CALS Farm and Industry Short Course Program: Farm Microbiology: Notes

SECTION 5.
Food Microbiology
including the specialized areas of Dairy and Silage Microbiology

PART ONE – GENERAL FOOD MICROBIOLOGY

1. Importance of microorganisms in foods.
   
   
   1. Spoilage of foods.  Problem of contaminating organisms causing undesirable changes in food that may be improperly kept.
      
      
   2. Production of some foods.  Use of bacteria and other microorganisms to cause desireable changes in a raw product such that the finished product benefits by longer shelf-life and improved taste and appearance.
      
      
   3. Food-borne illnesses.  Pathogenic organisms transmitted via food and also toxins produced by microorganisms in food which may be ingested.
      
      
   4. Indicator organisms.  For example, finding coliforms may indicate a contamination problem; E. coli would indicate fecal contamination.
      
      
2. Food spoilage.
   
   
   1. Proteins, fats and carbohydrates.  Excellent substrates for a variety of microorganisms. Meats, fish, poultry, eggs and milk are examples of high-protein foods. Milk, fruits and vegetables contain sugars which can be fermented.
      
      
   2. Foods containing very high levels of sugar.  High concentrations of sugars can be inhibitory to a variety of organisms, but certain yeasts and molds may require such a high concentration in order to grow. Such organisms are called osmophiles and may contaminate jams, jellies, honey, etc. over a period of time.
      
      
   3. Dry foods.  Generally resistant to microbial activities. What little H₂O there may be can be saturated with solutes and thus be inhibitory. With increasing H₂O such as high humidity, molds tend to be the initial spoilage organisms.
      
      
3. Food preservation – i.e., prevention of spoilage.
   
   
   1. Principles.
      
      
      1. Keep microorganisms out.
         
      2. Inhibit microbial growth and activity.
         
      3. Removal of microorganisms.
         
      4. Kill microorganisms.
         
      5. Inactivate enzymes in foods.
         
         
   2. Methods to control microbial growth in foods.
      
      
      1. Keeping organisms out.  Keeping utensils and food preparation areas clean. Preventing cross-contamination between raw and cooked food. As complete a separation as possible of highly contaminated parts of animals (skin, intestines) from meat in slaughterhouses. Cleaning vegetables and fruits effectively. (Etc.)
         
         
      2. Removing organisms.  Filtration of liquid foods.
         
         
      3. Low temperatures.  Tend to inhibit microbial activity. Cold temperatures (including freezing) are not sterilization methods by any means.
         
         
      4. High temperatures.  Choice of a suitable time/temperature combination to kill pathogenic and spoilage organisms – depends on type of food, expected concentrations of organisms
         
         
      5. Drying.  Remember that H₂O is essential for microorganisms in that nutrients are taken in as a solution and all enzymatic activities take place in an aqueous environment. When H₂O is removed during the drying process, the concentration of solutes rises such that H₂O becomes no longer "available" to microorganisms, and the solutes come out of solution eventually.
         
         
      6. Inclusion of inhibitory chemicals in the recipe.  Salt (NaCl) is an often-used inhibitory ingredient.
         
         
      7. Inclusion of inhibitory chemicals in the basic food itself.  Many foods contain anti-microbial agents – e.g., benzoic acid in cranberries. A discussion of naturally-occuring antimicrobial agents can be found here.
         
         
      8. Addition of chemicals by microorganisms.  Acids are the most significant inhibitory agents – sometimes inhibitory to those that produce the acids!
         
         
      9. Ultraviolet and ionizing irradiation.
         
         
      10. Combination of methods and the Barrier Concept.  A combination of methods can be used to cause inhibition – by the synergistic effect of the various inhibitory agents with each other. Any agent may not be in high enough concentration to effect inhibition by itself (and it may cause some discomfort to the consumer if it were in such a high amount), but a combination of agents in lower concentrations may do the job. Called "barrier" or "hurdle" concept using the analogy of many small hurdles eventually wearing out a runner as would just one that is high enough. A good explanation of the barrier concept is here.
          

      A review of fermented food products and their additives can be found here.

