Food Microbiology Laboratory for the Food Science Student (PDF)

Cangliang Shen Yifan Zhang Food Microbiology Laboratory for the Food Science Student A Practical Approach Food Microbiology Laboratory for the Food Science Student Cangliang Shen Yifan Zhang Food Microbiology Laboratory for the Food Science Student A Practical Approach Cangliang Shen Yifan Zhang Davis College, Division of Animal Department of Nutrition and Food Science and Nutritional Sciences Wayne State University West Virginia University Detroit, MI, USA Morgantown, WV, USA ISBN 978-3-319-58370-9 ISBN 978-3-319-58371-6 (eBook) DOI 10.1007/978-3-319-58371-6 Library of Congress Control Number: 2017943814 © Springer International Publishing AG 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Preface In order to help food science students understand the relationship between microorganisms and food products, it is important to develop a food microbi- ology laboratory textbook. Currently, a very limited amount of food microbi- ology laboratory manuals is commercially available, which makes it difficult for students to have a useful food microbiology laboratory manual with them. The aim of this book is to supply the food microbiology lab course instructor with a useful lab manual to teach this course and food science students and food microbiologists who will be working in the food industry with a read- able and easily understood and followed laboratory manual. This book is designed to give students an understanding of the role of microorganisms in food processing and preservation; relation of microorganisms to food con- tamination, foodborne illness, and intoxication; general food processing and quality control; role of microorganisms in health promotion; and federal food-processing regulations. The listed laboratory exercises are aimed to pro- vide a hands-on opportunity for the student to practice and observe the prin- ciples of food microbiology especially enumeration, isolation, and identification of microorganisms in foods. Students will familiarize them- selves with techniques used to research, regulate, prevent, and control micro- organisms in food and understand the function of beneficial microorganisms during the food manufacturing process. This book includes almost all pic- tures of each step of lab work, and all the pictures are coming from real lab practice and lab results. This lab manual is typically fit for small land-grant institutions that teach food microbiology lab classes and for non-land-grant universities who plan to develop a food science course curriculum. Morgantown, WV, USA Cangliang Shen Detroit, MI, USA Yifan Zhang v Acknowledgment I appreciate my division director Dr. Robert Taylor who assigned me to develop and teach a food microbiology laboratory course when I joined West Virginia University. I got the inspiration of writing this food microbiology manual from my Ph.D. advisor Dr. John N. Sofos, university distinguished professor of Colorado State University. He taught me that “safety, quality, and quantity” are the three basic principles to conduct any food microbiology research almost 10 years ago when I was an amateur of food microbiology. I will pass this infor- mation to my future students and to the readers of this lab manual. I would love to greatly appreciate Ms. Lindsey Williams, communications manager at Davis College, West Virginia University, who has helped to take all pictures of all lab works and results in each chapter. I want to thank my graduate students Mr. KaWang Li and Ms. Lacey Lemonakis and my undergraduate teaching assis- tants Ms. Payton Southall, Jordan Garry, and Jessica Clegg for their generous help during the writing of this lab manual. The writing of Chaps. 10 and 11 obtained strong support from Mr. Mark Carlson at Western Kentucky University. Finally, I would love to thank my wife Dr. Yi Xu for her love and support. This lab manual is also dedicated to my newborn son Alex Shen. Cangliang Shen Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV vii Contents 1 Food Microbiology Laboratory Safety and Notebook Record............................................................................................ 1 2 Staining Technology and Bright-Field Microscope Use............ 9 3 Enumeration of Bacteria in Broth Suspension by Spread and Pour Plating......................................................... 15 4 Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating................................... 19 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice on Petrifilm................. 25 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads.......................................................................... 31 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters......................................... 37 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses................................... 43 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties................................... 51 10 Cultivation of Anaerobic Bacteria in Canned Food................... 59 11 Observation and Numeration of Molds from Spoiled Bread....................................................................... 65 12 PCR Identification of Listeria monocytogenes in Deli Meat................................................................................... 71 13 Cheese Making and Characterization......................................... 75 14 Wine and Pickle Making and Characterization......................... 79 15 Antimicrobial Resistance of Commensal Bacteria from the Environment.................................................................. 87 ix x Contents 16 Introduction of Oral Presentation and Job Interview Preparation.................................................................................... 91 Sample of Food Microbiology Lab Course Syllabus......................... 97 Index....................................................................................................... 101 Authors Cangliang Shen is an assistant professor/extension specialist at Davis College, Animal and Nutritional Science and Families and Health Center, at West Virginia University. Dr. Shen worked at IEH Laboratories and Consulting Group, USDA ARS, and Western Kentucky University as postdoctoral research microbiologist and assistant professor of microbiology after obtaining his Ph.D. from Colorado State University. Dr. Shen is currently teaching undergraduate-/graduate-level microbi- ology courses including general microbiology lecture with lab and food microbiology lab. Dr. Shen has 10 years of research experience in postharvest sanitizing procedures for reducing food safety risks on poultry meat products and fresh produce at both laboratory and pilot plant levels. Dr. Shen has published 23 papers in peer-reviewed food microbiology journals and has presented 25 times in national or international food science conferences. Yifan Zhang is an associate professor in the Department of Nutrition and Food Science at Wayne State University, Michigan. She is an edi- torial board member of the Journal of Food Protection and a member of the International Association for Food Protection (IAFP), Institute of Food Technologists (IFT), and American Society for Microbiology (ASM). She earned her Ph.D. in Food Science from University of Maryland, College Park. Prior to joining the Wayne State University faculty, Dr. Zhang has worked as a postdoctoral researcher at The Ohio State University. Dr. Zhang’s research is on the molecular characterization and risk analysis of foodborne bacteria, the food and agricultural reservoir of antimicrobial resistance, urban agriculture, and the development of novel food safety control. Dr. Zhang is currently teaching introductory food science and advanced food science at the undergraduate level and microbial food safety at the graduate level. xi Food Microbiology Laboratory Safety and Notebook Record 1 Abstract We will provide food microbiology laboratory training for all students taking this class and emphasize the importance of lab notebook record, and finally we will show students the examples of good lab notebook record. Keywords Lab safety training Notebook Record Three Principles of Conducting Food Microbiology Lab Work