and molds and if we modify those factors that can help us control or prevent microbial growth and spoilage so that is a lot about why we store things the way we do or why we treat things the way we do is to try and uh turn the odds in our favor um this little acronym is kind of fun fat tom i always think of the big fat tomcat sitting there um but basically these are some of the key variables that we can control and if we modify them appropriately we can try to prevent that spoilage or growth of microorganisms f starts food nutrients a for acidity or ph p for temperature t for time o for oxygen and m for moisture so those are the key variables those six key variables really can help us control microbes and food we'll talk for example looking at uh acidity of foods um the ph of foods is a scale that we use to ph uses a scale and everything to basically look at the acidity of something um ph seven and below is considered acidic ph 7 and above is alkaline it's the negative log of the concentration of hydrogen ions if you want to know about the science of it but from a simple standpoint anything below 7 is acidic and as we see there are relatively low acid foods where you might have higher growth so they're slightly acidic but not too much and we have for example some of those vegetables and corn potatoes that sort of thing as we go down into the higher acid foods where we're getting down to things like a plum or a raisin grate for example um we're getting into those lower ph's those higher acidities that are going to inhibit or destroy microbes or prevent them from growing so generally as the ph goes down we're going to see that microorganisms are going to be less effective at growing temperature we mentioned before as well is another factor most bacteria are growing are growing best at kind of warm temperatures majority of the bacteria that we're dealing with are called mesophiles or medium temperature growing microbes that growth is going to slow and ultimately stop when those temperatures are lower than the optimum so as we cool things off that's why we refrigerate a lot of our foods right it tends to slow down that microbial growth and of course if we heat things up so for cooking foods death tends to occur when those temperatures are 10 or 20 degrees fahrenheit above that optimum temperature for their growth right so that's the other side of the equation on temperature we heat foods or cook foods to kill microbes as well and these are very ancient methods uh that humans have used for years to try and make their food safer in the simplest terms keeping hot foods hot and cold foods cold is going to inhibit growth or prevent growth right so that's the main thing we look at with temperature a little bit about time and microbial growth we start with a single cell in 20 minutes typically under ideal conditions it can divide into two cells and this is how these things grow by binary fission one cell becomes two becomes four another 20 minutes we have four cells right and it goes on and on when those conditions are right the bacteria start to grow very fast after three hours or nine generations that one cell is turned into 500 and after 6 hours 18 generations you've got a half a million cells so under the ideal conditions microbes can really take off and grow very fast so we really want to reduce the time that that food is at the optimum conditions for microbial growth if we can do that then we have control hence the old term keep hot foods hot cold foods cold you know you don't want that cold food sitting out at room temperature for four or five six hours right because what could happen if there's microbes there that we're being controlled by that low temperature all of a sudden they're growing and they're just taking off and you know you're going to have a situation after you know it sat out all day long that you might have one cell that turned into a quarter million cells if that's salmonella you've got a big problem right we want to reduce that time that um the food is at optimum conditions for growth that's one of our key control factors another thing we can look at is is the level of oxygen it's interesting because some microbes are what are called aerobes they're like us we're air ropes we have to have oxygen you cut off our oxygen supply it's not going to be good for us right bacillus pseudomonas which is one of the common spoilage organisms you see when food gets kind of slimy if it's been in the fridge for too long it's typically pseudomonal growth those are air rubs so they must have oxygen just like us if we remove oxygen so we have like vacuum or modified atmosphere packaging we've taken out the oxygen that's going to reduce their ability to grow now some microbes grow without oxygen clostridium botulinum botulism right the whole idea of canning foods you put the food in there we we cook it it's sealed we've driven out the oxygen but we've also we've basically cooked this thing to the point that we've basically killed the organism that's in there and we sealed that thing up and so it's not going to be able to grow if um if you see for example a can that's starting to swell um that's something you shouldn't be eating right and why because we might have an anaerobe like clostridium that survived that cooking process and is actually starting to generate gas and grow inside and then you have some organisms that can go kind of either way yeasts and salmonella are a prime example of that they grow better with oxygen because oxygen is such a great fuel but they can also grow anaerobically sometimes as well it's an interesting thing oxygen is a very good fuel but it's also very destructive oxidative damage to biological molecules is a serious issue most organisms humans aerobic bacteria for example have enzymes in that in their biochemistry that basically enable them to destroy toxic byproducts of oxygen many of the anaerobic bacteria don't have that so when they're exposed to oxygen they die very readily so kind of an interesting interesting factor there moisture was another one of those things that we talked about as a control measure right typically we measure moisture as water activity and water activity really it's you're measuring how available is that water to be used for growth relative to pure water right so pure water has a water activity of one um and it's usually basically the percent moisture in a saturated chamber like that uh divided by a hundred so a scale of zero to one is your water activity most foods are above 0.9 so they're 90 of the way to pure water and really that is where many of the