THE HAMBURGLAR
PROBLEM:

You have a background in food microbiology and your local community has come to you for help. It seems there has been an outbreak of E. coli and the community leaders are not sure where the microbe is coming from. However, it is felt that hamburger meat may be the source. You must help the community before more people get sick.

BACKGROUND INFORMATION:

Coliforms are a group of bacteria, which are found in large numbers in the gastrointestinal tract of most animals, including man. Coliforms are gram-negative, non-sporeforming rods that are facultative anaerobes. Coliforms ferment sugars to produce an acid and a gas. Two species are of major interest to the food microbiologist: Escherichia coli and Enterobacter aerogenes. Both species occur naturally in the soil, in water and in many raw foods including milk. E. coli and E. aerogenes are easily killed by cooking temperatures, so their presence in cooked food is considered to be caused by contamination after thermal processing. The main reason to study coliforms is as an indicator of potential human health problems. Recent discovery of human hemorrhagic E. coli 0157:H7 (first recognized as a human pathogen in 1982) has increased interest in coliforms by the food industry.

Selective and differential media are utilized to isolate or identify particular organisms. Selective media allow certain types of organisms to grow and inhibit other organisms. Differential media are used to differentiate closely related organisms or groups of organisms. Owing to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic color changes or growth patterns that are used for identification or differentiation. A variety of selective and differential media are utilized in medicine, diagnostic laboratories, water pollution control and in food and dairy laboratories.

In this lab three different media are utilized. Nutrient agar is neither selective nor differential therefore it will allow almost all bacteria to grow. Brain Heart Infusion (BHI) agar can also be used if nutrient agar is not available. This agar is also non-selective and non-differential.

Eosin Methylene Blue (EMB) agar will be used and is selective for gram-negative bacteria and differential for E. coli . If E. coli is present, the colonies will have a green metallic sheen. If E. aerogenes is present, it will have pink colonies with occasional purple centers. EMB agar contains Eosin Y and Methylene blue, which are used as indicators. When lactose (a sugar) is fermented, acids are produced and a color change is the result (similar to acid/base indicators). With the exception of E. coli , pink colored colonies are a result of lactose fermentation. If the bacteria do not ferment lactose (that is, they are not coliforms), no acid is being produced and the colonies are basically colorless. Methylene blue acts as an inhibitor of gram-positive bacteria; therefore bacteria of this type cannot grow on this medium.

If using Petrifilm® the coliform count plate is a selective media for all coliforms and gram-positive bacteria will not grow because of the violet red bile nutrients. A tetrazolium indicator dye is present to cause the coliform colonies to be red. The E. coli count plate is selective for coliforms and differential in the case of E. coli . With E. coli Petrifilm®, the medium recipe is the same as the coliform Petrifilm®, with the exception of an added glucoronidase indicator that causes the E. coli colonies to be blue. Also, coliforms growing on both kinds of Petrifilm® will have gas bubbles around the colonies due to lactose fermentation.

0.1% peptone or 0.85% physiologic saline is used in this lab as a diluent and is an isotonic solution for the bacteria to grow. A hypertonic or hypotonic environment will stress or possibly kill bacteria due to the osmotic pressure and thus give false bacterial counts.

Aseptic technique or sterile technique is defined as the procedures designed to keep unwanted microorganisms from contaminating either sterile materials or pure cultures of microorganisms. It is important that students are shown how to perform aseptic technique. Aseptic technique should be followed throughout all procedures of this experiment.

See Appendix C for information on counting coliforms, ordering Petrifilm® and aseptic technique.

OBJECTIVE:

  • To familiarize the student with coliforms (members of the family Enterobacteriaceae ), in this case E. coli and E. aerogenes.
  • To detect and quantify coliforms in food, using plate count techniques.
  • To observe examples of certain biochemical characteristics as well as cellular and colony morphology that together can assist in the identification of bacteria, specifically coliforms.
  • Demonstrate knowledge of and consistently practice aseptic technique procedures when handling microorganisms.