Safety: Before doing anything in lab, ask yourself is it safe; otherwise do not do it. Quality: All lab work should be based on scientifically high quality. Quantity: Based on high quality, we will finish as much lab work as we can. Introduction of Biosafety Level (BSL) BSL-1: Microbial agents are not well known for causing disease and present minimal potential hazard to lab personnel, i.e., generic Escherichia coli, Pseudomonas spp. BSL-2: Microbial agents cause moderate hazard to lab personnel and environment, i.e., E.coli O157:H7, Salmonella spp. BSL-3: Microbial agents may cause serious or potentially lethal disease through the inhalation route of exposure, i.e., Clostridium botulinum. BSL-4: Microbial agents pose a high individual risk of aerosol-transmitted laboratory infections and life- threatening disease, i.e., Mycobacterium tuberculosis (TB). We only use BSL-1 and BSL-2 microbial organism in this lab manual. Lab Safety Rules 1. Always wash your hands with antimicrobial soap water before and after lab work. 2. Always wear a clean lab coat and gloves, and do not wear open toe shoes and very short pants when conducting lab work. © Springer International Publishing AG 2017 1 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_1 2 1 Food Microbiology Laboratory Safety and Notebook Record 3. Always wear eye protection (goggles) when staining or handling hazardous laboratory chemicals. 4. Clean and sanitize your working bench with a disinfection solution before and after lab work. 5. Report any lab accidents immediately. 6. Clearly label petri dishes, tubes, and flasks before lab work; label name, bench (seat) number, date, microorganism name, etc. 7. Place any biohazard trash into biohazard trash can. 8. Smoking, eating, drinking, or placing anything into your mouth is prohibited. 9. Do not handle any electronic devices (cell phone, ipad, laptop) while doing the lab work. 10. Please let your instructor know if you are pregnant or planning to be pregnant. 11. Know the location of nearest fire alarm pull station, fire extinguisher, eyewash station, and first aid kit. Lab Notebook Requirement Notebooks can be checked after each lab section before you leave the classroom. The front cover of your notebook will be labeled with the following information: your name, course name, professor’s name, and semester. Reserve the first three pages of your notebook for a table of contents. You will update the table of contents each week. Number all pages. Each notebook entry for any given lab period will be formatted in the following way: 1. The Title. 2. A Purpose statement: Describe the purpose of the exercise. Ask yourself “What are the goals of this exercise?” 3. A Materials and Methods section: The Materials and Methods for each lab will be described at the beginning of the period by the instructor. You will have an opportunity to copy this informa- tion into your notebook at the beginning of each lab period. You should describe what was needed and the steps taken (including any modifications that were made). Be as specific as possible. Be sure to use correct spelling for all microorganism names and italicize scientific names. 4. A Results section: Record all observations in your lab notebook. Colored pencils/pens should be used to illustrate results (i.e., observations made with the microscope) – all figures/ tables must have a title and legend (a description of what is being shown – label all relevant information). 5. A Discussion: Summarize your findings, and discuss how the exercise helped you understand the learning objectives. Describe why something may not have worked and what you would do differ- ently next time to improve the outcome. Each section must be labeled. 1 Food Microbiology Laboratory Safety and Notebook Record 3 Examples of Good Notebook Records (From Ms. Payton Southall) 4 1 Food Microbiology Laboratory Safety and Notebook Record Examples of Good Notebook Records (From Ms. Payton Southall) 1 Food Microbiology Laboratory Safety and Notebook Record 5 Examples of Good Notebook Records (From Ms. Jessica Clegg) 6 1 Food Microbiology Laboratory Safety and Notebook Record Examples of Good Notebook Records (From Ms. Jessica Clegg) 1 Food Microbiology Laboratory Safety and Notebook Record 7 Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Staining Technology and Bright- Field Microscope Use 2 Abstract We will introduce bright-field microscope use, practice Gram staining with foodborne pathogens, and practice endospore staining with Bacillus cereus. Keyword Bright-field microscope Gram staining Endospore staining Bacillus cereus Objective Practice Gram staining and endospore staining with common foodborne pathogens. Gram Stain Strain Culture Escherichia coli ATCC25822, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Staphylococcus aureus ATCC 25923 (non-MRSA strain) Endospore Stain Culture Bacillus cereus Major Experimental Materials Bright-field light microscope Gram stain reagents (crystal violet, Gram iodine, 95% alcohol, safranin) Endospore reagent (malachite green) Sterilized loop Glass slides Lens paper Bunsen burner Introduction of Gram Stain Gram stain was created by Hans Christian Gram (Danish microbiolo- gist), which differentiates all bacteria into two groups Gram positive (Blue/purple color) and Gram negative (Pink/red color). The theory of Gram stain differentiation is based on cell wall structure and lipid component; therefore, it comes out with two theories below: © Springer International Publishing AG 2017 9 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_2 10 2 Staining Technology and Bright-Field Microscope Use 1. Cell wall theory: Gram-positive bacteria have heavy peptidoglycan, which helps decolorizer (alcohol) to dehydrate the Gram-positive cell wall and traps the crystal violet complex inside the cell wall and maintains the purple color of crystal violet. 2. Lipids theory: Gram-negative bacteria have high lipid amount (10–15%) in the cell wall, which makes decolorizer (alcohol) easily to remove the crystal violet complex, and then colorless cell wall is stained with safranin and appears pink/red color. Gram stains need to be done on a very young fresh culture (within 24–48 h). Introduction of Bacterial Endospore Two bacteria genera that will generate endospore (germi- nation) under stressful environment (poor nutrition, low humidity, desiccation, and high tempera- ture) are Bacillus and Clostridium. The calcium-dipicolinic acid (DPA) stabilizes the endospore’ DNA along with small soluble DNA protein to protect the bacteria from stress environment. When the growth condition improves, the endospore will generate vegetative cells (sporulation). Endospore stain is intended to find the presence/absence of endospore and the location of endospore. The loca- tion of endospore is species specific which can be located in the middle of cells (central) and the end (terminal, i.e., Clostridium sporogenes) or between the end and the middle (subterminal, i.e., Clostridium botulism). Endospore stain needs to be done on old culture (>5–7 days). Gram Stain Procedure 1. Prepare a fixed smear: label two large circles (dime) using marker onto a clean glass slides, flip over slides, place one drop of sterilized water in the center of each circle, and use loop aseptically pick bacteria cells from stock culture and spread them in their drops of water to cover the whole circle of the dime (Fig. 2.1). Fig. 2.1 Clean slides and label LI EC Note: Please f lip over your glass slide, otherwise the marker may damage the microscope lens. 2 Staining Technology and Bright-Field Microscope Use 11 Fig. 2.2 Smear preparation, air-drying + heat fixing Note: Flipping over slides is very important; otherwise, the dye from marker may damage the lens of microscope later on. A smear needs to be thin. 2. Air-dry the smear for 5 min (Fig. 2.2). 3. Heat fix the cells on slides by passing three times back and forth through the Bunsen flame. Heat fixing will inactivate bacteria enzyme and stick cells onto glass slides. 