microbes are happiest growing most microbes are going to grow above 0.95 water activity as you start getting down into these lower drier conditions you're starting to see some bacteria inhibited right at 10 salt clustering botulinum is inhibited most pathogens salmonella listeria e coli are not going to grow above 0.85 down by 0.83 some of the yeasts are inhibited and if you go all the way down to 0.65 0.7 most yeasts and molds are inhibited remember we said that they can deal with dry conditions better and so as you get into those drier foods what are you going to see doing most of the spoilage it's typically molds and so really with dry foods that's what we're thinking about as we as we increase sugar we increase salt it also has the effect of drying that food it can either be dried just due to the lack of water or dry because there's a lot of sugar or salt there kind of binding up the water it has the same effect as dryness with regards to water activity it lowers it makes it harder for microbes to grow one thing to think about though if we're dealing with a dry food we're processing dry foods like tree nuts for example you need to practice what is called dry sanitation so you have to kind of keep the water out you have to use other practices vacuuming blowing wiping down with sanitizers and chemicals that basically don't have any water in them if we introduce water into a dry food it can actually make it more likely to support microbial growth so now that we've gone through some of these parameters let's think about how do we control microbial growth with regards to sanitation think about your facility microbes are going to enter with your personnel your raw products through pests that might get into the facility and so one of the first things you have to do is is put some control measures in place to keep those unintended yes those unintended microbes from getting into your facility right and these are some of the control measures that you probably have in your facility and this is one of the reasons you have them incoming material handling has to be done appropriately so that you don't increase your chances of contamination microbial contamination worker hygiene and practices right hand washing proper uh glove use proper smock use that sort of thing again proper use of restrooms this is all designed to keep the workers from unintentionally contaminating that food product proper equipment design and maintenance we have to maintain it so that there aren't places for material to build up and harborage or microbes to be um living or animals to be living you know if you have an area where insects are going to infest the facility they might carry pathogens with them this is all part of that design process of course cleaning and sanitation this is a an ongoing battle in any food facility as product goes through there and the day-to-day uh use of the facility we have to clean and sanitize it to try and get it back to a hygienic condition when we start the day pest control is critical both vertebrate tests uh insect pests again they can serve as vectors or conduits to bring microbes in and finally of course is in environmental monitoring which basically is a process where we're looking at how effective all of these these control measures are and we'll kind of end our day talking about a little about environmental monitoring this is basically where we're testing that processing environment for microbial contaminants and if we think about it it is the relative state of the raw ingredients the process itself and the processing environment they're going to determine the numbers and types of contaminants in that finished product so we're basically looking at all three of those things what ingredients do we start with what kind of process do we apply to those ingredients to reduce that risk and how can we keep our processing environment in such a state that we don't increase or add to that contamination why is that important it's basically your early warning system i show like a tsunami siren or an air raid siren there because is our sanitation program effective that's one of the first things you're going to be able to figure out by looking at that environmental monitoring program and you need to know that that is working properly another really important thing foods may not receive a kill step before going to the customer depending on when that food product is if it's a you know a fruit item for example uh it may just go straight to the customer and the customer may take it home and eat it right it may be a dried fruit they just open the package and eat it so that is very critical you have to understand what you're doing uh in terms of maintaining the cleanliness the hygienic nature of that facility because we don't want any contamination to land on that product before it's packaged and go to the consumer also it's your early warning system right just like those air raid sirens you find a problem early you take care of it before it causes a problem farther down the line because if you allow contamination to persist you can have spoilage issues food borne illness issues recalls all of which can be very costly and damaging not only to your company but to your consumers also we live in the age of fisma the food safety modernization act and environmental monitoring plays a key role in that and i would encourage everyone to look closely at that if they have not already it is a verification activity for your sanitation controls um it's an essential component of your plan for exposed ready-to-eat foods under the preventive controls rule the expectation is you will be looking to make sure that that environment stays hygienic as that food is exposed prior to final packaging pathogens of concern under the food safety modernization act include salmonella in low moisture ready to eat foods and listeria monocytogenes in high moisture ready to eat foods listeria does better under high moisture conditions again salmonella a very persistent organism under low moisture conditions when you start those programs you typically start with pathogens because they are the highest risk right those are the ones that can actually hurt people and fda of course is very very focused on pathogens that's their primary concern they're the folks that have to deal with foodborne illness outbreaks recalls etc but there are other areas and components of this too sanitation verification often includes other things non-traditional methods like atp or chemical means so those tools can also be very useful and of course you have to have some program