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Volume IV - 2.5 Food Pathogens and Indicator Organisms

DFS Pyramid Logo

Orientation and Training

Food and Drug Administration

DOCUMENT NO.:

IV-02
VERSION NO.: 1.6

Section 2 - Microbiology

EFFECTIVE DATE:
10-01-03
REVISED:
05-02-14

The objective of this section is to introduce the microbiologist trainee to the most common known food borne disease-causing organisms and to the types of methods used to recover them from food. The organisms of interest will change as more emerging pathogens are encountered. The methods will continue to be updated as new ideas and technologies bring forth more rapid and more sensitive procedures. The intention here is to give the trainee a sufficient number of training samples to learn typical analyses and then move immediately into analyses of regulatory samples. Supplemental information on pathogens is found in the Bad Bug Book,  a web site of the Center for Food Safety (CFSAN).

2.5.1  Salmonella

A. Objective

  1. To recover Salmonella in foods by using pre-enrichment and enrichment techniques.
  2. To screen samples for Salmonella using rapid methods.
  3. To identify Salmonella using biochemical and rapid methods.
  4. To examine causes and symptoms of Salmonella food borne disease.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at: http://www.cfsan.fda.gov/~ebam/bam-5.htm

  1. Using BAM/AOAC methods, analyze four different food items for Salmonella. Use foods that call for different pre-enrichment broths. Prepare at least one 15-sub composite. The trainer may spike some or all of the foods with different serotypes of Salmonella. Screen sample using VIDAS AOAC method for Salmonella.
  2. Identify isolates using conventional biochemicals and one or more rapid methods such as API 20, Vitek, or MICRO ID.

C. Questions

  1. Why are different pre enrichment media used for different foods?  Give examples. Why is a pre-enrichment step needed for the recovery of Salmonella?
  2. Why is more than one type of enrichment (Rappaport-Vassilades, selenite cystine and tetrathionate broths) used instead of just one? 
  3. Describe sugar reactions in triple sugar iron agar (TSI) tubes. 
  4. Why use three primary plating media instead of one?
  5. Which Salmonella species would we most likely find if we used only one of the media?  Why? 
  6. Which Salmonella species do not produce hydrogen sulfide? 
  7. What is indole? 
  8. Can we identify all groups of Salmonella with antisera?  Give a reason. 
  9. Describe symptoms and onset time of Salmonella food borne disease.
  10. Describe and give examples of foods in Food Category I, II and III. (See BAM Chapter 1 Food Sampling)
  11. Why is the pH of the enrichment broth adjusted after addition of sample?
  12. What does the VIDAS assay detect? 
  13. Why do we need to boil the sample before performing the VIDAS assay?
  14. Which organism is most likely to cause a VIDAS false positive result?

2.5.2  Listeria monocytogenes

A. Objective

  1. Evaluate what types of samples may be candidates to be analyzed for Listeria monocytogenes.
  2. Examine sample matrices and determine appropriate enrichment procedures. 
  3. Choose appropriate methodology involved in the detection of Listeria monocytogenes.
  4. Differentiate biochemical characteristics between Listeria monocytogenes and other Listeria spp.
  5. Develop skills in the use of rapid method test kits for the detection and differentiation of Listeria species including but not limited to DNA-Probe techniques VIDAS, VITEK, API20E, and MICRO-ID.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:  
    www.cfsan.fda.gov/~ebam/bam-10.htm

Analyze two independent samples for Listeria monocytogenes. Each sample should consist of 10 sub-samples. Samples should be analyzed using official methodology outlined in official compendia (i.e. BAM). The trainer should spike each sample with a minimum three Listeria spp. The trainee should be able to identify and recover Listeria monocytogenes from each sample using both conventional and rapid method techniques (i.e. verify using Listeria VIDAS or other applicable rapid methods). Suggested samples include soft cheese and smoked fish (hot or cold smoked).

C. Questions

  1. What is unusual about the appearance of the motility of Listeria?
  2. At what high and low temperatures will Listeria grow?
  3. Describe the CAMP reactions of different Listeria species. What do the letters "CAMP" stand for in the CAMP test?
  4. Why is Rhodococcus equi, and not another Rhodococcus species used in the CAMP test?
  5. Describe the symptoms and onset time of Listeria food borne disease.
  6. Describe the appearance of typical Listeria monocytogenes colonies on selective plating media including but not limited to Oxford, Palcam and BCM?
  7. Describe how acid production from various carbohydrates (mannitol, rhamnose, xylose) typically differs between Listeria monocytogenes and Listeria innocua.
  8. What role does the addition of selective agents in pre-enrichment and enrichment media play?
  9. Name two ß-hemolytic Listeria spp and two non ß-hemolytic Listeria spp.

