Cultivation of microorganisms such as fungi and bacteria is often not included in scientific inquiries conducted in school because of the difficulty of manufacturing a suitable medium. A method using dry rehydratable film to reduce the need to manufacture a suitable medium and shorten incubation time was developed as an efficient microbial testing method. Using this method, students can easily perform experiments on microorganisms in schools where time and space are limited. For example, we carried out an inquiry on the possibility of drinking refrigerated milk that is already past its expiration date. Through this activity, we could raise issues related to the current shelf-life labeling system implemented in Korea. In addition, the method can measure microorganisms in several ways through air, direct contact, and indirect contact, making the procedure easier to use in scientific activities at school.

From elementary school to college, various experimental activities are performed not only in the science curriculum but also in after-school programs such as science fairs. By doing experiments, students learn scientific practices such as asking questions, analyzing and interpreting data, and constructing scientific explanations and arguments – the core of K–12 science education (National Research Council, 2012).

In this article, we introduce a new experimental activity for microbiology, which science teachers can more easily administer in the classroom. Microbiology is one of the key concepts in the Korean science biology curriculum from the elementary to high school level (Ministry of Education, Science and Technology, 2011). However, students have difficulty conceptualizing microbiology because microorganisms such as bacteria are not observable in everyday life. In this sense, experimental activities for microbiology in the classroom are crucial for both learning microbiology and carrying out scientific practices. However, one of the limitations of administering experimental activities for microbiology is that experiments are too complex and difficult to be prepared in a school lab. For example, to cultivate microorganisms, preparation of agar media (plates) is necessary but difficult, especially in school settings. For this reason, newly developed dry rehydratable films for cultivating microorganisms have been utilized instead of media in various scientific activities at schools (Mach et al., 2000).

Used in microbiological testing methods to examine bacteria and fungi, dry rehydratable film is a biological film coated with nutrients for the growth of bacteria. When introduced into dry rehydratable film media, specimens absorb moisture and form gels like agar, which allows microorganisms to form a colony. This method is similar to the conventional agar method in that it uses nutrients like agar, but it is more efficient for conducting many experiments in a short time, reducing the need for a medium and minimizing incubation time.

Using dry rehydratable film, one can investigate microorganisms contained in plants, the air, and items used in daily life. Here, we introduce dry rehydratable film in experiments on the type of microorganisms that grow in milk after the expiration date. We also present some suggestions that can be used in scientific inquiries at school.

## Microbiology Knowledge for This Activity

For this activity, students need to understand several aspects of microbiology. First, they need to understand that microorganisms such as bacteria and molds are able to grow quickly if given an adequate environment. For example, teachers may be able to show news stories on food safety during humid and hot weather. The growth of microorganisms causes an increase in the release products (e.g., lactic acid from milk), eventually resulting in foods becoming rotten.

The basic microbiology knowledge that the students need to possess before this activity is that (1) there are microorganisms in nonsterilized foods (e.g., milk in this activity), (2) microorganisms grow quickly if given an adequate environment, and (3) microorganisms can produce many kinds of biological products that might be harmful to human health.

## Dry Rehydratable Film

Dry rehydratable film is a dried film coated with nutrients so that specimens can absorb moisture and form gels, allowing microorganisms to grow. Korea-certified 3M Petrifilm (3M, St. Paul, MN; http://www.3m.com/) makes bacteria readings easier because an indicator is included in the dry rehydratable film media in consideration of the growth characteristics of microorganisms. Petrifilm is coated with nutrients required for microbial growth, cold-water-soluble gel, and indicator dye so that it can be used to examine microorganisms. Petrifilm is composed of two film structures as shown in Figure 1. The upper film consists of polypropylene film, adhesive plus indicator, and water-soluble gel; the lower film is made up of nutrients plus water-soluble gel, adhesive, and polypropylene film printed with a grid. There are many types of Petrifilm, including Aerobic count plates, Coliform count plates, E.coli count plates, Yeast and Mold count plates, Staph Express count plates, Rapid Coliform count plates, Enterobacteriaceae count plates, and High Sensitivity Coliform count plates (Figure 2). Petrifilms are available at the 3M online store. Prices vary, depending on the cultivated microorganism and where the Petrifilms are purchased. We purchased E. coli Petrifilms for $40 (25 sheets per box), Yeast and Mold Petrifilms and Enterobacteriaceae Petrifilms for$35 (25 sheets per box), and Aerobic Petrifilms for \$25 (25 sheets per box).

Figure 1.

Structure of 3M Petrifilm (3M, St. Paul, MN).

Figure 1.

Structure of 3M Petrifilm (3M, St. Paul, MN).

Figure 2.