4. Production of foods with the aid of microorganisms. (Fermented dairy products in next part.)
   
   
   1. Use of starter cultures vs. wild fermentation.  Starter cultures are added to effect a reliable fermentation in certain foods such as fermented milk products and many types of sausages. Some foods are best prepared by not adding any microorganisms – just letting the organisms that are expected to be in the raw food product do the job. This is a "wild fermentation" and is employed in the production of sauerkraut and some sausages.
      
      
   2. Sauerkraut.  This process also applies to cucumber pickles.
      
      
      1. Manufacture.  Cabbage leaves are shredded (discarding the core) and NaCl is added to a 2.5% total concentration and thoroughly mixed in. Considering that the cabbage leaves are about 50% H₂O and the NaCl is dissolved only in the H₂O (and microbial activity is only in the H₂O), the liquid component actually has about 5% NaCl which is inhibitory to many spoilage and disease-producing organisms. Mixture is placed in a container that will not admit air (oxygen). Eventual anaerobic conditions are also inhibitory to many organisms but not the desired fermenters which will function effectively in the 5% NaCl concentration.
         
         
      2. Obtaining anaerobic conditions.  This occurs when the oxygen is used up by respiration by the cabbage leaves and respiring microorganisms. This can take just a very short time – probably less than a day.
         
         
      3. Fermentation.  Sugar in the leaves is utilized as bacteria undergo fermentation, producing acid – the 3rd major inhibitory agent and the one most responsible for preservation of the product. (Acid and NaCl also contribute significantly to the taste.) During first few days, coliforms predominate and produce acid and gas; they then decrease in numbers as the acid increases. Leuconostoc is the "second wave," producing more acid (and gas) over the next several days – eventually becoming inhibited by the increasing acid. Third organism to achieve high concentration is Lactobacillus which continues acid production (lactic acid with no gas) which may drive pH below 4. (Both Leuconostoc and Lactobacillus are in the "lactic acid bacteria" group which are included in the table in Section 3.) When various kinds of bacteria participate in a process at different stages in the process – a certain organism predominating at a given stage – the term floral succession applies.
         
         
      4. Defects.  Sometimes may get a slime-forming strain of Lactobacillus. If not anaerobic, molds will readily grow on the surface – easily tolerating the high amount of acid. Sometimes may get a yeast which may impart some off flavors. Another yeast can cause a pink color.
         
         
   3. Silage.
      
      
      1. Phases of the fermentation/manufacturing process.  Process is similar to that of sauerkraut. A useful web page on the subject can be found here.
         
         
         1. Aerobic phase.  After the chopped forage material is thoroughly packed into the silo – which is then sealed – the O₂ is used up by microbial and plant respiration with the release of CO₂ and heat.
            
            
         2. Lag phase.  During this period, plant cell membranes break down, releasing sugars and other materials usable as nutrients for microorganisms.
            
            
         3. Fermentation phase.  Ideally, bacteria which produce acetic acid start off this phase, followed by lactic acid bacteria (usually Lactobacillus) at about the third day.
            
            
         4. Stable phase.  When so much acid has been produced that microbial activity ceases – usually after 3-4 weeks – this phase is achieved and the silage remains stable barring the entrance of oxygen.
            
            
      2. Defects.
         
         
         1. Due to too much oxygen.  Excess heat may be produced along with too much degredation of plant tissue with loss of nutrients. Very high temperatures cause browning and undesirable taste and texture.
            
            
         2. Due to silage being too wet.  Anaerobic conditions are achieved too soon, before desirable temperature for acetic acid-producing bacteria is reached. Organisms such as certain species of Clostridium which produce butyric acid can take over, resulting in repulsive taste and odor.
            
            
         3. Due to excess contamination by soil.  Enough Clostridium (a common soil organism) may be present such that the undesirable butyric acid fermentation can take place.
            
            
      3. Addition of NPN (non-protein nitrogen).  These are compounds such as urea and anhydrous ammonia (NH₃) which are meant to increase the nitrogen content of the silage by becoming nutrients for bacteria whose growth ultimately augments the overall protein content. More about this subject can be found here.
         
         
         1. At beginning of silage production process.  The pH starts a bit higher due to the alkaline effect of the NH₃, but microbial growth is enhanced and the final pH is still low enough to preserve the silage. On the surface, the additional NH₃ can be inhibitory to some microorganisms like molds.
            
            
         2. When fed to livestock.  Rumen bacteria use these compounds, resulting in additional microbial protein ultimately used by the animal.
            