4. Major steps of Gram stain: (a) Place your slide on staining rack, and put one to two drops of crystal violet onto the smear for 60 s, and then gently rinse with water. (b) Add one to two drops of Gram iodine (a mordant to help crystal violet stain strong) for 60s, and then gently rinse with water. (c) Decolor by adding one to two drops of 95% alcohol for 10–15 sec, and then gently rinse with water. (d) Counterstain by adding one to two drops of safranin, and then gently rinse with water. 5. Drain off excess water, blot dry with paper towel, and air-dry slide. 6. Observe your stained smear with low power 10X, and then switch to 100X with oil immersion; record your results in the notebook. 7. Gram staining results should include three items: (1) Gram reaction, positive or negative; (2) morphol- ogy, rods, cocci, etc.; and (3) arrangement, single, chain, grape shaped, tetrad, etc. 8. Please refer to Fig. 2.3 as a Gram staining result. Endospore Stain Procedure 1. Smear preparation including air-drying and heat fixing is the same as Gram stain. 2. Flood the smear with malachite green (cover the whole slides with the dye, Fig. 2.4). 3. Heat steam slides for 3–5 min using the flame of burner back and forth a couple of times, but do not let the slides dry out. 4. Cool down slides, remove dye, and gently rinse with water. 5. Add safranin onto the slide. 6. Drain off excess water, blot dry with paper towel, and air-dry slide. 7. Observe your stained smear with low power 10X, and then switch to 100X with oil immersion; record your results in the notebook. 12 2 Staining Technology and Bright-Field Microscope Use Fig. 2.3 Examples of Gram staining result (from previous students’ lab work) 2 Staining Technology and Bright-Field Microscope Use 13 Fig. 2.4 Flooding with dye (only for endospore staining) Bright-Field Microscope Use Microscope structure Brief Procedure 1. Stage down with course focus (large and thick wheel). 2. Spin/rotate objective lens. 3. Place 10X objective lens in position. 4. Adjust light to “low” or “10X” with condenser (iris diagram). 5. Bring stage up as high as it can reach. 6. Look into 10X ocular lens and find specimen. 7. Slightly switch 10X objective lens, and add one drop of oil. (Do not take microscope out of focus.) 8. Adjust fine focus (small and thin wheel) to find specimen. Questions for Review 1. Why are Gram-positive stains purple and Gram-negative stains pink in color? 2. Describe two conditions in which bacteria organism will stain gram variable (showing Gram- negative results). 3. Is bacterial endosporulation a reproductive mechanism? Why? 4. Why is the location of endospore a very important information? 14 2 Staining Technology and Bright-Field Microscope Use Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Enumeration of Bacteria in Broth Suspension by Spread and Pour 3 Plating Abstract We will practice spread plating and pour plating using 10- or 100-fold dilution technique to enumerate bacteria cells in solutions on agars. Keywords Spread plating Pour plating Dilution technique Escherichia coli Objective Understand serial dilution technique, and practice spread and pour plating. Bacterial Strain Escherichia coli ATCC25822 grown in Tryptic Soy Broth at 35 °C after 24 h. Major Experimental Materials Tryptic Soy Agar Buffered peptone water (0.1%), 15 ml dilution tubes Water bath (50–55 °C) with melted agar (25 ml) in glass bottles Empty petri dishes Sterilized spreaders and pipette tips (200 μl and 1000 μl) Introduction Most bacteria that grow in support medium like Tryptic Soy Broth incubating at 35 °C for 24 h will generate around 109 cells/ml (=9.0 log10 colony-forming unit/ml), which is too much to put onto agar medium to manually count the colony. Therefore, we need to do tenfold or 100-fold serial dilution and then add 0.1 ml of the diluted solution from the final dilution tube onto agar medium through spreading or pour plating and count bacterial colonies after incubation. For tenfold and 100-fold dilution, we usually use 9.0 or 9.9 ml 0.1% buffered peptone water (BPW). Spread plating is conducted by spread liquid solution on agar medium using sterilized plastic spreader or flamed glass spreader. Pour plating is conducted by adding liquid solution onto an empty petri dish followed by pouring 20–25 ml of melted agar, rotating the petri dish, and mixing very well. Due to the use of melted agar (50–55 °C), heat-sensitive bacteria will be killed by the mild heat; the number of colonies of pour plating will be slightly lower than spread plating. © Springer International Publishing AG 2017 15 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_3 16 3 Enumeration of Bacteria in Broth Suspension by Spread and Pour Plating Dilution technique figures (tenfold serial dilution, assume 0.1 ml of each dilution tube adding onto agar plates) 1 ml 1 ml 1 ml 1 ml 1 ml 9.0ml 9.0ml 9.0ml 9.0ml 9.0ml Bac. BPW BPW BPW BPW BPW broth (0.1%) (0.1%) (0.1%) (0.1%) (0.1%) Diluon factor of tubes “0” 10 -1 10-2 10 -3 10 -4 10 -5 Final diluon factor of plates “0” 10 -2 10-3 10-4 10-5 10-6 “0” Dilution Averagely spreading 1.0 ml original solution onto three agar plates, adding all colony- forming unit (CFU) of three agars after incubation. Dilution Procedure 1. The original bacterial broth is considered to be at a dilution factor of “0.” 2. Each dilution blank contains 9.0 ml of sterile 0.1% BPW. 3. To make a 1/10 dilution, transfer 1.0 ml of sample into a 9.0 ml sterile dilution blank (0.1%BPW). This is a 10−1 dilution. 4. Alternately, you can transfer 1.0 ml of diluent into a 9.0 ml blank and make a 10−2 dilution. 5. Continue to make dilutions as necessary. Note: We also can add 0.1 ml into 9.9 ml of diluent to make a 10−2 dilution. Dilution technique figures (100-fold serial dilution, assume 0.1 ml of each dilution tube adding onto agar plates). 0.1 ml 0.1 ml 0.1 ml 9.9ml 9.9ml 9.9ml Bac. BPW BPW BPW broth (0.1%) (0.1%) (0.1%) Diluon factor of tubes “0” 10-2 10-4 10-6 Final diluon factor of plates “0” 10-3 10-5 10-7 3 Enumeration of Bacteria in Broth Suspension by Spread and Pour Plating 17 Numeration After spread and pour plating, incubate plates inverted at 35 °C for 24–48 h, and manually count colonies on the agar plates. The acceptable and countable zone is 30–300 CFU/plate. CFU = colony- forming unit. If colonies are 300, record as TNTC (too numerous to count). Work figure of dilution procedure of plating ~ 109 CFU/ml E. coli solution onto agar plates. 0.1 ml 0.1 ml -6 1 0 f inal 9.0ml 0.1 ml dilution BPW (0.1 %) factor E.coli 9.9ml 9.9ml BPW BPW broth (0.1 %) (0.1 %) 0.1 ml 9.9 ml 10-7 final BPW 0.1 ml dilution (0.1 %) factor -5 10 final dilution factor Method 10−5 10−6 10−7 Reported count Spread plating Pour plating Review Questions 1. A pure bacterial culture was diluted by adding a 0.1 mL aliquot to 9.9 mL water. Then, 0.1 mL of this dilution was plated out by spread plating, yielding 50 colonies. Calculate the CFU per mL in the original culture. _____________ 2. A pure bacterial culture was diluted by adding a 1.0 mL aliquot to 9.0 mL water. Then, mL of this dilution was plated out by pour plating, yielding 82 colonies. Calculate the CFU/mL in the original culture. ______________________ 18 3 Enumeration of Bacteria in Broth Suspension by Spread and Pour Plating Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Isolation of Foodborne Pathogens on Selective, Differential, 4 and Enriched Medium by Streak Plating Abstract We will introduce the selective, differential, and enriched medium for several foodborne pathogens, practice streak plating to get bacterial single colonies on various agars, and observe bacterial colony morphology. Keywords Streak plating Single colony Selective Differentiate Enrich Blood agar MacConkey agar Mannitol salt agar Modified Oxford Agar XLT-4 agar HardyCHROM agar Objective Practice streak plating of foodborne pathogens on selective, differential, and enriched medium and observe colony morphology. Bacterial Strain Generic Escherichia coli ATCC25822, Listeria innocua ATCC33090, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus ATCC 25923 (non-MRSA strain). Medium Blood agar, MacConkey agar, mannitol salt agar, Modified Oxford Agar, XLT-4 agar, and HardyCHROM agar. Introduction of Selective and Differential Medium Selective medium: Contains one or more components (chemical or antibiotics) that suppress the growth of some microorganisms without affecting the ability of wanted organism to grow. Differential medium: Contains one or more chemical