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Michigan Department of Agriculture For an
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Nutrients
Atmosphere
Objectives
Appendices
This module is designed to provide information and demonstrate the application of basic microbiology. Participants will enhance their ability to identify potential food hazards, evaluate the adequacy of and discuss the proper control methods for these hazards. On completion of this module, participants will be able to: Microbiology is a broad term that covers the study of organisms that were not observed before the advent of the microscope. For our purposes in this course, this means bacteria, yeast, mold, viruses, and parasites. This module on general microbiology will address mainly bacteria but much of the information provided also applies to controlling yeast and molds. This course will focus on pathogens, those organisms that are capable of causing disease. Specific bacteria, virus, and parasites will be addressed. Foodborne illness takes quite a toll. There may be as many as 33 million cases of foodborne illness in the United States annually, with an estimated 9,000 deaths. Knowledge of microbiology is essential to you as sanitarians and your role in preventing foodborne disease. An understanding of microbial growth and the factors influencing growth will allow you to assess whether appropriate controls are present to prevent foodborne illness. Not all microorganisms are alike. While some are pathogens, others cause spoilage, which results in objectionable textures and odors in a food. And some organisms are actually beneficial; they are used to make products like cheese, bread, pickles, yogurt, beer and wine. Microorganisms are so small that most of them must be magnified about 1,000 times before they can be seen through a microscope. Using an average-sized bacterium as an example, about 1,000 could be placed side-by-side on the period at the end of a sentence. Consider a drop of milk. In spoiled milk there are about 50 million organisms per milliliter, or a total population of about 50 billion organisms in a quart. You can put all 50 billion in that one-drop. Yeasts and molds are collectively called fungi. These organisms grow under conditions in which many bacteria cannot, such as low pH and low water activity. Molds have many cells that make up a tangled mass of thread like structures called mycelium. Individual threads are called hypha. The most common molds grow by elongation of the hyphae and reproduce by fragmentation of the hyphae or production of spores. Spores are a dormant form of a microorganism that are generally formed in response to adverse environmental conditions. Some bacteria produce spores too, and some of these are of great significance in the food industry due to their highly resistant nature. While some molds are used in food processing, as in the manufacture of specialty cheeses such as Blue cheese, molds are also involved in food spoilage and some species produce mycotoxins, poisonous substances that can have serious health consequences. Yeasts are single cells and typically larger than bacteria. Most reproduce by budding. Yeasts are used to ferment wine and beer and leaven bread. Fortunately, they are not associated with foodborne disease but do cause spoilage problems in foods such as sauerkraut, fruit juices, syrups, molasses, jellies, meats, beer and wine. Bacteria are also single cells and generally come in two forms in foods spherical (cocci), or rod-shaped (bacilli). In addition, bacteria can be divided into two groups on the basis of their ability to form, or not to form spores. Spores are a dormant stage in the life cycle of the organisms. They are often compared to a plant seed that will germinate and grow when conditions are favorable. In general spores are extremely resistant to heat, cold, and chemical agents. Most food preservation techniques used by processors employ knowledge of factors that affect the growth of bacteria. Nutrients, temperature, water activity, pH, chemical inhibitors, and atmosphere all can be used to control growth. Each of these will be addressed individually. Factors Affecting Growth: Nutrients Inhibitors Temperature pH Water Activity Atmosphere Bacteria, like any living organism require food and water to carry on their life processes. Nutrients must be in solution before they can be transported into the cells, so water is essential. In general, bacteria also require sources of carbon, nitrogen, sulfur and phosphorous. Some microorganisms have the necessary enzyme systems to transform these few simple materials into the complex substances required for their life processes, while other microorganisms require certain preformed compounds. The specifics of the nutrient requirements and the actual mechanisms involved in nutrient transport are important and interesting areas of study. But, unless you are a microbiologist or biochemist the details of this can be rather complicated and tedious. From a practical standpoint, since microorganisms require nutrients to grow and proliferate, proper sanitation is essential to eliminate food residues, especially on food contact surfaces. Additionally, since microorganisms require nutrients to be in solution for transport into the cell, it is important that the food-processing environment is constructed to prevent the accumulation of standing water. Bacteria have phenomenal growth rates. They grow through a process of binary fission - splitting in two every 20 to 30 minutes under optimal conditions. We can follow bacterial growth through a 4-phase growth cycle. Lag Phase: This is the first phase, where cells may increase in size but the actual number of cells does not