Examples of the different types of Petrifilm. (A) Aerobic count plate. (B) Yeast and mold count plate. (C) E.coli coliform plate. (D) Enterobacteriaceae count plate. Arrows indicate the colonies of microorganisms.

Figure 2.

Examples of the different types of Petrifilm. (A) Aerobic count plate. (B) Yeast and mold count plate. (C) E.coli coliform plate. (D) Enterobacteriaceae count plate. Arrows indicate the colonies of microorganisms.

## Activity: Measuring the Amount of Microorganisms in Milk

Using 3M Petrifilm, we surveyed microorganisms in milk after the expiration date with the help of four 9th-grade students who participated in Research & Education. Milk can be easily spoiled, but it plays an important part in our diet. According to the Korea Consumer Agency, it is safe to drink milk that is 50 days past the expiration date if it is refrigerated at 0–10°C as suggested in product storage tips (Sim, 2011). In a related message, the agency pointed out that the current food shelf-life labeling system leads to excessive food waste. Consumers tend to throw away food that is still edible because they think that the expiration date determines the exact shelf life of the food. For this reason, there is support for the European and U.S. practice of dividing the expiration date into “use by” and “best before” dates.

In reality, the expiration date officially indicates the period within which sale of the food item to consumers is allowed. The Food Sanitation Act requires food manufacturers and processors to conduct experiments considering raw materials, manufacturing, and distribution methods; set the expiration date; and report such to the director of the Korea Food & Drug Administration. In general, food companies set the shelf life shorter than the actual expiration date obtained from experimental results using the safety factor (0.7). In the present study, we investigated changes in milk quality by examining the amount of microorganisms present in expired milk.

## Tips for Student-Driven Activities

Inquiry activities need to be student-driven, not teacher-driven, so that students can learn authentic science practices (Gibson & Chase, 2002). For this activity, teachers need to be aware of several points in order to avoid teacher-driven activities. First, teachers need to teach how to use the experimental equipment in advance so that students can actively participate in the experiment. Second, teachers should allow students to have a chance to generate research questions from the problems at hand. Students often face unexpected results. For example, they may observe very diverse microorganisms as a result of bacterial contamination of the experimental equipment or lack of good sterile technique. When students observe unexpected results, teachers should provide plenty of time so that they can discuss these results with their peers, find the problem, and conduct new experiments.

## Process

• Refrigerate 6 types of commercially available milk (we used 2% low fat and whole milk in this activity).

• Collect 5 mL of milk on the day of shelf life and put the milk in distilled water to obtain 50 mL.

• Inoculate 1 mL of the diluted milk into Petrifilm for bacteria (e.g., Escherichia coli and Enterobacteriaceae), yeasts, and molds.

• Inoculate 1 mL of 10-fold diluted milk that is 3, 6, 9, 12, 15, 18, and 21 days past the expiration date into four kinds of dry rehydratable films.

• Measure the number of colonies after compressing and cultivating four kinds of dry rehydratable films inoculated with milk at room temperature for 48 hours.

• The method of measuring the number of bacteria is as follows:

CFU/mL = Dilution factor × average colony- forming unit (CFU)

## Results

I was really surprised that the expired milk did not have large numbers of bacteria. Moreover, the pattern was quite unexpected…. I was not sure why this result happened but I would like to do this experiment again because I might have made a mistake. I would also like to compare the number of bacteria between the refrigerated but opened milk and in the milk that we used…. [from a participant’s note]

We found that many participants were satisfied with this activity. Especially, they were satisfied because they were able to easily conduct the experiments using the Petrifilms and observe many types of microorganisms in milk. We also found that many students failed in their first time doing the experiments because of their carelessness and wanted to do the experiment again. Indeed, such repeated experiments are very important in understanding scientific activities in the real world. In addition, students were able to generate many types of new research questions based on their previous findings. Some of them wanted to conduct new experiments based on their new research questions.

Table 1 shows the results of analysis on the changes in bacteria measured at intervals of 3 days for a period of 3 weeks from the expiration date. After the number of bacteria in milk decreased, it increased and decreased again. In other words, the amount of bacteria in milk was rapidly changing. Except for bacteria, other components such as yeasts, molds, E. coli, and Enterobacteriaceae were rarely found even 3 weeks after the expiration date.

Table 1.

Results of analysis on the changes in microorganisms measured at intervals of 3 days for a period of 3 weeks from the expiration date.