2.5.3 Escherichia coli

A. Objective

  1. To enumerate and identify E. coli in foods.
  2. To familiarize the trainee with the different pathogenic strains of E. coli.
  3. To understand the MPN technique and how it is calculated.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to Escherichia coli official sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-4.htm

  1. Analyze at least 10 sub samples of a frozen food. The trainer will spike some of the subs with E. coli, other coliforms, etc. Use the "Frozen Food Method".
  2. Analyze at least 10 portions (sub samples) of oysters or clams. Use the "Shellfish Method".
  3. Analyze at least 10 sub samples of finished product shelled walnuts or pecans. Use the "Tree Nut Method”.
  4. The trainer will discuss the methods used for E. coli pathogenicity.
  5. Find the correct MPN of several MPN problems provided by the trainer.

C. Questions

  1. Describe different types of E. coli that cause food borne disease. List methods used for their determination.
  2. Is there an acceptable way to minimize foaming when shellfish are homogenized? 
  3. What percent foam is aspirated when pipetting sample from homogenate?  Would this affect the results? 
  4. In the Tree Nut Method, why is there a "rest period" between shaking of the original dilution? 
  5. Describe how a Gram stain is performed and the meaning of the results.
  6. Describe the quadrant streaking technique to obtain isolated colonies.
  7. What is the IMViC pattern for E. coli
  8. What does a typical E. coli isolate look like on L-EMB?
  9. When is it appropriate to use LST-MUG?  How does LST-MUG work?
  10. What time period is allowable between sample shaking and inoculation of tubes?

2.5.3.1  Enterohemorrhagic E. coli  (EHEC)

A. Objective

  1. To isolate and identify EHEC from food samples.
  2. To introduce PCR assay for detection of Shiga-like toxin genes in EHEC.
  3. To examine causes and symptoms of hemorrhagic colitis.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-4.htm

  1. Examine 2 different food samples (animal feed and cheese) of 10 sub samples using BAM/AOAC methods. The trainer will spike with some of subs with E.coli, EHEC and another coliform.
  2. Examine two foods for Shiga-like toxin genes in EHEC, using PCR method. The trainer will spike foods with an organism (or organisms) possessing one or both of the genes.

C. Questions

  1. Do all E. coli strains ferment sorbitol?
  2. What does TC SMAC stand for?
  3. Why is TC SMAC a better medium than HC agar for detecting E. coli 0157:H7?
  4. What is the advantage of streaking at 6 and 24hr? 
  5. Describe the symptoms of hemorrhagic colitis and the onset time.

2.5.4 Staphylococcus aureus

A. Objective

  1. To analyze foods for S. aureus using both the MPN and direct plating techniques.
  2. Identify and differentiate coagulase positive versus coagulase negative staphylococci.
  3. Differentiate biochemical characteristics between Staphylococcus spp
  4. To introduce ELISA techniques for toxin detection.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-12.htm

Analyze at least four foods for S. aureus using the MPN and the direct plating methods. The trainer will spike the foods with coagulase positive and coagulase negative staphylococci. Evaluate and compare the two methods.

Test one spiked food for staphylococcal enterotoxin using an ELISA based assay. If a test kit is not found, the trainer is responsible for explaining the theory and principles involved.

C. Questions

  1. Describe the symptoms and onset time of staphylococcal food borne disease.
  2. Describe the difference between intoxication and infection.  Which one is associated with S. aureus?
  3. Which ingredient/s of Baird Parker medium help injured organisms grow?
  4. What are typical observations of coagulase positive Staphylococcus aureus when plated on Baird Parker medium?  What is indicated by the presence or absence of a halo around an isolated colony on Baird Parker medium?
  5. Name the staphylococcal enterotoxin types and tell which is the most common cause of food borne disease.

2.5.5  Coliforms

A. Objective

To analyze foods and water for coliforms and fecal coliforms

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-4.htm

  1. Use foods prepared for E. coli analysis above for the coliform analysis.
  2. Test two water samples for coliforms using a five tube MPN technique. Read and use procedures in Standard Methods for Water and Wastewater, current edition.

C. Questions

  1. Why is sodium thiosulfate added to jars used to collect water samples? (see Investigations Operations Manual (IOM), current  edition.)
  2. Explain the difference between coliforms and fecal coliforms.
  3. What organisms are considered coliforms?