increase. Here the bacteria adjust their metabolism to the environment. This occurs when there is a drastic change in temperature or when the bacteria are moved from one medium to another. Logarithmic Growth Phase: Also known as the Log Phase. Cells actively divide by simple fission: one cell becomes two. During this phase the bacteria experience rapid exponential growth provided the necessary conditions of moisture, warmth and nutrients are present. The time required for a cell to grow and then divide into two cells is termed the generation time or doubling time. Stationary Phase: Cell numbers remain constant. Cell growth and cell death are in balance because they are beginning to deplete nutrients and accumulate waste products. Death Phase: Cell numbers begin to decline as a result of ongoing depletion of nutrients and the accumulation of toxic metabolic by-products. The lag phase is very important. If food is handled properly, the bacteria are kept in this phase and not allowed to multiply. Proper sanitation is important to limit available nutrients and thereby prevent bacterial growth. Another essential factor that affects the growth of bacteria is temperature. Microbial growth can occur over a wide range of temperatures from about 14°F to 194°F. Organisms are divided into three groups based on their temperature growth range:
It is not just the temperature that is the problem; it is the total time of exposure at these temperatures that needs to be controlled. The goal is to minimize the time of exposure of foods to temperatures in the mesophilic range. It is recommended that foods be kept below 40°F or above 140 °F. In many situations it may be impossible to completely avoid product exposure to mesophilic temperatures.
Table 1 Table 1 Principal Groups of Foods Based on aw
Foods can contain chemicals that are either natural or added that restrict or prevent the growth of microorganisms. Salt is a good example of an added chemical that can inhibit the growth of bacteria. Chemical preservatives like sodium nitrite, sodium benzoate and calcium propionate can also inhibit the growth of microorganisms. Another factor that can control the growth of bacteria is pH. pH is expressed as the negative logarithm of the hydrogen ion concentration. [pH = (-log of the [H+])] If you do not understand that, pH shows how acid a food is. Most bacteria don't grow very well in acid foods. pH ranges from 0 to 14, with 7 being neutral. Foods with a pH of 4.6 and below are considered acid foods, like most fruit juices. Foods with a pH above 4.6 are said to be low acid, like meats and vegetables.
Microorganisms can only grow at certain pH levels. Mold and yeast can grow over a broad range of pH. Bacteria are more restricted. Gram positive bacteria grow in a pH range of 4 to 8.5 and Gram negative grows between 4.5 and 9.0. So you can use pH to control the growth of bacteria. Food is considered to be in a safe pH range - or shelf stable - when the final pH is 4.6 or below. "Gram positive" and "Gram negative" are designations that microbiologists use to distinguish different types of bacteria. Different bacteria have different cell walls. To make bacteria stand out under a microscope, a stain is used. Bacteria with different cell walls take up the stain differently. Gram positive bacteria appear blue, and Gram negative appear red. This tells the microbiologist some things about the bacteria present in a food. For example, in the pH chart you can see that Gram positive bacteria are a little more tolerant of acid conditions. These bacteria are generally a little more resistant to heat too. Some of them are spore formers. In general, Gram negative bacteria include those that are associated with intestinal illness. That brings up something very important. That is how the bacteria associated with foodborne illness affect the body. It relates to how fast a person becomes ill after consuming a food and helps the inspector determine which food and which pathogen may have caused an illness. Some bacteria require a specific type of atmosphere for growth. Microorganisms are categorized as aerobes, anaerobes, facultative anaerobes and microaerophilic. Aerobes require oxygen and include such bacterial genera as Bacillus. Anaerobes grow only in the absence of molecular oxygen. These organisms include clostridia. Facultative anaerobes, which include most of the other food borne pathogens, can grow whether the environment has oxygen or not. Microaerophilic is a term applied to organisms, which grow only in reduced oxygen environments. It is important to understand that microbial pathogens are associated with all of the groups mentioned above. Knowledge of the atmospheres surrounding the food is an especially important consideration in determining which pathogens are likely to be a problem. Many of the organisms that spoil foods are aerobic. Technologies that are used to extend shelf life do so by altering the atmospheric environment of the food package to prevent the growth of aerobic organisms. These technologies include vacuum packaging, controlled atmosphere packaging, and modified atmospheric packaging. Most pathogens are facultative anaerobes so attempts to control spoilage by changing the atmosphere from aerobic to anaerobic or somewhere in between can be potentially dangerous because by eliminating the competing aerobic flora it can select for pathogens and inhibit those microorganisms that give the tell tale signs of spoilage. A product may not appear spoiled but may be toxic. There are two types of foodborne illness. One is an infection and the other is an intoxication. The bacteria that cause these are different. They're all considered pathogens but the way they make a person sick is different.
