Minimizing Bacteria in the Thawing and Cooking of Meat

Objective

The purpose of this project is to determine which method of defrosting meat is safest and which method of cooking kills the most bacteria.

Introduction

Which is the safer way to thaw frozen meat: at room temperature or in the refrigerator? For cooking meat safely, is a microwave as good as a conventional oven?

One way to find out is to measure how many viable bacteria are present in samples of meat that have been thawed or cooked by the different methods mentioned above. How do you measure the number of viable bacteria? One way is to homogenize a sample of meat in a blender, dilute the sample, and then plate it on a bacterial culture plate. The plate is then incubated overnight (or longer), and visible colonies of bacteria are then counted. The goal is to dilute the sample sufficiently so that individual bacteria are separated from one another on the plate, meaning that each colony will have arisen from an individual bacteria—referred to as a colony forming unit or CFU.

Typical laboratory cultures have between 10⁶ and 10⁹ bacteria/mL, and for plating bacteria, you typically use a volume of 100 μL (which is the same as 0.1 mL). So if you simply took your sample straight from the culture, you'd expect to have between 10⁵ and 10⁸ (100,000 to 100,000,000) bacteria in your 100 μL sample. Obviously, you would end up with far too many colonies to count! In fact, the plate would be so densely covered that you wouldn't be able to distinguish individual colonies.

To get around this problem, the obvious solution is to dilute the sample. If you wanted to end up counting about 100 colonies per plate, then you'd need to dilute between 1,000– and 1,000,000-fold. It's not practical to make such large dilutions in a single step, so a good way to do this is by using serial dilutions. The diagram in Figure 1, below, illustrates the process. Each tube starts out with 9 mL of sterile water. 1 mL of the bacterial culture solution is added to the first tube, and mixed. This dilutes the bacterial solution by a factor of 10. Then, 1 mL of the solution from the first tube is removed, and added to the 9 mL of sterile water in the second tube. This is another 10-fold dilution, making a 100-fold dilution of the original solution.

Figure 1. Serial 10-fold dilutions of a bacterial sample.

The serial dilutions are continued in a similar manner until the desired final dilution is achieved. In order to calculate how many bacteria were present in the original solution, you count colonies on the plate, and then multiply by the total dilution factor. You can see that it is important to make the volume measurements accurately and reproducibly for this process. Errors in measurement will cause errors in the bacterial count.

In this project, you'll see how you can apply this method to figure out how many viable bacteria there are in samples of meat that have been exposed to different thawing and cooking conditions.

Terms, Concepts and Questions to Start Background Research

To do this project, you should do research that enables you to understand the following terms and concepts:

• colony forming unit (CFU),
• serial dilution.

Questions

• Why are the samples diluted so many times before plating them on agar?
• What are some potential sources for error in the bacterial counts in this experiment?
• What can you do to minimize those sources of error?
• For more advanced students, what can you do to estimate the size of the error in the bacterial counts? (See Variation 1, below.)

Bibliography

• The first part of this webpage describes the method used in this project for estimating the number of bacteria in samples. More advanced students can read the entire page and learn about chi-square curve-fitting:
  Kirkman, T., date unknown. "Chi-Square Curve Fitting," Department of Physics, College of St. Benedict & St. John's University [accessed September 14, 2006] http://www.physics.csbsju.edu/stats/chi_fit.html.
• This webpage has background information on bacterial growth. It is from an online textbook of bacteriology, which can be an excellent source of further information on bacteria:
  Todar, K., 2002. "Growth of Bacterial Populations," Todar's Online Textbook of Bacteriology, Department of Bacteriology, University of Wisconsin, Madison [accessed September 14, 2006] http://textbookofbacteriology.net/growth.html.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• 10 ml graduated cylinder,
• 1 mL automatic pipettor with sterile tips,
• 200 μL automatic pipettor with sterile tips,
• 10 sterile, disposable 10 mL pipettes,
• digital scale (grams),
• 100 sterile, disposable 15 mL test tubes,
• 4 L sterile water,
• styrofoam chest,
• 2 100°C thermometers,
• blender,
• 40 tryptic-soy agar plates,
• bent glass rod for spreading bacteria on plates,
• bunsen burner,
• ethanol,
• aluminum foil,
• plastic wrap,
• microwave oven,
• standard oven,
• stereo microscope,
• 2 lbs. boneless, skinless meat (e.g., beef, chicken, or pork).