2.5.6  Aerobic Plate Count 

A. Objectives

Training should be consistent with methodology utilized in the home laboratory.

  1. To analyze foods for number of aerobic organisms that grow at 35 C.
  2. Demonstrate effective preparation of decimal dilutions from food homogenate, milk, cosmetic or product rinse.
  3. Apply conventional pour plate technique.
  4. Apply spiral plate count (SPLC) method.
  5. Distinguish significant figures when calculating APCs or SPLCs.
  6. Calculate reporting results for APCs in common and uncommon cases.
  7. Utilize proper aseptic technique and quality control principles for conventional or SPLC methodology.
  8. Apply proper plating procedure.
  9. Implement proper sterility controls.
  10. Calibrate spiral plater.
  11. Examine and interpret results on the SPLC method.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at: 

     http://www.cfsan.fda.gov/~ebam/bam-3.htm

Analyze at least four foods for APC/g or APC/ml. The trainer will supply foods with a count range between 101 and 106. The trainee will report results in correct significant figures.

Also read Association of Official Analytical Chemists International (AOACI) , American Public Health Association (APHA), Standard Methods for the Examination of Dairy Products, and the International Dairy Foundation (IDF) sections regarding the Conventional Plate count methodology and Spiral Plate Count (SPLC) methodology.

C. Questions

  1. Name foods that may have natural high counts. Name foods that should have low counts.
     
  2. Why are we interested in an APC count?
  3. Why do "spreaders" sometimes form when doing an APC?  At the air-agar interface? At the agar-glass interface?
  4. How long and at what temperature can one "thaw" a frozen food?   Discuss. 
  5. Will there be a change to PAC results with repeated freezing and thawing of product?
  6. What is the proper procedure for manually mixing dilution blanks?
  7. At what temperature should pour plates be dispensed?
  8. How should the analyst interpret plates with more than 250 colony-forming units (CFUs)?
  9. How should the analyst interpret plates with less than 25 CFUs ?

2.5.7  Yeast and Mold Count

A. Objective

  1. To enumerate colonies of yeasts and molds in foods.
  2. To introduce dilution and spread plate techniques. 
  3. To revisit reporting in significant figures.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-18.htm

Analyze at least four foods for (col)/g or (col)/mL. The trainer will supply foods with a count range between 101 and 106. The trainee will report results in correct significant figures.

C. Questions

  1. Why are we interested in yeast and mold counts?   
  2. What is the medium of choice for yeast and mold analysis? 
  3. What medium is especially useful for analyzing samples containing "spreader" molds? 
  4. What agent or agents are added to the agar to inhibit bacterial growth?
  5. Why should the plates be left undisturbed until the incubation period is complete?

2.5.8  Vibrios

A. Objective

  1. To recover and identify Vibrio cholerae, V. vulnificus and V. parahaemolyticus.
  2. To introduce polymerase chain reaction (PCR) techniques.
  3. To introduce non-radioactive gene probe methods.
  4. To introduce enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) methods.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at: 
     http://www.cfsan.fda.gov/~ebam/bam-9.htm 
     http://www.cfsan.fda.gov/~ebam/bam-28.htm

  1. Examine at least one oyster sample for V. cholerae, V. vulnificus, and V. parahaemolyticus. The trainer will spike the sample with various Vibrio spp.
  2. Examine one food for toxigenic V. cholerae using the PCR method.

C. Questions

  1. Describe the food borne illness and onset times of the Vibrio spp. that were studied.
  2. What are the advantages and disadvantages of using a PCR method?
  3. Why is NaCl added to media used for Vibrio spp?
  4. Why do some methods enumerate the organism and other methods only check for the presence of the organism?
  5. What are the characteristics of V. mimicus? What are the similarities and differences of this organism compared to V. cholerae?
  6. What are the major factors in the pathogenesis of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus?

2.5.9  Bacillus cereus

A. Objective

  1. To recover and identify B. cereus group organisms from foods.
  2. Differentiate biochemical characteristics between various Bacillus spp.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

      http://www.cfsan.fda.gov/~ebam/bam-14.htm

The trainer should spike at least four dry foods with B. cereus. In addition, the trainer should spike foods with B. thuringiensis and B. cereus var. mycoides.