    MATERIALS:

  • Sterile screw cap test tubes
  • 1-ml sterile pipettes
  • Blender
  • Hamburger
  • E. coli cultures (Order active or freeze dried from a biological supply company such as Flinn)
  • E. auerogenes cultures (Order active or freeze dried from a biological supply company such as Flinn)
  • 0.1% peptone or 0.85% saline solution
  • Autoclave
  • Petrifilm® Coliform count plate
  • Petrifilm® E. coli count plate
  • Eosin Methylene Blue (EMB) agar
  • Nutrient agar
  • Sterile petri plates or petri dishes if no Petrifilm® is available
  • Hand counter to count bacteria (approximately $12.00)
  • 90-ml sterile test tube
  • Sharpie®
  • Stomacher bag with filter (order from biological supply company)
  • Bent glass rod for spread platting
  • Beaker 100-ml
  • Aluminum foil

    SAFETY AND DISPOSAL:

    Potential human pathogenic organisms will be used in this lab. Aseptic technique is mandatory to prevent infection. KEEP HANDS AND OTHER OBJECTS AWAY FROM YOUR MOUTH. NO MOUTH PIPETTING. If a spill occurs, report it to your instructor and wash the area with 70% ethanol. Place all pipettes and test tubes in the appropriate container as instructed by your teacher. Wash lab counter before and after experimentation with 70% ethanol solution. Wash hands thoroughly with bactericidal soap before and after experiment.

    TIME:

    Preparation- Bacterial cultures must be prepared the night before beginning lab. A few minutes are required to inoculate bacteria, assuming you have prepared a working stock or previously bought active or freeze dried cultures (The instructions on how to work with the active and freeze dried cultures should accompany the order.).

    Activity- One class period to perform serial dilutions and prepare culture plates. 18-24 hours are needed to grow bacteria. One class period to count plates.

    PROCEDURE:

    1. To be done by the instructor:
      1. At least two days before the lab, you need to have all media, including peptone blanks, sterilized and ready to go. At least two sterile broth tubes (nutrient or BHI) will be needed to inoculate prepared cultures.
      2. Obtain test tubes with screw caps. Students will need 9 for each group. Place 9 ml of 0.1% peptone or 0.85% physiologic saline in each test tube with a screw cap. Sterilize in the autoclave at 121C for 15 minutes at 15 pounds per square inch of pressure.
      3. The day of lab, prepare hamburger solution by placing 10 g of hamburger meat in a blender. Add 90 ml of sterile 0.1% peptone or 0.85% physiologic saline, and mix for two minutes on low. Add 1 ml of E. coli and 1 ml of E. aerogenes. This solution will be the 10 -1 dilution and should be given to the students in the test tube labeled 10 -1 . 2 ml of this solution should be plenty per group. It is easier to obtain the 2-ml of solution if a stomacher is available. This contains a filter, which will leave solid material behind that might clog up the pipette.
      4. Instructors should explain and demonstrate how to perform serial dilutions, inoculations, Petrifilm® and spread plating technique. Schematic drawings are provided in Appendix C.
    2. To be done by the student (All materials have been sterilized prior to the lab. Take care to use aseptic technique):
      1. You have been given 9 test tubes label 10 -2 through 10 -10 . The test tube labeled 10 -1 contains a hamburger dilution (10:1) that has been given to you by your instructor. It is your job to determine if the hamburger meat has fecal contamination.
      2. To avoid mistakes start with all test tubes on the back row of your test tube rack. Once you have performed the first dilution, place that tube up one row. You will do this with each subsequent dilution.
      3. Using aseptic technique for all procedures, take 1 ml of the 10 -1 dilution and place it into the test tube labeled 10 -2 . Shake vigorously 20-30 times using full-arm swings over approximately a 0.5-meter arc. You can use a vortexer if one is available instead of shaking. This should be done with each subsequent dilution.
      4. Take 1 ml from the 10 -2 test tube and place it in the 10 -3 test tube.
      5. Take 1 ml from the 10 -3 test tube and place it in the 10 -4 test tube. This is the process for serial dilutions and should be continued until the last test tube (10 -10 ).
      6. Depending on the experiment used, plating can be done a couple of ways: (Using Sharpies® have students label their plates and Petrifilm® on the edges as not to obscure the view when counting colonies.)
        1. When using Petrifilm®:
          • Obtain 1 ml of dilution in test tube labeled 10 -5 . Place this onto the Petrifilm® coliform count plate labeled 10 -5 using the plastic spreader. Place Petrifilm®, invaginated sides down, over the inoculum and press gently. The inoculum should form a circle under the top layer of the Petrifilm® coliform count plate. Continue this process for test tubes labeled 10 -6 through 10 -10 .
          • To differentiate between the number of E. coli and E. aerogenes , have the students repeat the above process using Petrifilm® E. coli count plate, which will identify the number of E. coli present.
          • Incubate Petrifilm® upright in stacks of no more than 20 at 37C overnight.
        2. When using petri dishes or plates:
          • Obtain 0.1 ml of test tube labeled 10 -4 . Spread plate this on nutrient agar or (BHI) agar. Label the plate 10 -5 . Continue this for all test tubes labeled 10 -5 through 10 -9 .
          • To differentiate between the number of E. coli and E. aerogenes , have the students repeat the above process using Eosin Methylene Blue (EMB) agar, which will show a green metallic sheen where E. coli is present and pink with possible purple centers for E. aerogenes.