A foodborne infection occurs when the microorganism itself is ingested with the food. The organism establishes itself in the host's body and multiplies. Since the infection is a consequence of growth in the body, the time from ingestion until symptoms occur is relatively long.
A foodborne intoxication occurs when specific pathogenic bacteria grow in the food and releases toxins into the food that is subsequently consumed. It is the toxin that makes the person sick. Since the illness is a consequence of absorption of the preformed toxin by the intestinal tract, and not microbial growth in the host's body, symptoms of intoxication have a much more rapid onset than foodborne infections.
A third type is called toxicoinfection, which combines the two. Toxicoinfections are characterized by bacteria that are non-invasive and cause illness by producing toxins while growing in the human intestines. The times of onset are generally, but not always, longer than those for intoxications, but less than those for infections. Very often microbiologists test for indicator organisms as a substitute for testing for pathogens. The ideal indicator organism should be present when pathogens are present, absent when there are no pathogens, occur in greater numbers than the pathogens to provide a safety margin, and be easy to detect. Indicator Organisms: Fecal coliform Staphylococci Geotrichum candidum One group of indicator organisms is called the coliform group. Members of this group that grow at elevated temperature are called fecal coliforms. These organisms are found in the gastrointestinal tract of humans and warm blooded animals and have been used as an indicator of human fecal pollution in shellfish and their growing waters, as well as other food commodities. Other examples of indicator groups include staphylococci as an indicator of handling abuse, and Geotrichum candidum, the machinery mold, as an indicator of plant insanitation and contaminated equipment. In the laboratory, the microbiologist isolates and identifies the bacteria present. There are specific steps that must be taken to do this. Generally, there are three steps in detecting and identifying bacteria: Enrichment Selective agar Biochemical tests An enrichment medium is used to favor the growth of the organism you are looking for and to give it a chance to increase in number. You enrich the sample by placing the food product in a medium that has the nutrients discussed earlier that are specific to the type of organism you are trying to isolate. Then, you place a portion of the enrichment into a medium that selects for the desired organism. This medium contains some of the control mechanisms that were mentioned above (salt, an adjusted pH, or other chemicals or antibiotics) which will select for the organism you want, and not allow quite so many other organisms to grow. You then streak the selective medium onto an agar plate to isolate a pure colony, one that grew from a single cell. This pure colony is essential for subsequent tests since you need to deal with one organism at a time. The next step is to subject the isolated colony to biochemical tests that are specific to the type of organism you are looking for and that will confirm that you have the organism you think you do. That is the conventional method of looking for organisms. Each step requires time so it takes a while - usually several days - to get results from the laboratory. You also may hear references to "Aerobic Plate Count", also called "Standard Plate Count". This method provides an estimate of the total number of viable aerobic bacteria in a food, rather than a specific organism. It is generally used to determine food quality. In milk products, high counts may indicate that the milk was handled under insanitary conditions. This procedure is based on the assumption that each microbial cell in a sample will form a visible separate colony when mixed with an agar medium and permitted to grow. The food is diluted and placed in the agar medium in petri dishes so that the colonies can be counted. Microbial populations are at best an estimate. They are reported as Colony Forming Units or CFU per gram. Another approach to counting bacteria is a statistical method based on probability theory called the "Most probable number" or "MPN". The test material is diluted in a series of dilutions to reach a point where not even a single cell remains in the