            
   4. Fermented sausage.
      
      
      1. Manufacture.  Meat is mixed with NaCl, sugar and a small amount of nitrite. NaCl and nitrite inhibitory to a number of spoilage and disease-causing organisms – nitrite effective against Clostridium botulinum. Nitrite also helps in ultimate development of desirable color.
         
         
      2. Fermentation.  Initial addition of a starter culture (Lactobacillus or Pediococcus – both lactic acid bacteria) results in a more-reliable fermentation in which one expects considerable acid to be produced. Acid important in preservation, taste and "tightening up" the meat protein. – resulting in a relatively hard product. Acid also helps (with nitrite) in ultimate development of desirable color.
         
         
      3. Defects.  Too much nitrite can eventually cause a green discoloration. This can happen if nitrate is added and there is too much nitrate reduction to nitrite by indigenous microorganisms. Also, if starter culture is not added, there may not be enough indigenous lactic acid bacteria to carry on fermentation; less acid leads to more spoilage potential.
         
         
   5. Bread.
      
      
      1. Functions of yeast.  Ferment carbohydrates to CO₂ and ethanol. CO₂ necessary for the rising of the dough. Ethanol burns off in the baking process.
         
         
      2. Sources of flavor.  Yeast cells and their products can impart considerable flavor. (Nucleotide component of yeast is used as a flavoring agent.)
         
         
      3. Spoilage.  Chiefly molds.
         
         
   6. Some other examples.
      
      
      1. Green olives.  Similar fermentation to that of sauerkraut. Olives initially soaked in lye to modify the taste. Lye must not be washed out too much or soluble nutrients may also be lost.
         
         
      2. Vinegar.  Basically an alcoholic fermentation which has been allowed to oxidize – i.e., ethanol is oxidized to acetic acid by certain bacteria such as Acetobacter. Use of the word "vinegar" implies product made from fermented apples (also called apple vinegar or cider vinegar) unless modified otherwise – such as "wine vinegar" which is made from fermented grapes. Other substrates for fermentation and subsequent oxidation include various fruits, cereals, molasses, honey and coconut.
         
         
      3. Soy sauce.  Soybeans and roasted wheat are mixed and inoculated with a mold. Brine is added. Mold enzymes break down carbohydrates and proteins. Bacteria lower pH to around 5 which favors yeast growth and fermentation with production of ethanol.
         
         
      4. Alcoholic beverages.
         
         
5. Food-borne Illnesses.
   
   
   1. The Bad Bug Book.
      
      
   2. Definitions of food poisoning and food infection.
      
      
      1. Food poisoning.  Ingestion of toxin which had been made during microbial growth in the food.
         
         
      2. Food infection.  Actual growth of disease-causing organisms in the body after ingestion of sufficient numbers of them in a food product.
         
         
   3. Food poisoning (food intoxication).
      
      
      1. Botulism.
         
         
         1. The organism: Clostridium botulinum.  Gram-positive, strictly anaerobic, endospore-forming rod. Found in many soils.
            
            
         2. Types of botulism.  Various types of toxin produced during growth of the organism – given letter names (A, B, etc.); four types affect humans.
            
            
         3. Conditions for toxin production.  Growth of organism in the food (usually anaerobic canned product) with consequent toxin production. More of a problem with home-processed canned foods than those produced commercially.
            
            
         4. The disease.  Although first symptoms are gastrointestinal (nausea, vomiting, pain and abdominal distension), toxin is a neurotoxin and paralysis begins with face and eyes and moves downward toward chest and extremeties. Death caused by asphyxiation in 3-6 days in fatal cases.
            
            
         5. Prevention of outbreaks.  Sufficient pressure-cooking to destroy endospores and preclude growth and toxin production of organism. (Need minimum time and temperature.) Also, the toxin is heat-sensitive, so sufficient cooking will destroy toxin.
            
            
      2. Staphylococcal food poisoning.
         
         
         1. The organism: Staphylococcus aureus.  Gram-positive, facultatively anaerobic coccus. (Looks like bunches of grapes in gram-stained smear, but so do many other species of Staphylococcus and various other genera.) Growth generally associated with warm-blooded animals and humans. About one-third of general population has S. aureus growing in the nasal region.
            