components which can differentiate different microorganism growth based on the color and morphology of colony. Note: A medium can be both a selective and a differential medium. Introduction of Medium for Isolating Foodborne Pathogens MacConkey agar, containing neutral red, crystal violet, and bile salts, inhibits Gram-positive organ- isms (due to crystal violet) and supports Gram-negative organisms to grow. Colonies of E. coli O157:H7 are pink/red colony (due to lactose fermentation) surrounded by a bile salt precipitation © Springer International Publishing AG 2017 19 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_4 20 4 Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating zone. Non-lactose fermentation colony is colorless (e.g., non-O157 Shiga toxin-producing E.coli). Mannitol salt agar, containing 7.5% salt, 6 carbon mannitol, and phenol red, is used for isolating S. aureus (yellow colony). The 7.5% salt inhibits most bacteria other than staphylococci. Mannitol can be fermented by S. aureus which cause phenol red to turn yellow color at acidic pH. Modified Oxford Agar (MOX) contains oxford agar base plus selective ingredients, used for isolating Listeria spp. (black colony). Agar base provides basic nutrition of nitrogen, carbon, amino acids, and vitamins. Listeria spp. hydrolyze esculin to form 6,7-dihydroxycoumarin (esculetin), which reacts with ferric ions (ferric ammonium citrate) to form black colony. Lithium chloride inhibits growth of enterococci other than Listeria spp. (high salt tolerance). Selectivity is increased by add- ing colistin sulfate and moxalactam to the base and completely inhibits Gram-negative organisms and most Gram-positive organisms after 24 h of incubation. XLT-4 agar is used for isolating non-typhi Salmonella (red colony with black center), which can fer- ment multiple sugar including xylose, lactose, sucrose, and decarboxylase of lysine and generate hydrogen sulfide. Hydrogen sulfide production (black color) is detected by the addition of ferric ions. Sodium thiosulfate is added as a source of inorganic sulfur. Sodium chloride maintains the osmotic balance of the medium. Phenol red is added as a pH indicator. XLT-4 supplement – Tergitol 4 – is added to inhibit growth of non-Salmonella organisms. HardyCHROM Agar: HardyCHROM™ Salmonella agar facilitates the isolation and differentiation of Salmonella spp. from other members of the family Enterobacteriaceae. Peptones in the medium supply the necessary nutrients. Selective agents inhibit the growth of Gram-positive organisms. Artificial substrates (chromogens) are broken down by specific microbial enzymes which release insoluble colored compounds. Salmonella species break down only one of the chromogens and will produce deep pink- to magenta-colored colonies. Bacteria other than Salmonella spp. may break down the other chromogenic substrates and produce blue colonies. If none of the substrates are u tilized, natural- or white-colored colonies will be present. Blood agar, a Tryptic Soy Agar containing 5% sheep blood, differentiates pathogen growth based on the hemolysis of the red blood cells caused by hemolysin (exotoxin from L. monocytogenes). Practice of Streak Plating Streak plating is to “dilute” microorganism onto solid agar medium and then obtain pure culture (single colony represents one genera name and one species name) on agar surface: Step 1. Use flamed loop to pick bacteria (agar, slant, or broth) and begin with inoculating from the first quadrant of the agar surface. Light touch without damaging the agar surfaces. Step 2. Flame loop and cool down by touching the un-inoculated area of the agar surface. Step 3. Rotate the plate, picking up area from the quadrant one, and streak again. Step 4. Flame loop, rotate plate, and repeat procedure for quadrants three and four. Step 5. Incubate plate inverted at incubator for 35 °C for 24 to 48 h. Figure of Streak Plating 4 Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating 21 Lab Task Two students/group share seven agars and finish streak plating of assigned pathogen on a specific agar and “spot” plating with four pathogens on blood agar. 1. Streak-plating Generic Escherichia coli ATCC25822 onto MacConkey agar, and incubate plates inverted at 35 °C for 24 h. 2. Streak-plating Staphylococcus aureus ATCC 25923 onto mannitol salt agar, and incubate plates inverted at 35 °C for 48 h. 3. Streak-plating Listeria innocua ATCC33090 onto MOX agar, and incubate plates inverted at 35 °C for 48 h. 4. Streak-plating Salmonella enterica serovar Typhimurium onto XLT-4 agar and HardyCHROM agar, and incubate plates inverted at 35 °C for 24 h. 5. “Spot” plating of foodborne pathogens onto blood agar. Divide a blood agar into four sections and label each section with one assigned culture. LI EC SA SAL LI, L. innocua; EC, E.coli; SA, S. aureus; SAL, S. Typhimurium Use your loop and “spot inoculate” each section with pathogen. Incubate plates inverted at 35 °C for 24 h. Question for Review 1. _________Mannitol salt agar (MSA) only allows the growth of halophiles (salt-loving microbes); nonhalophiles will not grow. Among the halophiles, mannitol fermenters will produce acid that turns the pH indicator yellow; mannitol nonfermenters leave the medium red. Onto MSA you inoculate a halophilic mannitol fermenter and a halophilic mannitol nonfermenter. In this case, the medium is acting as (a) __________ medium(s). A. Selective B. Differential C. Selective and differential D. Enriched 2. Briefly explain why Salmonella growing on XLT-4 agar is red colony with black center. 3. __________Phenol red is utilized in MacConkey agar to: A. Inhibit microscopic organisms B. Detect the production of gas by-products C. Conduct the sulfate reduction test D. Detect the acid reaction of lactose fermentation 22 4 Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating Part of Result Figure (from Previous Lab Students) 4 Isolation of Foodborne Pathogens on Selective, Differential, and Enriched Medium by Streak Plating 23 Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia 5 coli of Organic Fruit Juice on Petrifilm Abstract We will practice using Petrifilm to enumerate food microbial quality index including aerobic plate counts, coliform, and E. coli from organic fruit juice. Keywords Petrifilm APCs Coliform E. coli Juice Objective Organic juice made from whole foods will be plating upon 3M@ Petrifilm to test aerobic plate counts (APCs), E. coli, and total coliform populations. Major Experimental Materials Organic fruit juice APC, ECC/TCC 3M@ Petrifilm Buffered peptone water (0.1%), 15 ml dilution tubes Sterilized pipette tips (200 μl and 1000 μl) Incubator Introduction Indicator microorganism: Indicator organisms of foodborne pathogens are present in high number and easy to detect. Their growth and survival characteristics are similar to those pathogens. Coliforms: They are most commonly used as indicator organisms, usually from the intestine of warm-blooded animals. They are Gram-negative, non-endospore-forming, facultative, fermenta- tion lactose generating acid and gas and members of Enterobacteriaceae family. E. coli: A member of the coliform group; the presence of E. coli may have more accurate correlation to the presence of pathogens than coliforms and indicates fecal contamination. Petrifilm plates: Created by Food Safety Division of 3M Corporation, which contain a foam barrier accommodating the plating surface itself (a circular area of about 20 cm2) and a top film. Petrifilm has © Springer International Publishing AG 2017 25 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_5 26 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice… a cold-water-soluble gelling agent containing dehydrated nutrients and indicators for microorganism activity and enumeration. Dilution factor APCs, 10−1 and 10−3; coliform/E. coli, 0 and 10−2; total coliform, 0, 10−1, and 10−3. Dilution Protocol APCs and total 9.0 ml BPW 1.0 ml coliform petrifilm (0.1 %) (1 0-1 dilution) Orange Juice APCs and total 9.0ml 9.9 ml BPW coliform petrif ilm 1.0 ml BPW 1.0 ml 1.0 ml (0.1 %) (10-3 dilution) (0.1 %) Coliform/ E. coli and total coliform petrifilm (0 diluon) 1.0 ml Coliform/ E. coli petrifilm (1 0-2 dilution) Procedure of Plating upon Petrifilm 1. Label each Petrifilm. 