final dilution. If bacteria are present, the medium in the tube is cloudy, or positive. If no bacteria are present, the medium is clear. The pattern of positive and negative tubes at the different dilutions is used to estimate the concentration of bacteria in the original sample. The microbiologist compares the observed pattern of results with a table of statistical values. Some people are surprised that conventional methods take so long. They think everything is high tech now, providing instant results. Not so, traditional methods are still very much in use. However, it is true that recent advances in biotechnology have dramatically altered the diagnostic procedures used in microbiology. These new "rapid methods" provide simpler and often more sensitive and rapid detection of pathogens and their toxins. The term "Rapid Method" describes a large variety of detection and identification tests, including those that take a few minutes to perform to those that require days. Basically "rapid" means they're faster than conventional microbiological methods. The use of rapid methods in foods has some limitations. Foods are so complex, and each one's different. Proteins, fats, oils, and other factors can interfere with the tests. The normal bacteria in a food can also interfere with how well a test works. Low numbers of pathogens in foods are hard to detect. Processing of the food changes the bacterial flora and composition of foods. Most of these problems can be remedied by enriching the sample but that takes time which means it's not as rapid. Each method must be fully evaluated before it can be applied to food testing. There is a process for doing that but even so the rapid methods that are approved can be used only for presumptive screening of foods, a negative result stands but a positive result must be confirmed using standard methods. The Bacteriological Analytical Manual (BAM) contains all the laboratory methods used by FDA in isolating bacteria from foods. If you want to detect bacteria, rapid methods can only be used after the food sample has been through cultural enrichment. If you want to identify bacteria, rapid methods are used only after a pure culture isolate has been obtained from the sample. One type of rapid method is a "miniaturized biochemical identification" device. They are disposable devices that perform 15 to 24 biochemical tests at one time. They are designed to identify specific bacterial species. The microbiologist must work with a pure culture. Some provide results in 4 hours; most within 24 hours. These units simplify the conventional procedure by eliminating tubes and plate media. Other rapid method kits speed up standard microbiological methods by using special substrates, enzymes or other apparatus. For example, a Petrifilm plate count card contains prepared media. You just add your sample at the appropriate dilution and incubate it. You can then count the bacteria present in the sample. It is disposable and eliminates the need to make the agar plates we talked about earlier. With a positive MUG test kit a special chemical reaction alerts the microbiologist that the organism he's looking for is present. One type of MUG test kit is called a Colicomplete test. The discs are impregnated with two chemicals that react in the presence of coliforms and E. coli. You inoculate the tube, add one of the discs and incubate. If a blue color develops you have a presumptive positive for coliforms. You then shine a UV light on the tube. If the tube fluoresces you have a presumptive positive for E. coli. Some of the rapid methods involve using antibodies, nucleic acids or robotics to detect pathogens and toxins. Of these, the antibodies are most versatile and are used in various test kits. They take advantage of antibody-antigen interactions that are specific to a particular pathogen. A latex agglutination test works that way. If the reaction is positive, the latex beads cause the bacteria to clump. The ELISA test stands for "enzyme linked immunosorbent assay". It is another test that relies on antibody antigen interaction. The final result shows as a color change that can be easily read by the microbiologist. ELISA tests can be used to detect and quantify pathogens and toxins. A system called Polymerase Chain Reaction or PCR uses an enzyme to replicate a portion of a target pathogen's DNA. The reaction involves attaching a marker to the DNA so that it is easily detected. The advantage of this test is that you can detect very small numbers of a particular pathogen. Unfortunately, it does not differentiate between live and dead pathogens. These are only a few examples of what rapid method kits are and can do. The selection of a rapid method test kits depends in part on the organism of concern the food product being tested and the intended purpose of the test. Ward, D.R. 1997. "Basic Food Microbiology", Food Microbiological Control. FDA. Feng, Peter. 1997. "Rapid Methods", Food Microbiological Control. FDA