            
         2. Conditions for toxin production.  Organism can grow with consequent toxin production in many foods – can break down proteins, fats, carbohydrates. Toxin will persist in food after population of organism diminishes.
            
            
         3. The disease.  Nausea, vomiting, cramps, diarrhea – usually within 2-4 hours after ingesting food.
            
            
         4. Prevention of outbreaks.  Keep infected persons away from preparing food – i.e., those with open sores and/or dribbling noses. Keep foods either "too hot" or "too cold" for growth – i.e., above 46°C or below 4°C. (Church picnics where food is kept outside on warm sunny days have been notorious for staphylococcal food poisoning outbreks.)
            
            
      3. Fungal toxins (mycotoxins).
         
         
         1. Aflatoxins.  Caused by Aspergillus flavus and A. parasiticus (molds). Foods affected are usually relatively dry foods (nuts, bakery products, etc.) although any food capable of supporting growth of these molds can subsequently contain the toxin. There can be carryover from field to food product, such as wheat to flour to pasta and bakery products. Toxin can cause toxic, mutagenic and carcinogenic effects on a wide range of organisms including humans, animals and plants.
            
            
         2. Mushroom toxins.  Responsible for many human cases annually.
            
            
   4. Food infections.
      
      
      1. Salmonella infections (human salmonellosis and other diseases).  Gram-negative, facultatively anaerobic rod belonging to the enteric group. Associated with many animals including reptiles. After ingestion of contaminated food and passage through stomach (where most cells may be killed by the low pH) bacteria infect epithelial cells of lower small intestine; inflammatory response releases fluid into bowel, causing diarrhea, the major symptom of salmonellosis. Occasionally enteric fever can develop when cells of some strains of Salmonella preferentially enter blood stream, ultimately invading tissues throughout the body and causing abscesses and shock and also little or no diarrhea; most severe enteric fever in humans is typhoid fever.
         
         
      2. E. coli infections.  Gram-negative, facultatively anaerobic rod belonging to the enteric group. Most strains are non-pathogenic and are common inhabitants of the intestinal canal. There are several diarrhea-causing types of E. coli – each affecting the small or large intestine and producing invasive or toxic effects ultimately producing a variety of symptoms. The "enterohemorrhagic" type produces fever, cramps and bloody diarrhea due to release of toxin into large intestine; most cases are caused by serotype O157:H7.
         
         
      3. Listeriosis.  Caused by Listeria, a gram-positive, non-endospore-forming rod. Found throughout nature in water and on plants. Can grow in refrigerated food. Cells enter bloodstream after contaminated food is eaten, causing fever, muscle pain and sometimes meningitis. Nonpasteurized milk and coleslaw are often implicated. As with any foodborne disease pathogen, one must exercise care in handling food (cleaning and cooking thoroughly, preventing cross-contamination between raw and cooked product, keeping either "too hot" or "too cold" for microbial growth, etc.)
         
         
      4. Infant botulism.  Intraintestinal growth of Clostridium botulinum with toxin production. Organism may came from endospores in house dust and honey.
         
         
      5. Hepatitis.  Several types, each caused by a distinctly different virus. As name of disease implies, liver damage is a significant part of disease. Hepatitis A spread by the fecal-to-oral route via water, food (including raw shellfish) and hands; symptoms include fatigue, fever, nausea and jaundice with full recovery usually taking place within 2 months (sometimes up to 6 months).
         
         
      6. Trichinosis.  Caused by roundworm, Trichinella spiralis that lives in the small intestine of meat-eating animals. Disease contracted by eating improperly-cooked meat (pork implicated most often) from which the roundworms penetrate host's tissues; abdominal pain and diarrhea persist for about a week and are followed by fever, muscle pain and facial swelling.
         

PART TWO – DAIRY MICROBIOLOGY

1. Milk.
   
   
   1. Definition of milk.  Liquid secreted by mammary glands of female mammals to nourish their young for a period soon after birth. Must not contain colostrum which is what appears immediately after birth. In US, the word "milk" implies cow's milk.
      
      
   2. Average gross composition of milk.  (Percentages are shown for cow's milk.)
      
      
      1. Water.  87.3%. (By comparison, reindeer milk contains 63.3% water.)
         
         
      2. Fat.  3.7%. (Reindeer milk contains 22.5% fat!) Fat appears as globules which are suspended in what is otherwise a solution. Globules rise to the top and form the cream layer. If skim off the cream or otherwise remove it, resulting milk is called skim milk if it contains less than 0.5% fat. If it contains 1-2% fat, it is called low-fat milk.
         