2. Make dilution according to the work protocol 0.1% BPW solution. Because 1 ml of solution will be plated onto Petrifilm, the final dilution is the same as tube dilution factor. 3. Pull up the film, and add 1 ml onto the center of Petrifilm with dehydrated medium. 4. Roll down the film and prevent air bubble. 5. Use plastic template to gently press down the sample to fill the area (coliform/E. coli film can skip this step). 6. Incubate at 35 °C for 24 to 48 h. Petrifilms cannot be stacked more than 20 pieces. 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice… 27 APCs, red colony; coliform, pink colony; E. coli, blue colony with gas. Review Question If you are a microbiologist working in a dairy farm and will test APCs and coliform/E. coli in unpasteurized raw milk, please write down your dilution flowchart in your note- book. The dilution factor will be APCs, 10−2 and 10−4; coliform/E. coli, 0, 10−1 and 10−2; and total coliform, 0, 10−1, and 10−3. 28 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice… 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice… 29 Part of Result Figures (Work from Previous Students) APC counts = 64 × 103 = 6.4 × 104 CFU/ml = 4.81 log10 CFU/ml. E. coli counts (less than 30 CFU on Petrifilm only estimated results) = [(28 + 23)/2] × 100 = 2550 = 3.41 log10 CFU/ml. Note: Coliforms are all E. coli cells in this lab result. Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ 30 5 Enumeration of Aerobic Plate Counts, Coliforms, and Escherichia coli of Organic Fruit Juice… ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Enumeration and Identification of Staphylococcus aureus 6 in Chicken Salads Abstract We will practice enumerating Staphylococcus aureus in chicken salads by inoculating, dilution technique, and spread plating onto selective medium. The presumptive colonies will be identified using Gram staining, catalase test, coagulase test, and latex agglutination test. Keywords Staphylococcus aureus Chicken salads Gram staining Catalase test Coagulase test Latex agglutination test Objective Test the population of Staphylococcus aureus in inoculated chicken salads using spread plating, and identify S. aureus colonies by Gram staining, catalase test, latex agglutination test, and coagulase test. Major Experimental Materials Staphylococcus aureus ATCC 25923 (non-MRSA strain) Chicken salads from commercial supermarket Buffered peptone water (100% and 0.1%), 15-ml dilution tubes Sterilized spreader Sterilized pipette tips Whirl@ filtered food sampling bags Stomacher Mannitol salt agar, Tryptic Soy Agar H2O2 reagent (for catalase test) Gram stain reagents StaphTex™ Blue latex agglutination text kit Coagulase plasma (6 × 3 ml tubes) © Springer International Publishing AG 2017 31 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_6 32 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads Introduction Staphylococcus aureus is the most commonly identified pathogen in all postsurgical infections with methicillin-resistant S. aureus (MRSA) becoming an emerging health problem. Staphylococcal food intoxication is a gastrointestinal illness caused by eating foods contaminated with S. aureus. Foods frequently incriminated include meat and poultry products, egg products, and salads such as egg, tuna, chicken, potato, and macaroni. Food handlers are usually the main source of food contamination in outbreaks. Symptoms develop within 6 h after eating contaminated foods, including nausea, vomiting, cramps, and diarrhea, and most often are self-limiting within 1 to 3 days. Enumeration of S. aureus in Chicken Salads Commercial chicken salads will be inoculated with S. aureus (your instructor will do this): 1. Inoculated chicken salads (200 g) will be sterilized transfer into filtered-whirl@ food sampling bag. 2. Add 200 ml of 0.1% buffered peptone water into the sample bag. 3. Stomach for 1 min. 4. Conduct serial dilution into 9.0 or 9.9 ml 0.1% BPW solution to obtain final dilution factor. 10−2, 10−3, and 10−5 and spread plating onto mannitol salt agar and Tryptic Soy Agar. Please draw your dilution diagram and check with instructor! 5. Incubating plates at 35 °C for 48 h. 6. Manually count colonies of agar plates and record onto notebook, and calculate as follows: Log10 CFU/g = Log10 [(Final CFU on plates/dilution factor)*(200 g + 200 ml)/200 g] For example, 50 CFU on plates with dilution factor 10−5, and then final Log10CFU/g = Log10 (50 × 105 × 2) = Log10107 = 7 log10CFU/g. 7. Typical colonies (yellow color) of S. aureus will be identified using the following tests: A. Gram stain Major steps of Gram stain 1. Place your slide on staining rack and put one to two drops of crystal violet onto the smear for 60 s, and then gently rinse with water. 2. Add one to two drops of Gram iodine (a mordant to help crystal violet stain strong) for 60 s, and then gently rinse with water. 3. Decolor by adding one to two drops of 95% alcohol for 10–15 s, and then gently rinse with water. 4. Counterstain by adding one to two drops of safranin and then gently rinse with water. B. Catalase test: positive (bubble) 1. Place one drop of H2O2 on a clean glass slides. 2. Using sterilized loop, pick presumptive colony from mannitol salt agar and slowly immerse with cells into H2O2. 3. A positive test is read immediately from the slide, showing oxygen gas bubbles. C. Coagulase test Transfer a presumptive colony from mannitol salt agar into the commercial coagulase plasma with EDTA and incubate at 35 °C and examine after 12 h for the clot formation. Firm and complete clot stays in place when tube is tilted or inverted. 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads 33 D. Latex agglutination test Latex agglutination test detects the agglutination (clumping) happened when the unknown organism isolated in a culture is mixed with an antiserum that contains antibodies specific for its antigen. The antibody (antiserum) usually is conjugated to a latex particle in order to enhance the visibility of the agglutination reaction. Procedure 1. Place one drop of saline solution onto two wells of the test card. 2. Using sterilized loop, pick presumptive colony from mannitol salt agar and mix with the saline solution. 3. Shake (very important!) the latex reagent bottle (control and test reagent) and place one free-falling drop into each well. 4. Use a new sterilized loop to mix the reagent with colony very well. 5. Rock the card for 1 min to observe the clumping. Review Question A chicken salad was picked from a local subway and brought to the laboratory. 200 grams were added to 200 ml of sterile buffered peptone water and homogenized for 2 min. The solution was chilled to prevent overheating. The final dilutions and the collected data are presented below. The plate medium is TSA agar. Answer parts (a), (b), and (c). (a) There are ______________ CFU per ml of homogenate. (b) There are_______________ CFU per gram of the chicken salad. (c) What does CFU stand for? ________________________________________. Plate Colonies observed Final dilution A Too numerous to count 10−1 B 564 10−2 C 275 10−3 D 19 10−4 34 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads 35 Part of Results Figures (Work from Previous Students) A. Gram staining results (×100 with oil immersion) Staphylococcus aureus is Gram positive, cocci, and grape shaped. B. Latex agglutination test C. Spead plating 36 6 Enumeration and Identification of Staphylococcus aureus in Chicken Salads Class Notes ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Enumeration and Identification of Listeria monocytogenes 7 on Ready-to-Eat (RTE) Frankfurters Abstract We will practice enumerating Listeria monocytogenes on commercial frankfurters by inoculating, dilution technique, and spread plating onto selective medium Modified Oxford Agar. The presumptive colonies will be identified using Gram staining, catalase test, and 12L test. In this chap- ter, it requires students to finish part of the work independently. Keywords Listeria monocytogenes Frankfurters Modified Oxford Agar Gram staining Catalase test 12L test Objective Test the population of Listeria monocytogenes on inoculated frankfurters by spread plat- ing, and identify L. monocytogenes using Gram staining, catalase test, and 12L@ biochemistry