This section contains background information on specific foodborne bacterial pathogens. An understanding of the growth characteristics and sources of bacterial pathogens in foods is essential to conducting a hazard analysis of a food product. The following bacterial pathogens will be discussed in this section: Pathogenic bacteria
Enterovirulent Escherichia coli Group (EEC Group) Module 1 provided an overview of microbiology. This module will be specific and discuss various foodborne bacterial pathogens. They will be broken into two groups: gram-negative rods, and gram-positive rods and cocci. The pathogens within each group have some similarities in addition to the way they stain. For example, gram negative rods are nonspore formers and tend to have a fecal source. On the other hand, gram-positive rods and cocci can be spore formers and are typically associated with environmental sources like soil and sediments. The gram-negative rods that will be covered in this module are: Campylobacter jejuni, Yersinia spp., Salmonella spp., Shigella spp., Escherichia coli and Vibrio spp. , Plesiomonas shigelloides
The gram-positive rods and cocci that will be covered in this module are: Bacillus cereus, Listeria monocytogenes, Clostridium perfingens, Clostridium botulinum, Staphylococcus, Streptococcus, Aeromonas hydrophila, Miscellaneous enterics
On completion of this section, participants will be able to:
Unlike bacteria, viruses are not alive. Viruses are much smaller than bacteria and consist of a protein coat, which encloses a nucleic acid core. They are what are called "obligate intracellular parasites". The virus attaches to a susceptible cell and injects its nucleic acid into the cell. It takes over the host cell producing millions of new viruses and destroys the cell in the process. Viruses only infect a particular type of cell in a particular species of animal. So the ones we have to worry about only infect human beings. Only a small number are needed to make someone ill. When viruses are in a food, they are simply there and do not replicate or increase in number. Viruses are extremely persistent and may remain in a contaminated food for long periods of time. To increase the number of virus particles to make them easier to detect, you have to grow them in a susceptible host cell. Currently, there are no susceptible host cells other than humans for the viruses associated with foodborne illness so detection is difficult. Foods are contaminated with viruses in four major ways:
Sewage-polluted estuarine waters can contaminate fish and shellfish. Oysters, clams and mussels, which are filter feeders, entrap the pathogens from the water in their mucous membranes and transfer them to their digestive tract. If the shellfish is consumed whole and raw, so are the viruses. The surfaces of other estuarine species can also get contaminated but most of these are not consumed raw. In order to be a problem, they must be recontaminated after cooking by use of equipment or utensils that had been contaminated through contact with raw seafood or infected employees. Contaminated irrigation water can deposit viruses on the surface of fruits and vegetables. Again, it is generally foods that are consumed raw that are of concern. Viruses can be introduced if contaminated drinking water is used to wash or transport food, or is used as an ingredient in the food, or if you just drink it. Viruses can be added to food by infected food handlers with fecal material on their hands, a result of poor personal hygiene practices. Sometimes such people are noticeably ill, but other times they are without symptoms, and are just carriers of the virus. Ready-to-eat products such as bakery and deli items are of particular concern but virtually any food may cause illness if it is contaminated with human fecal matter containing the virus.