         
      3. Lactose.  4.9%. Disaccharide composed of glucose and galactose. This sugar is found nowhere else but in milk.
         
         
      4. Proteins.  Casein (2.9%) and albumin (0.5%).
         
         
      5. Ash.  Minerals remaining after incineration (0.7%). Includes compounds of calcium and potassium.
         
         
      6. Minor constituents.  Includes B vitamins and also C and D. Milk is usually fortified with A and D for sale.
         
         
   3. Nutritional value.  Enough to satisfy all nutritional needs for infants of the species.
      
      
   4. Keeping quality (shelf-life).  Like leaving a bacteriological medium exposed to the air. Will quickly get contaminated with fast-growing organisms.
      
      
2. Sources of microorganisms in milk.
   
   
   1. Interior of the udder.  Considered sterile in healthy animals except near the tip.
      
      
   2. Exterior of the udder.
      
      
   3. Coat of the cow.
      
      
   4. Utensils.
      
      
   5. Air.
      
      
   6. Flies and other insects.
      
      
   7. Humans.  Can serve as source of disease-producing microorganisms.
      
      
3. Changes in milk resulting from microbial growth.  Note that pasteurized milk can be easily recontaminated. In fact, spoilage is usually caused by organisms recontaminating the milk after pasteurization.
   
   
   1. What happens if milk is allowed to spoil.  There is usually a 4-stage microbial succession over the following two weeks: (1) First Streptococcus and (2) then Lactobacillus ferment the lactose and produce acid. (3) Yeasts and molds take off at resulting the low pH and consume some of the acid, raising the pH. Then, at a more moderate/neutral pH level, (4) Bacillus and other protein degraders predominate and the milk becomes watery (less milky in appearance) as the protein is hydrolyzed.
      
      
   2. Fermentation.  Acid generally produced by the lactic acid bacteria, causing a sour taste and eventually precipitation of the casein (curd formation). Sometimes gas is also produced by these and other organisms (CO₂ or a combination of H₂ and CO₂).
      
      
   3. Proteolysis.  Breaking down of the casein to peptides and individual amino acids, resulting in a watery condition.
      
      
   4. Alkali production.  Production of ammonia and amines from decomposition of amino acids.
      
      
   5. Ropiness or sliminess.  Slime-forming bacteria sometimes produce a ropy condition where the slime can be drawn out to a considerable length.
      
      
   6. Changes in butterfat.  Lipolytic (fat-degrading) organisms which convert fats to fatty acids.
      
      
   7. Flavor and aroma changes.  Acid production from fermentation causes a sour taste. Environmental coliforms developing into a high concentration can cause a fecal aroma. (Fecal aroma can also be absorbed into milk directly from the environment.) Fatty acids can be degraded to produce a rancid taste.
      
      
   8. Color changes.  Certain organisms have been known to produce green (Pseudomonas), red (Serratia) and purple (Chromobacterium) coloration.
      
      
4. Procedures to reduce or minimize changes in milk.
   
   
   1. Asepsis.  Minimizing contamination before and after pasteurization.
      
      
   2. Use of low temperatures.  Keeping milk just above freezing is best (0-4°C).
      
      
   3. Use of heat.
      
      
      1. Pasteurization.  Time and temperature combination necessary to kill pathogens and also significantly reduce spoilage organisms – 63°C for 30 minutes or higher temperatures for shorter times.
         
         
      2. Boiling and steam under pressure (autoclaving).  Done for very short time, probably not long enough to reduce endospore numbers.
         
         
   4. Other methods of preservation.
      
      
      1. Condensing.  Removing much or most of the water. Microorganisms are inhibited by the increased solute concentration.
         
         
      2. Drying.  Removal of all water with cessation of bacterial metabolism and reproduction.
         
         
      3. Adding preservatives.  As a rule for commercially-available milk, this is not done. Milk is usually shown as consisting only of milk, perhaps with added vitamin(s).
         
         
      4. Preservation by fermentation.  Practiced for thousands of years, probably originating with attempts to duplicate conditions where a wild fermentation happened to result in a pleasing, long-lasting product. Art became a science in recent centuries.
         