management interpretation once you generate all of this data you need to know how to intelligently use it when you establish a program your first step is going to be picking your team and it's just kind of like your asset came when you design your house a plan you want sanitation your quality folks production maintenance any consultants that you might need to bring outside from the outside if you lack that expertise in-house you look at your process flows and you evaluate your risks looking for things like recontamination threats you know that finished product ready to eat going into the packaging is there a potential for recontamination with raw materials or or other environmental concerns you go through and identify some of the hygienic areas in your facility um are there any post-process pre-packaged areas basic gmps uh or their non-process areas they're all gonna have different levels of concern um if you have a post-process pre-package area that's probably your highest hygienic concern right because it's exposed right before going into finished packaging you really want that area to be very very clean and sanitary basic gmp areas the food production area prior to processing if you have like a kill step you want to make sure that you're not doing anything that increases the contamination of that product or makes it worse so you have to practice good manufacturing practices in that area non-process areas may be of lesser concern offices things of that nature or loading docks but you want to make sure that those areas still stay clean and don't serve as a hard bridge for things like pests once you've gone through and kind of done this evaluation you want to select your sampling sites and really i have this simple swabbing equation the greater the risk the greater the frequency in terms of the number of samples you take and how often you look okay the higher the risk the more you have to check it it's just common sense one of the things that helps people in site selection is the concept of zoning and this is well established in environmental monitoring so basically um if you look at your zones your highest sensitivity zone or zone one is the product contact sites so slicers peelers dicers hands if you have hand sorting going on and you can see the person doing swapping here on this bucket elevator for example so the direct contact with the product is your highest sensitivity that's your zone one zone two might be an area next to that so you see a person with a little q-tip style swab zone uh swabbing the edge of this piece of equipment next to the belt these are areas in close proximity if they're not sanitized properly or there's a harbridge spot there like a hollow roller or you know a weld spot that isn't smooth or there's a maybe an area where something was bolted on the bolts removed and you've got a hole in there that's an area where things might build up and you might have a hard bridge of organisms that could then contaminate zone one of the product zone threes are going to be farther out in that plant environment things like your floors walls drains forklifts things that are out in that general plant environment you probably don't clean and sanitize them as often as you do other areas but they can still be a potential source of cross-contamination and finally again into those were more remote zones like zone 4 and you see this woman swabbing out and looks like in a warehouse area on the floor again these areas are more remote from processing but we want to still keep them in fairly good repair and fairly clean because we don't want them to serve as a harbridge and potential for contamination that could then be stay tracked by a forklift into the process floor and maybe ultimately make its way onto the product when we sample and test there's a number of things we can do the samples can be a variety of things surface swabs both sponges and q-tip style dust or scrapings water or air samples as well can be taken of that plant environment again on your product sites zone ones you're typically looking for what they call indicator organisms like aerobic plate count coliforms the enterics you typically aren't looking for the pathogens they're often very difficult to find anyway and you want to be looking for something that will generate a number so that you have some sort of a metric to be able to look at how effective your sanitation programs are as you go out into the plant environment say zones two through four you're going to be looking for those pathogens right you're probably going to be large surface area samplings looking for salmonella listeria these are areas where the organism can become established on a daily basis we're probably cleaning zone ones very effectively if we're doing anything in our sanitation program we're focusing on zone one but that other plant environment the wall or the drains may not get the same attention and these are areas where those pathogens can actually start to accumulate maybe grow and it's more likely that we'll actually be able to detect them there the tools we would use include both those sponges and the q-tip style swabbing tools which actually have a medium on them to preserve that micro kill it gets to the lab we can also use scoops or spatulas or sample cups if we find a build up of product debris for example we may want to take that in and sample it as well when you do those sample collections you're typically working from zone one to zone four zone one is your most hygienic area so we don't wanna be trooping something from the loading dock all the way into the product contact site right we wanna work from the cleanest area out and your sampler should be practicing very good hygiene wash sanitize your hands put on sterile gloves before handling that swab you want to change gloves and or sanitize those gloves between swabs so that you're not cross-contaminating from one side to the next and remember the only non-sterile surface the swab should touch is the sample site we want to make sure that you're not sampling what's on your hand but sampling in fact what's in the facility again those sample collections can vary um typically we're sampling a larger area for pathogens versus the indicators because the indicators we're typically going to get some some numbers we're going to be counting those and and we'll see higher numbers with the pathogens again they're very difficult to find sometimes so you really want to be uh sampling a larger area if it's a zone one sampling site you're going to try to wipe that down with some sort of sanitizer um after you've done your