Pathogenic viruses that enter shellfish waters tend to accumulate in sediments, where they can persist for months. They survive better at lower, winter temperatures, which is when most of the shellfish are harvested for human consumption. Pathogenic viruses have been isolated from both "opened" and "closed" waters, and from shellfish harvested from each. Once taken in by shellfish, the viruses may persist for months. Illegal harvesting of shellfish from unapproved waters may exacerbate the problem of shellfish-borne viral illness. In clean seawater, contaminated molluscan shellfish naturally eliminate pathogens from their digestive tracts through normal feeding, digestion, and excretion. In a process called relaying, shellfish from contaminated waters are transferred to clean waters where they filter feed for a predetermined period of time to eliminate bacteria and viruses from their systems. In depuration, shellfish are placed in tanks with purified flowing water or circulating seawater and are allowed to filter-feed. Depuration conditions are closely controlled so the process usually takes two to three days, while relaying can take two or more weeks. Generally, the removal of viruses takes longer than removal of bacteria. So, elimination of bacteria is not a reliable indicator of viral elimination. Both Hepatitis A virus and the Norwalk agent are resistant to extremes of pH and are extremely stable at both refrigeration and freezing temperatures. There appears to be resistance to heat and radiation treatments as well. Most control measures have been evaluated in shellfish only. One interesting note is that shellfish tissue is quite protective and therefore pathogenic viruses are fairly heat resistant there. Transmission of human viral disease by consumption of cooked shellfish has been documented epidemiologically. Hepatitis A virus is still infective when treated at 133° F for 30 minutes in shellfish. Cooking conditions such as frying, steaming, baking, and stewing result in only a ten-fold reduction of viruses. Heat treatments necessary to completely inactivate viruses in shellfish generally result in a product that is organoleptically unacceptable. Other products that are heated to temperatures of 180°F should be free of the virus. On the other hand, chlorine is an effective agent to inactivate these viruses in waters, provided the water is relatively clear prior to chlorination. Control of viruses:
The most effective control for viruses is preventing contamination of food products in the first place. Shellfish must be harvested from waters that are not contaminated by sewage. Crops must not be irrigated with fecal contaminated water. Drinking water must be from a safe source, or properly treated. And employees must conform to hygienic practices. Vaccination for HAV is available to the general public. It has been suggested that HAV vaccination be required of all food handlers. Passive immunization with gamma globulin following exposure to HAV or in anticipation of possible exposure continues to be done. However this must occur in a timely manner. It does not provide immunity. It is expensive, and there are difficulties in identifying all exposed individuals. Norwalk and SRSV's immunity is temporary and vaccination efficacy will most likely be limited. Jaykus, Lee-Ann. 1997. "Viruses", Food Microbiological Control. FDA
On completion of this section, participants will be able to:
The following are the general concepts for this section.
This section will discuss some of the more important parasitic associated foodborne illness. The discussion will cover some of the most important illnesses caused by protozoa and parasitic worms including: At the completion of this section, participants will be able to:
Up to this point, microbiological hazards associated with food products has been discussed. But not all food hazards are directly caused by microorganisms, some are chemical hazards that are caused by byproducts from microorganisms or that occur naturally in the food source. The natural toxins discussed in this section include:
In fish, naturally occurring marine toxins present some unique food hazards. We need to be concerned with these toxins. The toxins found in fish are some of the most poisonous substances found on earth. Some are toxic at extremely low levels. In addition, many are heat stable and not normally destroyed by cooking. These toxins can be detected, but not easily. The presence of these toxins is usually detectable only through involved analytical methods. The affected fish look, smell, and often taste normal. Special attention needs to be paid to a group of seafood products called molluscan shellfish. Those include oysters, mussels, and clams. There are specific toxins that are associated with this group of filter feeders. The toxins that have been involved in human illnesses caused by shellfish poisonings include: Paralytic Shellfish Poisoning or PSP; Diarrhetic Shellfish Poisoning or DSP, Neurotoxic Shellfish Poisoning or NSP, and Amnesiac Shellfish Poisoning or ASP.
In addition to the toxins found in marine species there are naturally occurring toxins that are found in agricultural commodities. In general, these toxins are grouped under the heading of mycotoxins. Mycotoxins are produced by fungi, which wide spread in nature and therefore has the potential for appearing in most types of agricultural commodities. Not all fungus are toxic, and those that are only produce toxin if environmental conditions like water activity, temperature, availability of oxygen and other conditions are right. If all the conditions are correct, they can enter the foods directly, for example as a result of growth on a cereal grain like corn or wheat. Mycotoxins can also enter the food chain indirectly as a result of using a contaminated food ingredient for animal food. In that instance, the mycotoxins may be passed on into animal products like milk and cheese.
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