Microbiological Media for the

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A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

H A N D B O O K O F

Microbiological Media for the

Examination of Food

By

R O N A L D M . AT L A S

S E C O N D E D I T I O N

Boca Raton London New York

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Published in 2006 by CRC Press

Taylor & Francis Group

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International Standard Book Number-10: 0-8493-3561-2 (Hardcover) International Standard Book Number-13: 978-0-8493-3561-7 (Hardcover) Library of Congress Card Number 2005054896

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Library of Congress Cataloging-in-Publication Data

Atlas, Ronald M. 1946-

Handbook of microbiological media for the examination of food / by Ronald M. Atlas.--2nd ed.

p. cm.

Includes bibliographical references and index.

ISBN 0-8493-3561-2 (alk. paper)

1. Food--Microbiology--Laboratory manuals. 2. Culture media (Biology)--Laboratory manuals. I. Title:

Microbiological medica for the examination of food. II. Title.

QR115.A84 2006

664'.001'579--dc22 2005054896

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Preface

Increased emphasis on the microbiological safety of foods has led to greater testing of foods for potentially pathogenic microorganisms. Careful quality control in the food industry has become essential for preventing outbreaks of foodborne disease and for controlling food spoilage. Many foods are routinely examined for the presence of disease-causing and food spoilage microorganisms. Many of these quality control procedures involve the cultivation of microorganisms using a variety of media that are described in the second edition of the Handbook of Microbiological Media for the Examination of Food.

Given that an estimated 14 million cases of foodborne disease with known microbial pathogens occur every year in the United States alone, the Food and Drug Administration and U.S. Department of Agriculture have been implementing new regula- tions and guidance for the microbial testing of a variety of foods. Similarly, Europe and other regions of the developed world are implementing stricter oversight of food safety to ensure that foods are free of potentially dangerous pathogens, e.g., Salmonella spp, Vibrio spp., Listeria monocytogenes, Campylo- bacter jejuni, etc.

Emerging diseases caused by foodborne pathogens, such as Escherichia coli O157:H7, that have high mortality rates have raised special concerns. This has encouraged the development of new media for the cultivation of bacteria, fungi, and viruses that can aid in the rapid detection of microorganisms in foods and the protection of the food supply. Many of these new media include chromogenic or fluoro- genic substrates that permit the specific rapid detection of pathogens of concern.

The second edition of the Handbook of Microbiolog- ical Media for the Examination of Food provides descriptions of the media used to cultivate microorganisms from foods. Almost 1,400 media are described in the Handbook, including all the media recommended by the Food and Drug Administration for the detection of microorganisms in foods as well as media used elsewhere in the world. The Hand- book includes descriptions of the media used to cul- tivate microorganisms used for food production as well as those that are important for quality control, i.e., the cultivation of potential pathogens and food- spoilage microorganisms.

The second edition of the Handbook of Microbiolog- ical Media for the Examination of Food compiles in one place in a consistent style the formulations,

methods of preparation, and uses of each medium. It lists the common synonyms for the various media and the commercial sources when they are available.

The index provides a convenient tool for finding the media by names and application for the detection of specific microorganisms of interest.

Importantly, the Handbook of Microbiological Media for the Examination of Food includes descriptions of expected results as they apply to microorganisms of importance for the examination of foods. The compositions of various media can be compared so that alternate media can be used. Simi- lar or identical media described by different names that actually have the same formulations can be readily identified.

Using the second edition of the Handbook of Micro- biological Media for the Examination of Food should save time and effort for anyone cultivating microorganisms. The media are organized alphabeti- cally. Each medium includes the composition, instructions for the preparation, and uses of each of the media.

The information needed to prepare media for the cultivation of microorganisms can easily be found in the Handbook. It should be a valuable resource for anyone working in the area of food microbiology.

About the Author

Ronald M. Atlas is Professor of Biology at the Uni- versity of Louisville. He received his B.S. from the State University of New York at Stony Brook in 1968 and his M.S. and Ph.D. from Rutgers in 1970 and 1972, respectively. Dr. Atlas has received a number of honors including: The University of Louisville Excellence in Research Award, the Johnson and Johnson Fellowship for Biology, and the ASM Award in Applied and Environmental Microbiology.