            NOTICE: 10 -4 becomes 10 -5 on the plate. 10 -5 becomes 10 -6 , etc.

          • Incubate petri plates in an inverted position so as to prevent condensation from dripping on the agar surface and smearing together the colonies.
          • After all spread plating is completed, incubate plates at 37C for 24 hours.
      7. After incubation is finished, count the number of colonies growing on the agar plates and Petrifilm® by using a Sharpie® to mark the colonies counted. If a hand counter is available, the counting process is much easier. If plates have a number too high to be counted (greater than 250-300), label them as TNTC (to numerous to count). Record all data in Table 1.
      8. Using Table 1, collect all plate and film count data from all groups in the class. Average the data from the class.
      9. When the experiment is completed and all colonies are counted, place plates or Petrifilm® in an orange biobag and autoclave. When autoclaving is finished, mark as treated waste and place the orange biobag in a black trash bag.

    QUESTIONS:

    1. When comparing petri dishes with Petrifilm®, compare10 -5 on the film to 10 -5 on the plate. Were there any discrepancies in the number of CPF/g?


    2. What factors may have caused differences in colony counts between the two techniques?


    3. What is the significance of using aseptic technique?


    4. How did the total colony count compare to the coliform count?


    5. Do you think the hamburger is safe to use?


    6. If, in your opinion, the hamburger is tainted, can it still be used? Explain.





    7. Did your group get plate counts similar to the class mean? Why or why not.





    FOR ADDITIONAL INFORMATION CONTACT:

    Dr. Michael Johnson
    Robert Story - Microbiology, Food Science Department, U of A- Fayetteville,
    rstory@comp.uark.edu

    TEACHER'S GUIDE:

    EXPECTED RESULTS:

    1. If dilutions are done correctly, there will be a noticeable 10-fold difference in the colony count of subsequent plates.

      EXAMPLE: If the 10 -7 plate had approximately 300 colonies the 10 -8 plate should have 30 colonies and the 10 -9 should have 3 colonies.

    2. Non-selective media (nutrient of BHI) will have higher counts compared to corresponding sample on the selective media (EMB and Petrifilm®).
    3. If a plate has over 250 colonies it is difficult to count the number and is expressed as too numerous to count (TNTC).
    4. After counting is completed and data is recorded in table 1:
      • Compare total counts (non-selective) with level of total coliforms (total counts on EMB, coliform and E. coli Petrifilm®).
      • Compare E. coli count on EMB vs. Petrifilm®.
      • Compare E. coli vs. other coliform count.
    5. Students will determine CFU/g

      EXAMPLE: If there are 115 colonies in the 10 -7 plate, then there are 115 x 10 7 CFU/g hamburger or 1.15 x 10 9 CFU/g hamburger.

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