sampling because there are chemicals on the swab to preserve it to keep those microbes alive so they get to the lab intact and we don't want those added to the food product so take an alcohol-based sanitizer or whatever sanitizer you use on your lines and clean that area after sampling again always submit a negative control swab just to make sure that that swab was in fact in good condition that we didn't have a contaminant on the swab itself and when you submit those samples you want to make sure that they are transported in a timely manner typically within 48 hours and kept cool until they can get to the lab for testing again just a quick summary of the number of samples and types of samples that you might take when you're doing this type of testing um pathogens are going to be out in zones two three and four and they're going to be uh typically done on a weekly or monthly basis um and there's probably going to be anywhere from 10 to 15 in zones two and three which are typically in that facility remote from the context from the contact cider from the zone one zones four those really remote areas like warehouses you know plant offices and that sort of thing you'll still do some monitoring but it may only be on a monthly basis zone one um it is often going to be post cleaning pre-sanitizer application is what they recommend looking for those indicator organisms and the number of samples you pull will kind of be line dependent depending on the product that you're using uh the process that you're using you really have to do your own kind of on-site risk assessment to figure out just how many samples are appropriate for that but from a microbiological sampling you probably want to do that at least weekly a little bit about some of the fisma program requirements procedures must be written and scientifically valid you have to identify the test organism that you're looking for either pathogen or indicator the location and number of test sites and it must be adequate to determine whether preventive controls are effective the test method used the testing lab identified they recommend using an accredited laboratory so our lab for example does iso 7.025 which is kind of the benchmark common standard for accreditation in food testing labs you want to make sure that that is the case with your lab and you want to identify the corrective action procedures you take if you find an issue two of the recurring programs we see issues we see with programs there are not enough samples being taken either there's too much time between the samplings or not enough samples per day or per zone and either one of those can potentially put your product at risk um you know because you may miss some key contamination issues in the facility if you're not taking enough samples when you're doing your sampling and if you're even if you're doing a lot of samples but you're leaving too much time in between your sampling events you could potentially see a situation where that second sampling event you have contamination and now you have to ask well how much of my product has been exposed you know you want to make sure you're doing this frequently enough so that if there is a contaminant you catch it right away and it doesn't impact as much of your product the other issue we commonly see is the positive results are not correctly dealt with um you cannot ignore finding a positive result especially on pathogens you have to do something immediately about that your sampling frequencies again you know sampling is is basically very intense to establish a baseline sometimes that might be anywhere from 25 to 50 swabs per zone per day for a month until you get enough data to get a baseline then once you have a baseline and you can kind of see what you can expect from your facility you can fall into that more routine sampling that we talked about before in that chart you also want to rotate your sights and allow your monitor some discretion and site selection you know so when that person is doing the monitoring of the swabbing they should be trained to understand the importance of what they're doing the basics of why they're doing it and you may want to test basically uh each sample site about four times a year because you can see some seasonal variations in contamination and that's important for you to know again a positive result for pathogens is you have to respond to that appropriately some of the typical corrective actions can include stopping production quarantine that area and the affected area if it's a product contact site like a zone one vector swabbing uh the site and adjacent sites if it's zone two or three is also something that you need to do so that's basically seeing where that could have spread to and how widespread the contamination is you may need to break down lines for inspection swabbing and cleaning um if it is a floor side for example you want to thoroughly clean that site maybe it's a 50 foot radius right around that area and then you increase your sampling frequency to daily until you get three negative results so you have a hit you go in there you do your vector swabbing immediate intensive swapping to see how far that contamination went you clean and sanitize that area swab it again if it worked great the next day same thing swab it again and you do that three times in a row and if you get three negatives you can be fairly certain that you've eliminated that potential side of contamination again positive result follow-ups if if zone ones were positive if you were doing investigations you did do swab swabbing for pathogens on zone one that product has to be placed on hold it can be reworked or condemned but if you do rework it it has to be a validated process only right you cannot be um just doing this and thinking that i think i killed it you typically have to make sure that it has gone through a validation you know that you're getting like a five blog kill if there's pop product that's contaminated often fda will weigh in and approve your reworking process testing alone is not a suitable means of clearing these products so you simply cannot just test your way out of this the levels of these pathogens can be so low and sporadic in a product that you might get a hit one time and then four four five or ten more tests are negative but that doesn't mean that it's still not there you may have just picked areas that were negative you cannot test your way out of that again documentation you want your procedures and methods your training records your assignment lists pre-operational inspection logs your corrective action records what