PeriodBacteriaYeasts & MoldsE. coliEnterobacteriaceae
2035.5 0.0
3 days 918.3 0.2
6 days 1062.6 0.2
9 days 1589.4 0.0
12 days 2291.3 0.3
15 days 1891.5 0.0
18 days 2117.4 0.0
21 days 3036.6 0.0
PeriodBacteriaYeasts & MoldsE. coliEnterobacteriaceae
2035.5 0.0
3 days 918.3 0.2
6 days 1062.6 0.2
9 days 1589.4 0.0
12 days 2291.3 0.3
15 days 1891.5 0.0
18 days 2117.4 0.0
21 days 3036.6 0.0

In general, milk is divided into Grade A, Grade B, and Grade C in the case of accredited raw milk. First, to meet the standards of Grade A raw milk, the number of flat colonies should be <50,000/mL using average direct microscopy. In the case of Grade B milk, the number of bacteria should not exceed 1 million/mL. The rest that does not meet the criteria of Grade B raw milk is classified as Grade C. From this, it is identified that refrigerated milk maintains Grade A status even after 3 weeks past the expiration date, which suggests that the milk is still safe to drink within 3 weeks if refrigerated (Kim, 2002). In addition, all milk remains safe to drink even after 3 weeks, according to standards for limiting the number of E. coli to <2/mL (Yim, 2003).

## Conclusion & Application

Milk and milk products currently marketed in Korea are processed using ultra-high-temperature pasteurization (UHT). This process, which sterilizes milk for 2 to 3 seconds at a high temperature (>130°C), is advantageous in that fewer milk components are destroyed and spore-forming bacteria are eliminated. Based on the assertion of the Korea Consumer Agency that it is safe to eat well-preserved products that comply with storage suggestions even after the expiration date, related experiments were carried out. The results found that expired milk is still drinkable when refrigerated.

This finding showed that Korea’s food shelf-life labeling system may contribute to excessive production of food waste. It is more reasonable to divide the expiration date into “best before” and “use by” dates as is done in Europe and the United States.

In addition to the method of measuring microorganisms in liquid, such as milk, that has passed its expiration date using dry rehydratable film, there are other ways to measure microorganisms in the air or through direct and indirect contact. They are as follows.

1. Measurement of microorganisms in the air for monitoring the environment: Drop a certain amount of distilled water into the lower dry rehydratable film. Fix it with a clip or tape to avoid contact with the hydrated part, and then cover the upper film after exposure to air within 15 minutes.

2. Measurement of microorganisms through direct contact: Drop a certain amount of distilled water into the dry rehydratable film so that direct contact with the rehydratable film is possible with coins or fingers. Bring the upper film bonded with gel into contact with the surface to be tested.

3. Measurement of microorganisms on the surface: Rub the surface to be tested with a cotton swab, in the case of handles and cell phones, with which direct contact with dry rehydratable film is impossible. Put the cotton swab in a sterilized beaker containing physiological saline and shake it. Drop 1 mL of the specimen into the lower film using an eye dropper. The cotton swab, eye dropper, and beaker should be sterilized before use.

4. Measurement of microorganisms in solid: Homogenize microorganisms in solid at low temperatures by adding sterile saline after cutting a certain amount using a sterile scissor and knife. Physiological saline is added to make a certain amount of test solution. In the case of butter or ice cream, prepare the test solution after dissolving it in hot water with a temperature <40°C.

After inoculating the specimen to be tested, cover it with the upper film and compress it. Cultivate it at room temperature for 24 to 48 hours and observe the number of colonies. Wash your hands after touching dry rehydratable film or if you come into contact with the specimen. Professional disposal services can be contracted for removal of biological waste such as inoculated Pertrifilm plates.

## Pedagogical Implications

Students can learn that they can solve real-world problems using microbiology knowledge from textbooks and well-designed experiments. This is authentic scientific practice that the new science framework emphasizes (National Research Council, 2012). Students can freely generate new questions based on their previous findings and conduct new experiments to test those questions. In addition, when students face unexpected findings for various reasons, they can freely redesign their experiments doing them again, essentially trouble-shooting their experiments through problem solving. This is a student-driven activity. 3M Petrifilm enables teachers to more easily administer experimental activities in their science classes. It also enables students to more easily and actively conduct experiments. In this sense, 3M Petrifilm is a very promising tool for teaching microbiology and for student-driven activities.

## Acknowledgments

The authors thank Kim Eun-ji, Kim Ju-seong, Seo Hyeong-whan, and Choi Ga-in, all of whom are 9th-grade students, for their participation in the R & E of Gifted Education Center at Kyungpook National University.

## References

References
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Ministry of Education, Science and Technology. (2011). Science Curriculum. Seoul: Korean Foundation for the Advancement of Science & Creativity.
National Research Council. (2012). A Framework for K–12 Science Education: Practices, Crosscutting Concepts, and Core Ideas. Washington, D.C.: National Academies Press.
Sim, S. (2011). A study of adequacy for intake marketing overed food (III). Korea Food & Drug Administration.
Yim, J. (2003). A status study of autonomization of shelf life in milk. Journal of Korean Dairy Technology and Science Association, 21, 120–124.