He has taught a variety of courses in microbiology including food and industrial microbiology and has authored several microbiology textbooks. He has conducted studies on the use of gene probes and the polymerase chain reaction for the detection of pathogens and indicator bacteria in water and food.

He has served on the editorial boards of Applied and Environmental Microbiology, BioScience, Bio- techniques, and Journal of Industrial Microbiology.

He is editor of CRC Critical Reviews in Microbiol- ogy. He also has served as President of the Ameri- can Society for Microbiology.

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Introduction to Food Microbiology...1

Food- and Waterborne Pathogens ...1

Food Spoilage ...6

Microbiological Production of Food ...7

A-1 Medium...11

AAM Medium...11

Abeyta-Hunt Bark Agar...11

ABY Agar ...12

AC Agar...12

AC Broth...12

Acetamide Agar ...12

Acetamide Agar ...13

Acetamide Broth ...13

Acetamide Medium...13

Acetate Agar ...13

Acetate Broth ...14

Acetate Differential Agar...14

Acetobacter Agar ...14

Acetobacter/Gluconobacter Agar ...15

Acetobacter Medium...15

Acetobacter xylinum Medium...15

Acetogen Medium...15

Achromobacter Choline Medium ...16

Achromobacter Choline Medium, Modified...16

Achromobacter Medium ...16

Achromobacter Medium ...17

Acid Broth...17

Acid Egg Medium...17

Acid Products Test Broth ...17

Acid Tomato Broth ...17

Acidic Tomato Medium for Leuconostoc ...18

Acidophilic Bacillus stearothermophilus Agar ...18

Acidophilic Bacillus stearothermophilus Broth ....18

Actidione Agar...18

AE Sporulation Medium, Modified ...18

Aeromonas Differential Agar...19

Aeromonas hydrophila Medium ...19

Aeromonas Medium...20

AFPA...20

Agar Medium for Differential Enumeration of Lactic Streptococci ...20

Agar Medium P...21

AK Agar No. 2...21

AKI Medium...21

Alcal Mannose Medium...22

Alcaligenes Agar...22

Alcaligenes Medium ...22

Alcaligenes N5 Medium ...23

Alcaligenes NA YE Medium ...23

Alcaligenes NB YE Agar...23

Alcaligenes NB YE Broth...23

Alcaligenes NB YE Medium ...23

Alginate Utilization Medium ...23

Alkaline Bacillus Medium...24

Alkaline Peptone Agar...24

Alkaline Peptone Salt Broth ...24

Alkaline Peptone Water ...24

Alkaline Peptone Water ...25

Alkaline Polypectate Agar...25

Alkaline Starch Agar ...25

Alkaline Xylan Agar...25

Alkalophile Medium...25

Alkvisco Medium ...25

Allantoin Agar ...26

ALOA Medium...26

ALOA Medium...27

Amino Acid Assay Medium ...28

Anaerobe Agar...28

Anaerobe Agar...29

Anaerobe Medium ...29

Anaerobic Agar...29

Anaerobic Agar...30

Anaerobic Broth...30

Anaerobic CNA Agar ...30

Anaerobic Egg Yolk Agar...30

Anaerobic Egg Yolk Agar...31

Anaerobic TVLS Medium ...31

Andersen’s Pork Pea Agar ...31

Andrade’s Broth...32

Andrade’s Carbohydrate Broth and Indicator...33

Anthranilic Acid Medium, Revised...33

Antibiotic Medium 1...34

Antibiotic Medium 1 with Tetracycline...34

Antibiotic Medium 1 with Tetracycline, Streptomycin, and Chloramphenicol...34

Antibiotic Medium 3 Plus...35

Antibiotic Sulfonamide Sensitivity Test Agar ...37

AOAC Letheen Broth ...38

Aolpha Medium ...38

Apple Juice Yeast Extract Medium ...39

Introduction to Food Microbiology

Food products serve not only as sources of nutrition for humans and other animals but also as substrates for the growth of microorganisms. The uncontrolled growth of microorganisms in food causes spoilage, a serious problem accounting for sizable losses of food products. Some disease-causing microorganisms also contaminate foods and potable waters.