did you do when you found a problem and you're holding release records and these are some of the general record-keeping requirements um again under the fisma regulation original records two copies are electronic accurate available legible contain actual observations and values concurrent with the activity identify the facility date and time of activity signature initials of the creator retain for at least two years and be retrievable within 24 hours of request a little about sanitation verification i know we're running a little late here and i apologize for that this is basically your pre-operational examination of the food processing equipment and facilities emphasizing zones one and two so the areas that are going to get the highest level of hygienic attention from the facility you're basically seeing if you're cleaning and sanitation have been effective your corrective actions are established to be taken if sanitation has been inadequate and it gives you documentation of your processes uh two ways that you can do this is simply looking and smelling right uh two of the oldest senses available looking for visible product residues or scraping for any kind of slime or biofilm layers smelling for any kind of spoilage or microbial growth there could be fermentation or rancidity if it's a high sugar product you might have fermentative spoilage which you can smell or if it's a high fat product there may be a kind of a rancid smell and that those are both indicators that there's probably product residues that aren't getting cleaned up in terms of the more technical methods of doing this we can use our microbiological swabs for our indicators we may even add some organisms as we're looking for sanitation we can look at yeast and mold as well because those are major spoilage organisms coliforms are ones that you can add to this because they are typically very easily killed by sanitizers so if your sanitizer is lost effectiveness uh you might see that with full thumb numbers and of course uh equal life you're dealing with tree nuts a lot of folks look for that one because there is a a limit as it were in uh tree nuts with regards to fda now microbial indicators are great and microbial verification techniques are an essential part of your your tool but waiting for those results can be a real disadvantage because the microbes have to grow that's why we have a number of chemical tools out there at our disposal the atp or bioluminescence technique you have about a limited detection of a thousand microbes from a microbial contamination standpoint but it also detects food residues as well allergen kits out there we have specific kits for different allergens we can also use protein swabs for allergens that we don't have a specific tip tip for again these chemical methods are great they're not super sensitive for microbes because they're so small but they're an excellent way to look for product residues and to see if sanitation has been effective um and it's the same phenomenon when you establish a verification program um you assemble your team you look at your sampling sites try to come up with your limits there may be industry standards uh that are available or you can collect your own baseline data based on how good the team thinks they can do in terms of cleaning and sanitizing set up your own routine monitoring typically it's weekly for the microbes right at least on a weekly basis see what your micro numbers you're doing and the chemical tools are often used after each sanitation cycle so that could actually be daily uh in your facility again if the results are above an acceptable limit that's a sanitation failure right a failing result requires a response the nice thing about those chemical tools we can go back repeat the sanitation and verification not only are atp or allergen or protein swabs but also our visual and sensory evaluation of those lines again that's a great powerful tool that you have there because you can actually go back fix the problem before any product goes over that line uh if there is a potential for product contamination you can have a product hold review those sanitation practices and product testing maybe if we need to before micro numbers indicate there's an issue we may have to do that again as with our environmental monitoring program documentation is critical success and sanitation really is going to be success in swabbing okay so you have to keep that in mind um sanitizing without cleaning is pointless i can't emphasize that enough if you have a dirty surface and you apply a sanitizer it's going to be inactivated by that soil before it ever kills the microbes hypochlorites are a prime example proteins in soil or in food products inactivate the hypochlorite it does not kill the microbe microbes can form a biofilm on a really dirty surface and so these are basically slime layers really resistant to sanitizers and you can see listeria in the in those scratches on that stainless steel surface and that one picture there which is kind of cool a biofilm is basically this big slime layer and it will you know microbes like to attach to surfaces even in the food production facility they'll produce the slime layer they'll stay attached periodically pieces of that will break off and you may see this in your product testing where suddenly you'll have enough you'll have no no counts a big count then no counts then another big count often that's a biofilm that's that's periodically breaking off and contaminating the product the scariest thing about biofilms is that they're highly resistant to sanitizers in this case for an hour and a half they soak this biofilm in a four part per million per million chloramine sanitizer solution and you can see through the staining technique they use the cells on the top are dead but the ones down below are still alive so if we wash that off and allow it to dry those red living cells on the bottom will recolonize that surface and you'll go right back to where you were with regards to contamination so we're at the end here again we apologize for our late start the concluding remarks environmental monitoring is really your early warning system it's going to measure the performance of your food safety programs and show regulators and customers that you're serious about food safety also it's going to provide you valuable legal protection because it will show that you are doing the best that you can you're doing your due diligence to produce a safe product and with that we are done so i thank you again for showing up here i thank you for persisting while we waited for you