         
5. Determining bacteriological quality of milk.
   
   
   1. Standard plate count.  Inoculating petri plates of a standard all-purpose medium (such as "Plate Count Agar") with known amounts of various dilutions of milk samples. Maximum numbers of colony-forming units per ml allowed such as 100,000-300,000 for raw milk and 20,000-30,000 for pasteurized milk. Higher levels allowed for milk used in cheese-making.
      
      
   2. Coliform count.  Done with appropriate media as discussed in Section 7. Maximum allowable number per ml in pasteurized milk is very low (10 or less).
      
      
   3. Direct microscopic count.  Can count certain number of microscopic fields and then multiply by appropriate factor to get no. of cells per ml.
      
      
   4. Dye reduction tests.  Time it takes to reduce (decolorize) an indicator dye such as methylene blue and resazurin decreases with an increased population of organisms. Can relate time to approximate numbers.
      
      
   5. Tests for abnormal (mastitic) milk.  Here one is detecting somatic cells in the milk which result from mastitis which is defined as the reaction of milk-secreting tissue to injury produced by bacteria, chemicals or physical force.
      
      
      1. The California and Wisconsin Mastitis Tests and the Modified Whiteside Test.
         
         
      2. Catalase test.  Somatic cells contain the enzyme catalase which will convert H₂O₂ to H₂O and O₂; the latter is detectable by its bubbles.
         
         
6. Presence of foreign chemicals in milk.
   
   
   1. Antibiotics.
      
      
      1. Sources of antibiotics in milk.  From purposeful treatment of mastitis or indiscriminate use not based on careful assessment of need.
         
         
      2. Detrimental effects of antibiotics in milk.  Allergies in consumers. Leaving antibiotic-resistant microbial cells to continue and predominate while the sensitive cells are inhibited or killed.
         
         
      3. How can the problem be minimized?  Use antibiotics only when appropriate. Let withdraw from animal before milk is taken.
         
         
   2. Pesticides of the chlorinated hydrocarbon type.  These include dioxin and DDT.
      
      
      1. Sources of pesticides in milk.  From treatment of dairy cattle and barns and also ingestion of treated feeds.
         
         
      2. Where pesticides accumulate.  Milk fat and tissues.
         
         
      3. Detrimental effects of pesticide residues.  Weight loss and other toxic effects which can be passed along in milk.
         
         
      4. How can the problem be minimized?  Treatment only as really needed. Allowing time between withdrawal of pesticide from animal and availability of milk – but this may take too long.
         
         
7. Examples of cultured dairy products.
   
   
   1. Yogurt.
      
      
      1. Preparation of the milk.  Milk is fortified with additional dry milk and heated to partially denature the protein. Denaturation of protein leads to a more even curd and less expression of whey.
         
         
      2. The fermentation process.  Cooled, "fortified" milk is inoculated with 2 or 3 species of lactic acid bacteria (usually Streptococcus thermophilus and Lactobacillus bulgaricus) and allowed to incubate for several hours at a relatively high temperature (45°C). Rapid growth of organisms produces substantial acid which coagulates the protein, forming a curd.
         
         
   2. Cheeses.
      
      
      1. Solidification of protein accomplished by:
         
         
         1. Acid from lactic acid fermentation.  One or more lactic acid bacteria allowed to ferment the lactose.
            
            
         2. Addition of coagulant (rennet).
            
            
      2. Variety of cheeses due to:
         
         
         1. Variety of organisms used for fermentation and/or flavoring.
            
            
         2. Sources of milk.
            
            
         3. Different aging processes.
            
            
      3. Examples.
         
         
         1. Swiss cheese.  Addition of Propionibacterium is responsible for development of propionic acid and holes (from CO₂ bubbles).
            
            
         2. Blue cheese.  Addition of the mold Penicillium roqueforti (which grows along channels in the curd made by piercing needles) results in the blue marbled appearance and the flavor of ketones produced by the mold from fatty acids.
            
            
8. Probiotics.
   
   
   
   

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Outline for this section. Farm Microbiology Home Page. CALS Farm and Industry Short Course Home Page. Bacteriology Department Web Site.

Page last modified on 3/7/04 at 6:30 PM, CST. John Lindquist, Dept. of Bacteriology, University of Wisconsin – Madison