test. Major Experimental Materials Listeria monocytogenes (BSL-2) Frankfurters (hotdog) from commercial supermarket Buffered peptone water (100% and 0.1%) Whirl@ food sampling bags Stomacher Modified Oxford Agar, Tryptic Soy Agar, and blood agar H2O2 reagent (for catalase test) Gram stain reagents 12L@ listeria test kit Introduction Listeria monocytogenes is a Gram-positive, non-endospore-forming, facultative bacte- rium that can survive/grow in a wide variety of foods, including dairy products, meat and poultry products, vegetables, and seafood. L. monocytogenes is a remarkably difficult organism to control in food-processing environments due to its psychrotrophic nature and ability to tolerate adverse growth © Springer International Publishing AG 2017 37 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_7 38 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters conditions. In the United States, from 1998 to 2002, ready-to-eat (RTE) meat products, including commercially cured ham, were involved in several multistate outbreaks of listeriosis. These outbreaks triggered the US Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS) to require RTE meat processors, starting from 2003, to select at least one of three alternatives for L. monocytogenes control: (i) both a post-lethality treatment (e.g., steam, pressure, or antimicrobial agent) and an antimicrobial process, (ii) either a post-lethality system or an antimicrobial process, and (iii) sanitation practices only, with more frequent microbial verification testing. Lab Work Procedure Inoculation: Two frankfurter links will be received by two students/group and will be inoculated (0.2 mL) by rolling-spreading L. monocytogenes on the surface of each frankfurter (56 cm2) with a sterile bent plastic rod on a foiled paper. The inoculated hotdogs were left at room temperature for 10 min to encourage attachment. Enumeration of L. monocytogenes on Frankfurters 1. Inoculated two frankfurters will be transferred into whirl@ food sampling bag. 2. Add 100 ml of 0.1% buffered peptone water into the sample bag and rolling bag. 3. Vigorously shake for 30 s (30 “Mississippi”). 4. Conduct serial dilution into 9.0 or 9.9 ml 0.1% BPW solution to obtain final dilution factor. 10−2 and 10−4 and spread plating onto Modified Oxford Agar and Tryptic Soy Agar. Please draw your dilution diagram in your notebook. 5. Incubating plates at 35 °C for 48 h. 6. Manually count colonies of agar plates and record onto notebook, and calculate as follows: Log10 CFU/g = Log10 [(Final CFU on plates /dilution factor)*100 ml)/112 cm2] For example, 50 CFU on plates with dilution factor 10−4, and then final Log10CFU/cm2 = Log10 (50 × 104 × 0.89) = Log10445, 000 = 5.65 log10CFU/g. 7. Typical colonies of L. monocytogenes will be identified using the following tests: A. Gram stain: Gram positive, single, large rod shaped Major steps of Gram stain 1. Place your slide on staining rack and put one to two drops of crystal violet onto the smear for 60 s, and then gently rinse with water. 2. Add one to two drops of Gram iodine (a mordant to help crystal violet stain strong) for 60 s, and then gently rinse with water. 3. Decolor by adding one to two drops of 95% alcohol for 10–15 s, and then gently rinse with water. 4. Counterstain by adding one to two drops of safranin and then gently rinse with water. B. Catalase test: positive (bubble) 1. Place one drop of H2O2 on a clean glass slides. 2. Using sterilized loop, pick presumptive colony from MOX Agar and slowly immerse with cells into H2O2. 3. A positive test is read immediately from the slide, showing oxygen gas bubbles. 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters 39 C. Hemolysis test: Streak-plating typical colony onto blood agar, and observe α-, β-, or γ- hemolytic. L. monocytogenes and L. innocua are β-hemolytic (transparent zone). D. 12L@ test: Multiple channel biochemistry test for Listeria spp.; please follow instruction in the fact sheet. Procedure 1. Pick four to five suspect colonies from MOX agar and suspend in Listeria suspending medium. 2. Place test strip in holding tray and remove lid. 3. Place four drops (100 μl) of bacterial suspension to each well. 4. Add one drop hemolysis to well 12. (We will do this on blood agar.) 5. Replace lid and incubate at 35 °C ± 2 °C for 24 h. 6. Record results on report forms and interpret using the color sheet. 12L positive and negative results table Well no. Substrate Negative reaction Positive reaction 1 Esculin (ESC) Pink or brown Black 2 Mannitol (MAN) Purple Yellow, brown, or straw 3 Xylose (XYL) Purple Yellow, brown, or straw 4 Arabitol (ARL) Purple Yellow, brown, or straw 5 Ribose (RIB) Purple Yellow, brown, or straw 6 Rhamnose (RHA) Purple Yellow, brown, or straw 7 Trehalose (TRE) Purple Yellow, brown, or straw 8 Tagatose (TAG) Purple Yellow, brown, or straw 9 Glucose-1-phosphate (G1P) Purple Yellow, brown, or straw 10 Methyl-D-glucose (MDG) Purple Yellow, brown, or straw 11 Methyl-D-mannose (MDM) Purple Yellow, brown, or straw 12 Hemolysin (HEM) Red cell deposit Partial or complete red cell lysis Note: well 12 can be done on the blood agar. Listeria spp. should be positive in well 1 (esculin), well 4 (arabitol), and well 7 (trehalose). 1ESC 2MAN 3XYL 4ARL 5RIB 6RHA 7TRE 8TAG 9G1P 10MDG 11MDM 12HEM L. monocytogenes + − − + − + + − − + + + Review Questions 1. Name the “three alternatives” for controlling Listeria monocytogenes on RTE meats. 2. There is a Listeria monocytogenes outbreak in a hotdog plant; as a microbiologist, your job is to isolate and identify this pathogen from hotdogs and describe the details how you will do this, and please be specific, including medium, test name, and possible results. 40 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters Part of Result Figures (Work from Previous Students) A. Positive L. monocytogenes by 12L test 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters 41 B. β-Hemolytic (transparent zone) of L. monocytogenes on blood agar C. Spread plating Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ 42 7 Enumeration and Identification of Listeria monocytogenes on Ready-to-Eat (RTE) Frankfurters ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Isolation and Identification of Salmonella and Campylobacter 8 spp. on Broiler Carcasses Abstract We will practice isolating and identifying Salmonella and Campylobacter spp. on commercial chicken broiler carcasses by modified USDA meth- ods. We will introduce the function of primary and secondary enrichment and practice growing Campylobacter in a microaerophilic environment. The presumptive colonies will be identified using Gram staining, latex agglutination test, and API 20e biochemistry test. In this chapter, it requires students to finish part of the work independently. Keywords Salmonella Campylobacter Broilers RV TT XLT-4 HardyCHROM Modified Campy-Cefex agar Bolton broth Gram staining Latex agglutination test API 20e test Objective Salmonella and Campylobacter spp. will be isolated from commercial broiler carcasses and identified using Gram staining, biochemistry test, and immunology test. Major Experimental Materials Raw chicken carcass Poultry sampling bag Buffered peptone water RV medium and TT medium XLT-4 and HardyCHROM agar Bolton broth and modified Campy-Cefex agar Microaerophilic jar and gas generator Campy-latex agglutination test kits API 20E biochemistry test kits Mineral oil Kovac reagent, 10% ferric chloride, 40% KOH, and 6% alpha-naphthol Gram stain reagents © Springer International Publishing AG 2017 43 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_8 44 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses Introduction Contaminated poultry meat represents the greatest public health impact among foods and is responsible for an estimated $2.4 billion in annual disease burden. Salmonella and Campylobacter spp. are the two most common foodborne pathogens associated with poultry meat, causing an esti- mated 9.4 million illnesses, 55,961 hospitalizations, and 1351 deaths annually in the United States. Starting in July 2011, USDA-FSIS established new performance standards in response to national baseline studies requiring routine testing for Salmonella and Campylobacter in all processing plants, where the