Experimental Procedure

Test 1 - Thawing Meat at Room Temperature vs. In Refrigerator

1. Cut, measure, and weigh 14 pieces of meat (approx. 5 cm × 5 cm × 2 cm, about 65 g each).
2. Set aside two pieces of meat for a baseline bacterial count.
3. Divide remaining meat pieces into two piles, wrap each in plastic wrap & foil, and freeze (up to 14 days).
4. Thaw one package of meat pieces at room temperature until the internal temperature of meat the reaches room temperature. Record how long this takes and the room temperature in your lab notebook.
5. Thaw the other package of meat pieces in the refrigerator until soft. Record how long this takes and the temperature in your lab notebook.
6. Measure bacterial count for two samples from each condition.

Test 2 - Cooking Meat in Microwave

1. Take two samples of meat thawed at room temperature and two samples thawed in the refrigerator and cook in microwave until the internal temperature of the meat reaches the recommended temperature: beef, 72°C; pork, 77°C; chicken, 85°C.
2. Measure bacterial counts of cooked meat samples.

Test 3 - Cooking Meat in Standard Oven

1. Take two samples of meat thawed at room temperature and two samples thawed in the refrigerator and cook using a standard oven heated to 190°C (375°F). Recommended cooking times are: beef, 20 min.; pork, 17 min.; chicken, 15 min.
2. Measure bacterial counts of cooked meat samples.

Measuring Number of Bacteria Present in a Meat Sample

1. Puree a piece of meat in sterilized blender with an small amount of sterile water (use the same amount of water each time). Note: to sterilize the blender, clean the jar and blade assembly with warm, soapy water and allow to dry. Wrap the blender jar and blade assembly only (not the base with the electric motor) in aluminum foil, and heat in an oven at 250°F for 30 minutes. Use oven mitts when removing it from the oven. Unwrap the foil just before use.
2. Do 10-fold serial dilutions (9–11 times) and then plate a 100 μL sample. You will have to determine how many times to dilute. We suggest plating 100 uL of solution from each of the last three dilutions (on three separate plates) to see which dilution works best for counting colonies.
3. Incubate the plates at 37°C, overnight or longer. Once you have determined the procedure (serial dilution and incubation time) that works best, stick with it for all of your counts.
4. Divide plate into 4 quadrants, and use the stereo microscope to count colonies in one quadrant. Multiply count by 4 to get total CFU/100 μL for the diluted sample. Then multiply by the total dilution factor to get CFU for each piece of pureed meat. You can then divide by the original weight of the meat to get CFU/g of meat.

Safe Disposal of Plates

At the conclusion of the experiment, all plates should be disinfected for safe disposal.

1. The best way to dispose of bacterial cultures is to pressure-sterilize (autoclave) them in a heat-stable biohazard bag.
2. If autoclaves or pressure cookers are not available, an alternative is to bleach the plates.
   1. Wear proper safety equipment (gloves, lab coat, eye protection) when working with the bleach solution; it is corrosive.
   2. Saturate the plates with a 20% household bleach solution (in other words, one part bleach and four parts water).
   3. Allow the plates to soak overnight in the bleach solution before disposing of them.
   4. Please note that the bleach solution is corrosive and needs to be thoroughly rinsed afterwards.

Variations

• For a more advanced project, you should try to determine the reliability of your bacterial counts. One way to do this would be to repeat the counts multiple times (minimum of three replicates). Since the serial dilution process can introduce considerable error into the counts, you would need to do a separate serial dilution for each count. Then you can take the average of your counts, and the standard deviation will give you an estimate of your error.
• You could expand the experiment to include different types of meat. Are some types of meat more prone to contamination than others?

• For more project ideas in this area of science, see Microbiology Project Ideas

Credits

Andrew Olson, Ph.D., Science Buddies

Sources

This project is based on:

• Karney-Grobe, S.L., 2004. "Meat That's Raw: How Do You Thaw?" California State Science Fair Abstract [accessed September 12, 2006] http://www.usc.edu/CSSF/History/2004/Projects/J1315.pdf.

Last edit date: 2007-03-22 22:00:00