C. Questions

  1. Describe the symptoms and onset times for the two types of B. cereus food borne diseases. What foods have been implicated in each type?
  2. Can B. thuringiensis produce diarrheal antigens?  Diarrheal disease?
  3. The symptomatic profile of emetic type of food poisoning produced by some strains of B. cereus most closely mimics that of Clostridium pefringens or Staphylococcus  aureus?
  4. The toxins of B. cereus most commonly associated with food poisoning are?
  5. Explain the purpose for each ingredient used in MYP agar and how B. cereus can be interpreted on MYP agar.
  6. When is the Plate Count Method recommended?  When is Most Probable Number (MPN) recommended?
  7. When interpreting test results in particular (motility, hemolytic activity, plating characteristics, and crystal production) what are typical results for each pertaining to B. cereus?

2.5.10  Campylobacter

A. Objective

  1. To recover and identify Campylobacter jejuni and C. coli from foods and water.
  2. To introduce microaerobic culturing techniques.
  3. To introduce the use of rapid methods for presumptive identification of Campylobacter.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-7.htm

Analyze three different products such as water, milk/dairy, and a poultry product for Campylobacter spp. Trainer will spike samples with C. jejuni and C. coli. Isolates will be tested with the Dryspot Campy Test or Alert for Campylobacter.

C. Questions

  1. Describe the symptoms and onset time of Campylobacter food borne disease.
  2. Describe methods used to obtain microaerobic conditions.
  3. What are the oxygen-quenching compounds added to Campylobacter media? 
  4. Which biochemical test is used to differentiate between C. jejuni and C. coli?
  5. What are the advantages and disadvantages of using rapid methods such as the Dryspot  Campy Test or Alert for Campylobacter?

2.5.11  Yersinia

A. Objective

  1. To learn the theoretical and analytical concepts related to Yersinia.
  2. To recover and identify Yersinia enterocolitica from food products.
  3. To introduce the theoretical concepts of pathogenicity testing.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-8.htm

Analyze two different products such as water and milk for Yersinia enterocolitica. Trainer will spike samples with Yersinia enterocolitica.

C. Questions

  1. Describe the symptoms and onset time of Yersinia enterocolitica illness.
  2. Does Yersinia grow and survive during refrigerated storage?
  3. What enrichment broths and selective media are used to culture Yersinia enterocolitica?
  4. Describe the biochemical characteristics of Yersinia enterocolitica.
  5. What is the relationship of plasmids and Yersinia?
  6. What types of tests are used to determine pathogenicity?

2.5.12  Clostridium perfringens

A. Objective

  1. Recover and identify C. perfringens from foods.
  2. Display anaerobic culture techniques.
  3. Differentiate biochemical characteristics between various Clostridium spp.
  4. Effectively utilize Reversed Passive Latex Agglutination (RPLA) test kit for the detection of enterotoxin.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-16.htm

The trainer should spike at least two foods for C. perfringens. The trainee will analyze the samples by plate count method using Tryptose-Sulfite-Cycloserine (TSC) without egg yolk and the alternative plating method using TSC with egg yolk. The trainee should also complete all presumptive confirmation and completed confirmation testing. If possible, the trainee should also be introduced to the RPLA enterotoxin test kit.

C. Questions

  1. Describe the symptoms and onset time for C. perfringens food borne disease.
  2. How would a microbiologist test the organism for toxin production?
  3. Tell when a microbiologist would use each enumeration method.
  4. How do anaerobe jar commercial systems produce anaerobic conditions?  How can you tell if it worked? 
  5. Describe stormy fermentation and how to test C. perfringens for stormy fermentation.
  6. Describe key characteristics of C. perfringens including motility, gram-reaction, nitrate reduction, and lecithinase activity.

2.5.13  Clostridium botulinum

A. Objective

  1. To present theory and methodology on recovery of C. botulinum from food.
  2. To introduce the theoretical concepts of the mouse bioassay.
  3. To discuss immunologic and other methods published and/or under collaboration to replace the use of animals.

B. Assignment

It is the trainer’s primary responsibility to transfer knowledge both practical and in theory related to sample analysis. This may be accomplished through a series of designated training samples. Once the trainer is confident that the trainee can successfully and independently perform the analysis, the trainee will be issued a series of training samples.

Read Bacteriological Analytical Manual (BAM) chapter, online at:

     http://www.cfsan.fda.gov/~ebam/bam-17.htm

Carry out the preparatory analytical procedure on a food product for isolating botulinum toxin. Trainer will spike food with C. sporogenes or C. botulinum depending on experience of trainee. 

C. Questions

  1. What are the symptoms and onset time of botulism?
  2. What is wound botulism and infant botulism? 
  3. Name the different types of toxins and which have been implicated in human botulism.
  4. Describe how the MLD is calculated.
  5. What is the difference between preformed and formed toxin?