percentage of Salmonella-positive samples must be below 7.5% and Campylobacter- positive samples should be less than 10.4%. Major Medium Used for Salmonella Isolation Rappaport Vassiliadis (RV) Salmonella Enrichment Broth: Malachite green is inhibitory to organ- isms other than Salmonella spp. The low pH (5.2) of the medium, combined with the presence of malachite green and magnesium chloride, selects for the highly resistant Salmonella spp. Tetrathionate (TT) Broth: Selectivity is accomplished by the combination of sodium thiosulfate and tet- rathionate, which suppresses commensal intestinal organisms. Tetrathionate is formed in the medium upon addition of the iodine and potassium iodide solution. Organisms containing the enzyme tetrathion- ate reductase will proliferate in the medium. Bile salt, a selective agent, suppresses coliform bacteria and inhibits Gram-positive organisms. Calcium carbonate neutralizes and absorbs toxic metabolites. Major Medium Used for Campylobacter Isolation Modified Campy-Cefex Agar: A Brucella agar (agar base plus horse blood and hemin) supplemented with cephalothin and amphotericin B for the selective isolation of cephalothin-resistant Campylobacter species such as C. jejuni, C. coli, and C. lari from food samples. Lab Work Procedure Today 1. Each group (two students) will pick up one broiler carcass. 2. The broiler carcass will be immersed into 400 ml buffered peptone water (BPW) in a poultry sam- pling bag, and vigorously shake for 60 s. Please do this within a large container; make sure the bags are not broken! 3. Transfer 25 ml of shaken BPW solution into 25 ml of Bolton broth in a tube, and mix very well. These mixtures will be incubated at 42 °C for 48 h under microaerophilic conditions (5.0% O2, 10% CO2, 85% N2) in a 2.5-liter microaerophilic jar. 4. The 370 ml BPW will be transferred into a sterilized bottle and incubated at 35 °C for 24 h. After 24 h 1. Subculture 0.1 ml of primary enriched BPW into 10-ml RV and 1.0 ml of enriched BPW into 10-ml TT medium, and incubate at 42 °C (RV) and 35 °C (TT) for 24 h. After 48 h 1. A loop of secondary enriched RV and TT solution will be streak plated onto XLT-4 and HardyCHROM agar, and incubate at 35 °C for 24 h to 48 h. 2. A loop of Bolton broth will be streaked on modified Campy-Cefex agar and incubated at 42 °C for 72 h under aforementioned microaerophilic conditions. 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses 45 Identification test 1. Presumptive Salmonella colonies (red colony with black center on XLT-4 agar and purple colonies on HardyCHROM agar) will be verified by Gram staining, Salmonella latex agglutination test, and API 20E test. 2. Presumptive Campylobacter colonies on the modified Campy-Cefex agar will be confirmed using the Campy-latex agglutination test and Gram staining (Gram-negative rods) to observe for cork- screw morphology. Since We Already Practiced Gram Staining and Latex Agglutination Test, Please Write Major Steps of Gram Staining and Latex Agglutination Test Below Gram Staining Latex Agglutination Test API 20E Test Procedure 1. Pick one to three presumptive Salmonella colonies from XLT-4 and HardyCHROM agar and suspend into a 5 ml of sterilized saline solution. 2. Use a bulb dropper to add the saline suspension into the cupules of the strip. 3. Fill the cupule with mineral oil for underlined test ADH, LDC, and URE. 4. Fill both the tube and cupule for boxed tests CIT, VP, and GEL. 5. Add 5 ml of water into the plastic base and place strip in the base. 6. Incubate the strip for 24 h at 35 °C. After 24 h 1. IND: Add one drop of Kovac reagent to record results in 2 min. 2. TDA: Add one drop of 10% ferric chloride immediately. 3. VP: Add one drop of 40% KOH and then one drop of 6% alpha-naphthol in 10 min. 4. Record the results (+ or -) on the worksheet. 5. Record the API number and add number up for the positive reaction, and search the code in the API reference book. 6. Record and confirm the identity of the isolation in Salmonella spp. 7. A possible Salmonella spp. code is 6704752 46 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses Figure of API 20 E Result Sheet Review Question Starting in July 2011, USDA-FSIS established new performance standards in response to national baseline studies requiring routine testing for Salmonella and Campylobacter in all processing plants. Questions: What are the performance standards? If you are a microbiologist working in a poultry company, what will you do for your company to meet this requirement, and if there are outbreaks happening, what will you do to isolate these two pathogens from contaminated chicken samples? API 20 E test result table of color change: Test name Positive Negative ONPG Yellow Colorless ADH Red or orange Yellow LDC Red or orange Yellow ODC Red or orange Yellow CIT Turquoise/dark blue Light green/yellow H2S Black deposit No black deposit URE Red or orange Yellow TDA Golden brown/red Yellow IND Pink-red ring Yellow VP Pink-red Colorless GEL Diffusion No diffusion GLU Yellow or gray Blue to green MAN, INO, SOR, RHA, SAC, MEL, AMY, ARA Yellow-yellow green Blue to blue green Positive Salmonella spp. Result Salmonella Typhimurium ATCC 14028 (positive control) 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses 47 A Salmonella spp. positive sample isolated from chicken carcass 48 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses 49 Part of Result Figures (Work from Previous Students) A. Latex agglutination test B. Gram staining results of presumptive Campylobacter spp. Gram-Negative, Single Rods with S Shape◻ 50 8 Isolation and Identification of Salmonella and Campylobacter spp. on Broiler Carcasses Class Notes ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________ Thermal Inactivation of Escherichia coli O157:H7 in Non-intact 9 Reconstructed Beef Patties Abstract We will be manufacturing non-intact reconstructed beef patties, inoculating it with E.coli O157:H7 and aerobically storing in a PVC film-covered foam tray. We will evaluate the cooking inactivation activity of E. coli O157:H7 in beef patties with various cooking time. We will be enumerat- ing survivals on support and selective medium and using USDA-IPMP 2013 software to analyze data and determine the fitted model. Finally, presumptive E. coli O157:H7 survivals will be identified using Enterotube II biochemistry test and latex agglutination test. In this chapter, it requires students to finish part of the work independently. Keywords E. coli O157:H7 Non-intact beef Thermal inactivation Thermocouple Latex agglutination test MacConkey agar Mug Enterotube II test Objectives Practice the cooking procedure to inactivate E. coli O157:H7 in manufactured non-intact reconstructed beef patties, and model the results using USDA-IPMP 2013 software. Major Experimental Materials Fresh ground beef E. coli O157:H7 strains Benchtop meat grounder Kitchen aid and manual hamburger patty maker Foam trays and air-permeable plastic film Griller and type-K thermocouple Buffered peptone water (100% and 0.1%) Filtered food sampling bag MacConkey agar, MacConkey agar with MUG, and Tryptic Soy Agar E. coliPRO™O157 Latex Kit Enterotube II © Springer International Publishing AG 2017 51 C. Shen, Y. Zhang, Food Microbiology Laboratory for the Food Science Student, DOI 10.1007/978-3-319-58371-6_9 52 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties Introduction As defined by the US Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS), non-intact beef products include ground beef, mechanically or chemically tenderized beef cuts, restructured entrees, and meat products that have been injected with brining solutions for enhance- ment of flavor and/or tenderness. Escherichia coli O157:H7 (ECOH) or non-O157-Shiga toxin-generating Escherichia coli (STEC) can generate Shiga toxin and cause severe hemolytic uremic syndrome, with as little as ten cells causing death in a formerly health person. Since 1999, ECOH has been considered an adulterant of raw, non-intact beef products. On November 2011, the USDA-FSIS announced that, as of June 2012, non-intact beef products would also be considered adulterated if they were contaminated with non-O157-STEC serogroups O26, O45, O103, O111, O121, or O145. Lab Work Procedure Inoculation and Beef