Related Links

• Science Fair Project Guide

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• All About Agar
• Interpreting Plates
• Inoculation: How to Put the Bacteria You Desire on a Petri Dish
• Microbiology Techniques & Troubleshooting
• Microorganisms Safety Guide

Project Summary

Difficulty	5  –  6
Time required	Average (about one week)
Prerequisites	To do this project, you will need access to a laboratory with facilities for culturing bacteria. You should be familiar with sterile technique and proper handling of bacterial cultures.
Material Availability	Specialty items
Cost	Average ($50 - $100)
Safety	Standard precautions for handling bacterial cultures.

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Objective

The purpose of this project is to determine the effective concentration for anti-microbial food preservatives.

Introduction

The problem of protecting food from spoilage has been with us since prehistoric times. The solutions to this problem have changed with advances in technology and knowledge about what causes food to spoil. This project will focus on retarding microbial growth, which is only one of the causes of food spoilage.

There are many ways that food can be spoiled. For example, oils in food can become oxidized, releasing free fatty acids that cause a bitter, rancid taste. Additionally, natural enzymes that take part in the ripening process of fruits and vegetables can remain active after harvest, causing spoilage. Different chemical preservatives have been developed to counteract each of these different mechanisms: "Preservatives can be categorized into three general types: antimicrobials that inhibit growth of bacteria, yeasts, or molds; antioxidants that slow air oxidation of fats and lipids, which leads to rancidity; and a third type that blocks the natural ripening and enzymatic processes that continue to occur in foodstuffs after harvest." (Dalton, 2002)

In order for an antimicrobial preservative to work, it must be used at the right concentration. Ideally, it will disrupt microbial growth while at the same time preserving most of the nutritional value of the food.

To do this project, you should first do background research on methods of food preservation. Then, select an antimicrobial preservation method to test for your experiment. As an example, this project will test one of the oldest preservation methods by adding different concentrations of salt.

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:

• bacteria,
• conditions for bacterial growth,
  • minimum, maximum, and optimum values for temperature, pH, osmolarity.
• preservatives,
• food additives,
• food preservation.

Questions

• What is meant by "effective concentration" for a chemical compound?
• What are the effective concentrations for the additives you intend to test?

Bibliography

• This article provides a general overview for methods of food preservation:
  Brain, M., date unknown. "How Food Preservation Works," HowStuffWorks.com [accessed September 20, 2006] http://home.howstuffworks.com/food-preservation.htm.
• These articles provide an introduction to the different types of food preservatives, and what each is used for:
  
  • Dalton, L., 2002. "What's That Stuff? Food Preservatives: Antimicrobials, Antioxidants, and Metal Chelators Keep Food Fresh," Chemical & Engineering News, American Chemical Society 80 (45): 40, available online [accessed September 20, 2006] http://pubs.acs.org/cen/science/8045/8045sci2.html.
  • Foulke, J.E., 1998. "A Fresh Look at Food Preservatives," FDA Consumer, U.S. Food and Drug Administration [accessed September 20, 2006] http://www.cfsan.fda.gov/~dms/fdpreser.html.
• This webpage has background information on nutritional requirements for bacterial growth. It is from an online textbook of bacteriology, which can be an excellent source of further information on bacteria:
  Todar, K., 2002. "Nutrition and Growth of Bacteria," Todar's Online Textbook of Bacteriology, Department of Bacteriology, University of Wisconsin, Madison [accessed September 20, 2006] http://textbookofbacteriology.net/nutgro.html.

Materials and Equipment

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

• 4 low-salt chicken boullion cubes,
• 500 mL hot water,
• 10 glass jars with lids,
• anti-microbial preservative; for example, you could try one of the following:
  • salt,
  • sugar,
  • vinegar,
• electronic balance,
• masking tape,
• marking pen,
• 40 nutrient agar plates.

Experimental Procedure

1. Dissolve 2 chicken broth cubes in 500 mL of hot water.
2. Divide the solution into 10 glass jars (50 mL/jar).
3. Following the table below, add preservative at 4 different concentrations, and add nothing for the control condition. Make two replicates of each condition (10 jars total). Be sure that the salt you add is fully dissolved.

   Label	Amount Broth (mL)	Amount Salt (g)
   Control	50	0
   #1  2.5%	48.75	1.25
   #2  5%	47.5	2.5
   #3  10%	45	5.0
   #4  20%	40	10

4. If you use a preservative other than salt, you should do background research to come up with an estimate of the effective concentration. Make the effective concentration your #3 test condition. Condition #'s 1, 2, and 4 should be 1/4, 1/2, and 2 times this concentration, respectively.
5. Take samples on 1st, 3rd, 5th and 7th days, and streak onto agar plates. Use the quadrant streaking method (Inoculation: How to Put the Bacteria You Desire on a Petri Dish) when plating samples.
6. Be sure to properly label all plates with the test solution and day number.
7. Tape plates closed, incubate (inverted) overnight, and count bacterial colonies.
8. Does the number of colonies decrease as concentration of the preservative increases?

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

• Compare refrigerated and/or frozen vs. room-temperature broth.
• Try additional preservatives. What are their effective concentrations?
• Try preservatives with solid food, e.g., salt meat or pickled vegetables. See the Science Buddies project, Minimizing Bacteria in the Thawing and Cooking of Meat for a method of pureeing solid food in order to assess bacterial content. Be sure to establish baseline bacterial content by pureeing samples before treatment with preservatives.
• Compare starting sterile vs. not. If you have a pressure cooker, here is an experiment you can try. Follow canning procedures for three jars. For the other three, use normal conditions for cooking and left-over food storage in three separate jars. Test by plating samples from successive jars from each condition at 1 day, 1 week, 2 weeks.

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

Credits

Andrew Olson, Ph.D., Science Buddies

Sources

• Youssefian, A., 2003. "How Do Additives Affect the Growth of Microorganisms?" California State Science Fair Abstract [accessed September 20, 2006] http://www.usc.edu/CSSF/History/2003/Projects/J1337.pdf.

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

Career Focus

If you like this project, you might want to think about career opportunities in Microbiology.

Microorganisms (bacteria, viruses, algae, and fungi) are the most common life-forms on Earth. They help us digest nutrients; make foods like yogurt, bread, and olives; and create antibiotics. Some microbes also cause diseases. Microbiologists study the growth, structure, development, and general characteristics of microorganisms to promote health, industry, and a basic understanding of cellular functions. Learn more about this career: Microbiologist.