Foodborne and waterborne pathogens remain a major source for the spread of disease worldwide. A major concern in food microbiology is the preserva- tion of food products and the prevention of foodborne and waterborne spread of disease. Careful quality control in the food industry is essential for preventing outbreaks of foodborne disease and for controlling food spoilage. Many foods are routinely examined for the presence of disease-causing and food spoilage microorganisms. Many of these quality control procedures involve the cultivation of microorganisms using a variety of media.

Food- and Waterborne Pathogens

The ingestion of foods containing toxins and human pathogens can cause a variety of diseases. It is essential to prevent contamination of food products with human pathogens and to control the potential proliferation of toxin-producing microorganisms, which can result in food poisoning and the transmission of foodborne pathogens. In some cases, the growth of pathogenic microorganisms in a food is accompanied by obvious signs of spoilage, such as the production of gas or foul-smelling compounds, which give a clear indication that the product should not be eaten. In other cases, there are no obvious signs of spoilage to indicate the presence of pathogens or toxins. It is therefore necessary to carry out inspection programs aimed at detecting the possible contamination of food products with pathogenic microorganisms. As a consequence of the high quality control procedures employed in the food industry, few occurrences of foodborne disease are traced back to the food processor. Rather, most outbreaks of food poisoning are caused by improper postpro- cess handling of the food, often as a result of improper preparation and/or handling at home or in a food service setting.

Quality control laboratories in the food industry routinely perform tests to detect pathogens in food products and to ensure that the numbers and types of microorganisms associated with a food are not likely to cause serious food spoilage and/or health prob- lems. In some cases, quality control procedures are aimed at detecting the presence of specific microorganisms, but in most cases the tests consist of exam-

ining the food for indicator organisms. For example, coliform counts are routinely performed on repre- sentative samples of many food products as an indication of possible fecal contamination, since food contaminated with fecal material has a relatively high probability of containing human pathogens.

Foods such as hamburger often have 100,000total bacteria per gram, but as long as they do not contain any Salmonella or other pathogens, they are consid- ered safe for human consumption.

In the United States, agencies involved in the sur- veillance and regulation of foods are the Department of Agriculture (USDA), the Food and Drug Admin- istration (FDA), and state boards of health. Stan- dards based on the presence or absence of pathogens, such as those set by Congress and regulated by the USDA and FDA, do provide the safeguards needed for assurance of the safety of food products.

On the international front, standards for the microbial testing of foods are established by the Interna- tional Organization for Standardization (ISO). Both the FDA and ISO standards specify the media and procedures that are to be employed for the microbial analyses of specific foods.

Microorganisms routinely enter the gastrointestinal tract in association with ingested food and water. The large resident microbiota that develops in the human intestinal tract after birth is important for the mainte- nance of good health and is usually not involved in disease processes. This microbiota is normally nonin- vasive and is associated with the surface tissues and ingested food material. Some pathogenic microorganisms, however, possess toxigenic or invasive proper- ties that permit them to cause disease when they enter the gastrointestinal tract.

There are two distinct processes that can initiate disease through the gastrointestinal tract. In the first type, microorganisms growing in food or water can produce toxins, and their ingestion initiates a disease process. Such diseases are classified as food poison- ing or intoxication because the etiological agents of the disease need not grow within the body; that is, there is no true infectious process. Toxins absorbed through the gastrointestinal tract can cause neural damage and death in some cases, as well as localized inflammation and gastrointestinal upset in others. In the second type of disease-causing process, invasive pathogens establish an initial infection through the gastrointestinal tract and cause localized gastrointestinal upset or systemic disease symptomatology.

Generally, the establishment of infection through the gastrointestinal tract requires a relatively large infec-

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2 Food- and Waterborne Pathogens

tious dose; that is, a relatively large number of pathogenic microorganisms is required to success- fully overcome the inherent defense mechanisms of the gastrointestinal tract. Quite different measures are required to prevent and treat infectious gastrointestinal diseases compared to those for specific microorganisms responsible for food poisoning.