Patty Preparation: The meat will be manually cut into trimmings and then coarse grounded in a meat grinder (Gander Mountain #5 Electric Meat Grinder, Saint Paul, MN). Ground meat will be then mixed with 40 mL of the E. coli O157:H7 inoculum cocktail in a bowl-lift stand mixer (Kitchen Aid Professional 600, Benton Harbor, MI) at medium speed for 2 min to ensure even distribution of the inoculum into the sample, which simulates E. coli O157:H7 contamination during non-intact beef preparation. A manual hamburger patty maker (mainstays 6-ounce patty maker, Walmart, Bentonville, AR) will be then used to make beef or veal patties with 170–180 g of grounded meat. Packaging: The reconstructed beef patties will be packaged aerobically in foam trays and covered using air-permeable plastic film and stored at 4.0 °C for 5 days. Cooking: After the 5-day storage, the beef steaks will be taken out from their packages, weighed and double panbroiled in a Farberware@ griller with setup temperature of 177 °C (or 350 °F) for 0, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 7, and 10 min. A type-K thermocouple was attached to the geometric center of the patty to monitor the internal temperature throughout the cooking with using PicoLog, a real time data recording software. Enumeration 1. Cooked beef steaks (need to be weighted) will be sterilized and transferred into filtered-whirl food sampling bag immediately. 2. Add 200 ml of 0.1% buffered peptone water into the sample bag. 3. Stomach for 1 min. 4. Conduct serial dilution into 9.0 or 9.9 ml 0.1% BPW solution to obtain final dilution factor 10−1 and 10−3, and spread plating onto MacConkey agar and Tryptic Soy Agar. Please draw your dilu- tion diagram in your notebook. 5. Incubate plates at 35 °C for 48 h. 6. Manually count colonies of agar plates and record onto your notebook; calculate as follows: Log10 CFU/g = Log10 [(Final CFU on plates/dilution factor)*(beef steaks gram + 200 ml)/beef steaks gram] For example, after cooking, the beef is 100 gram, 50 CFU on plates with dilution factor 10−1, and then final Log10CFU/g = Log10 (50 × 101 × 3) = Log101,500 = 3.2 log10CFU/g. 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties 53 Please draw your dilution procedure below: 54 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties Identification of E. coli O157:H7 Pick a typical presumptive E. coli O157:H7 from MacConkey agar (pink colony due to lactose fer- mentation positive) to conduct the following test: 1. Streak-plate onto sorbitol MacConkey agar with MUG and incubate at 35 °C for 24 h. Note: MUG is 4-methylumbel-liferyl-β-D-glucuronide. E. coli O157:H7 cannot ferment sorbi- tol, and it does not have enzyme β-D-glucuronidase; therefore, it is nonfluorescent white colony under UV light. 2. Latex agglutination test (E. coliPRO™O157 Latex Kit) (a) Place one drop of saline solution onto two wells of the test card. (b) Using sterilized loop, pick presumptive colony from MacConkey agar and mix with the saline solution. (c) Shake (very important!) the latex reagent bottle (control and test reagent) and place one free failing drop into each well. (d) Use a new sterilized loop to mix the regent with colony very well. (e) Rock the card for 1 min to observe the clumping. 3. Enterotube II test Enterotube II (picture below) is a tube of 12 compartmentalized, conventional agar media that can be inoculated rapidly from a single isolated colony on an agar plate. The media provided indicate whether the organism ferments the carbohydrates such as glucose, lactose, adonitol, arabinose, sorbi- tol, and dulcitol; produces H2S and/or indole; produces acetylmethylcarbinol; deaminates phenylala- nine; splits urea; decarboxylates lysine and/or ornithine; and can use citrate when it is the sole source of carbon in the medium. The Enterotube II is an example of a rapid, multi-test system used in the identification of unknown oxidase-negative, Gram-negative, rod-shaped bacteria of the family Enterobacteriaceae. The Enterobacteriaceae is a family of bacteria normally present in the intestinal tract of humans and animals. All members of the family ferment glucose and are oxidase-negative, facultatively anaerobic, Gram-negative rods with simple nutritional requirements. Procedure Step 1. Remove the screw caps from both ends; aseptically pick one presumptive E. coli O157:H7 colony from MacConkey agar with the sharp end (white end). Step 2. Twist the needle and pass it through the 12 wells and put it back to the original end, and screw the white cap back. Step 3. Bend and break the needle at the blue cap end; break the “window” (punch the plastic film) of ADO, LAC, ARB, SOR, VP, DUL/PA, URE, and CIT compartments. Screw the blue cap back. Step 4. Incubate the Enterotube II at 35 °C and read results in 24 h. Step 5. Interpret the results of your Enterotube II using the instructions below. Interpreting the Compartments: Circle the numerical value of each positive reaction. 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties 55 Compartment 1. Interpret the results of glucose fermentation: any yellow = +; red or orange = − If positive, circle the number 2 under glucose on your Results pad. Compartment 1. Interpret the results of gas production: wax lifted from agar = +; wax not lifted from agar = − If positive, circle the number 1 under gas on your Results pad. Compartment 2. Interpret the results of lysine decarboxylase: any purple = +; yellow = − If positive, circle the number 4 under lysine on your Results pad. Compartment 3. Interpret the results of ornithine decarboxylase. any purple = +; yellow = − If positive, circle the number 2 under ornithine on your Results pad. Compartment 4. Interpret the results of H2S production: true black = +; beige = − If positive, circle the number 1 under H2S on your Results pad. Compartment 4. Indole production. Your instructor will give you the Indole test results of your unknown. Compartment 5. Interpret the results of adonitol fermentation. Compartment 6. Interpret the results of lactose fermentation. Compartment 7. Interpret the results of arabinose fermentation. Compartment 8. Interpret the results of sorbitol fermentation: any yellow = +; red or orange = − If positive, circle the points granted for each carbohydrate given on your Results pad. Compartment 9. Voges-Proskauer test. This test is not used unless a final VP confirming test is later called for. Compartment 10. Interpret the results of dulcitol fermentation: yellow = +; any other color = − If positive, circle the number 1 under dulcitol on your Results pad. Compartment 10. Interpret the results of PA deaminase: black or smoky gray = +; any other color = − If positive, circle the number 4 under PA on your Results pad. Compartment 11. Interpret the results of urea hydrolysis: red or purple = +; beige = − If positive, circle the number 2 under urea on your Results pad. Compartment 12. Interpret the results of citrate utilization: any blue = +; green = − If positive, circle the number 1 under citrate on your Results pad. Once you have completed your biochemical readout, be sure you: Add all the circled numbers in each bracketed section and enter the sum in the space provided below the arrow on your Results page. You now have a five-digit reference number. Locate the five-digit number in the interpretation guide booklet and find the best identification in the column entitled “ID Value.” Results pad for calculating identification code number: You may use the Results pad above to keep in your laboratory notebook. E. coli O157:H7 code should be 75340. 56 9 Thermal Inactivation of Escherichia coli O157:H7 in Non-intact Reconstructed Beef Patties USDA-Integrated-Predictive-Modeling-Program Software IPMP 2013 is a new-generation predictive microbiology tool. It is designed to analyze experimental data commonly encountered in predictive microbiology and for the development of predictive models. In this chapter, we will use survival models to analyze the results. Brief procedures: Step 1. Download and install “USDA-IPMP 2013 software” into your computer: https://www.ars.usda.gov/northeast-area/wyndmoor-p a/eastern-regional-research-center/docs/ integrated-pathogen-modeling-program-ipmp-2013/ Then,

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