Many outbreaks of gastroenteritis involve the foodborne or waterborne transmission of viruses that can replicate within the human gastrointestinal tract and cause an inflammation of the lining of the gastrointestinal tract. Contamination of food with fecal matter is an important route of transmission of viral gastroenteritis. Several different viruses cause viral gastroenteritis, including adenoviruses, coxsackievi- ruses, polioviruses, and members of the ECHO virus group. The Norwalk agent, a small DNA virus identified as being responsible for an outbreak of “winter vomiting disease” that occurred in Norwalk, Ohio, in 1968, appears to be an important etiological agent of various viral gastroenteritis outbreaks. Rotavirus, a large RNA virus, also appears to be a very common etiological agent of diarrhea in infants, particularly in socioeconomically depressed regions of the world. It is difficult to detect the diversity of viruses causing this disease and foods and waters are only rarely examined for their presence.

Hepatitis is a systemic viral infection that primarily affects the liver, caused by several types of hepatitis viruses, designated types A, B, C, D, and non-A, non-B. Type A hepatitis virus is normally associated with foodborne and waterborne cases of hepatitis.

Type A hepatitis virus normally enters the body via the gastrointestinal tract and causes infectious hepatitis. Although there are some documented cases of foodborne hepatitis B, types B, D and non-A, non-B hepatitis viruses, these viruses normally enter the body through skin punctures and cause serum hepatitis. Hepatitis type A virus is usually transmitted by the fecal-oral route and is prevalent in areas with inadequate sewage treatment. Several outbreaks of viral hepatitis have been associated with contaminated shellfish, such as oysters, that have concen- trated viruses from sewage effluents. There have also been outbreaks from vegetables contaminated with infected fecal matter used as fertilizer. Contam- inated potable water supplies are probably the most common source of hepatitis A viral infections.

Polioviruses may enter the body through either the gastrointestinal or respiratory tracts. Transmission through the ingestion of food and water containing polioviruses is considered very important. Polio is effectively controlled through the use of vaccines.

This has been responsible for the decline of this dis-

ease, particularly in developed nations. Global vac- cination programs are aimed at eliminating this disease worldwide.

Several Clostridium species cause foodborne dis- ease. Canned foods are often the sources of clostridia causing human disease because Clostrid- ium species are endospore forming anaerobes.

Hence they are resistant to elevated temperatures, sometimes surviving the canning process high temperature killing, and can grow in the can because of the absence of air. Botulism, the most serious form of bacterial food poisoning, is caused by neurotoxins produced by Clostridium botulinum. The toxins are absorbed from the intestinal tract and transported via the circulatory system to motor nerve synapses, where their action blocks normal neural transmis- sions. Various strains of C. botulinum elaborate dif- ferent toxins. Types A, B, and E toxins cause food poisoning of humans. Type E toxins are associated with the growth of C. botulinum in fish or fish prod- ucts, and most outbreaks of botulism in Japan are caused by type E toxins because large amounts of fish are consumed there. Type A is the predominant toxin in cases of botulism in the United States, and type B toxin is most prevalent in Europe. C. botuli- num cultures form three distinct physiological groups that can be detected by culture methods. C.

botulinum types C and D are nonproteolytic and do not digest coagulated egg white or meat. All type A and some type B and F strains of C. botulinum are proteolytic whereas all type E strains and the remaining type B strains are nonproteolytic. Type G strains of C. botulinum show slow proteolysis. The optimum temperature for toxin production by proteolytic strains is approximately 35°C and for nonproteolytic strains is about 27°C. Nonproteolytic strains of C. botulinum types B, E, and F produce toxin at 4°C.

Over 90% of the cases of botulism involve improp- erly home-canned food. Of 236 outbreaks of this disease in the United States between 1899 and 1974, 57% were caused by contaminated vegetables, 15%

by contaminated fish, and 12% by contaminated fruit.

Certain canned foods provide an optimal anaerobic environment for the growth of C. botulinum that results in the release of toxin into the food. C. botuli- num, though, cannot grow and produce toxin at low pH and thus is not a problem in acidic food products.

Another clostridial species, Clostridium perfringens, is a major cause of a less severe form of food poison- ing. C. perfringens generally accounts for over 10%

of the outbreaks of foodborne disease in the United States. The ingestion of food containing toxin pro- duced by C. perfringens and the adsorption of the toxin into the cells lining the gastrointestinal tract ini-

Microbiological Media for the

H A N D B O O K O F

Microbiological Media for the

Examination of Food

By

R O N A L D M . AT L A S

S E C O N D E D I T I O N

Preface

About the Author

Table of Contents

Introduction to Food Microbiology

Food- and Waterborne Pathogens