Do packaged pre-sliced apples have germs?


Answer: Yes! They are full of bacteria! They have so much more bacteria than a freshly cut apple. Even a freshly cut apple sitting in a Ziplock bag in the fridge for a week has far less bacteria than a packaged pre-sliced apple. Check out the amazing results that my son got when he tested apples for his 7th grade science fair project. This is an awesome project that your child can do for his/her science fair project. You just need to buy some of these agar plates. Here is one result but keep reading for all of the results. My son tested 9 different kinds of packaged sliced apples. 


Comparing Apples To Apples


By Jon Pryor

7th Grade

January 2018




Abstract

Pre-sliced apples have been a very popular snack since the invention of NatureSeal. NatureSeal is a blend of calcium and vitamin C that keeps apples from turning brown so they last longer. However, the additional handling and processing that pre-sliced packaged apples go through can increase the risk of contamination with bacteria. Pre-sliced packaged apples have been recalled several times, most recently on December 26, 2017, due to contamination from the dangerous bacteria Listeria monocytogenes. For my project, I tested whether pre-sliced apples have more bacteria than freshly sliced apples. I pressed the apple slices on agar plates, let any bacteria grow, and counted the resulting colonies. My results show that the pre-sliced apples have significantly more bacteria than the freshly cut apples.


Hypothesis

I think that the pre-sliced packaged apples will have more bacteria than freshly cut apples.

 

Experimental Procedure

1. Pre-sliced apples and whole apples were purchased from Walmart, Kroger, Trader Joe's, Target, and McDonalds. The expiration dates were checked and the pre-sliced apples were promptly refrigerated. The fresh whole apples were not refrigerated. All apples were tested well before the "use by" dates. 


2. Some of the whole apples were washed for 15 seconds with cool water and dried with paper towel. Some whole apples were tested unwashed.



3. The pairing knife was washed with Dawn dish soap and rinsed in hot tap water before starting and in between the cutting of each apple. The knife was always cool before it was used to cut the apples.

4. Agar plates were labeled for each apple.


5. The whole apples were sliced and 2 slices were rubbed all over an agar plate. 



6. The pre-sliced apple packs were opened and 2 slices were carefully placed on the plate and gently pressed down. I wanted the colonies to grow up in an apple wedge shape. Some slices were rubbed all over the agar plate. I changed my gloves after handling each apple. 


7. A negative control was done by running my clean gloved hand on an agar plate.


8. A positive control was done by swabbing dog drool from my dog Luke and rubbing it onto an agar plate.



The plates were incubated in a warm incubator for 48 hours at about 32 degrees Celsius. The incubator was made with a plastic box, a clamp lamp, and a heat light bulb.


10. After 48 hours the plates were analyzed for bacterial growth, photographed, and compared. Colonies were counted whenever possible using a marker to put one dot over each colony. 

11. The plates were disposed of by pouring 3% hydrogen peroxide on the colonies/agar, sealing them in a Ziplock bag, and throwing them in the trash.


Results

Freshly Cut Apple Slices

All 17 of the freshly cut apples produced very few colonies when rubbed on an agar plate. It didn’t seem to matter whether the apples were washed, unwashed, regular, or organic. Not every kind of bacteria can grow on this type of agar plate, and viruses do not grow on agar plates. So, if a plate looks like it has no colonies, that doesn’t necessarily mean that no bacteria were present. Also, fungus and yeast can also grow on these plates.







Pre-sliced Packaged Apples

I tested 9 different types of pre-sliced packaged apples which included 6 different brands from 5 different stores. The pre-sliced apples that I tested all listed calcium ascorbate (a blend of calcium and vitamin C) on the nutrition label, except for the Walmart Apple Rings which had no nutritional information. I called the Crunch Pak company, and they confirmed that they use the actual Natureseal product to preserve their apples. I tested all of these apples well-before the “use by” dates on the packages. Crunch Pak also told me that the “use by” dates on their apples are 22 days after production. So, based on the “use by” dates, most of these apples are 11-15 days old. A you can see, all 8 kinds of pre-sliced apples that I tested grew countless colonies on the agar plates.





McDonald’s Apple Slices
















Walmart Apple Rings


Crunch Pak Apple Slices





Foodles Apple Slices


Crunch Pak Organic Apple Slices

Crunch Pak Dipperz


Trader Joe’s Organic Sliced Apples




Marketside Apple Slices















Chiquita







Pre-sliced apples that were rubbed all over agar plates



Positive and Negative Controls




Microscopic Examination

Since this type of agar plate can grow bacteria, fungus and yeast, I wanted to be sure that the microorganisms growing on my agar plates were bacteria. They did not look like fungus, which looks fuzzy. I went to Bellbrook High School and used a microscope to look at the bacteria on the agar plates. Mr. Reinhart helped me prepare slides, stain them with iodine, and look at them. We saw lots of spherical and rod shaped bacteria at 1000X magnification. They were moving around. We also looked at bakers yeast to compare. The yeast were visible at 100X magnification. The microorganisms on my plates were much smaller than yeast and looked different than the yeast. Mr. Reinhart and I agreed that the microorganisms growing on the my apple agar plates were bacteria.

View of Bacteria Under a Microscope



Chart and Graph

I counted the numbers of the colonies on the agar plates to make my chart and graph. For the freshly cut apples, the positive control, and the negative control, I counted the exact number of colonies that were on the plates. I used a permanent marker to put a dot over each colony so I didn’t lose my place when counting. However, all of the pre-sliced apples had too many colonies to count. For those plates, I estimated that there were 1,000 colonies based on the number of colonies on the positive control. The actual number may be more than 1,000. Knowing the exact number of colonies on the pre-sliced apple plates is not that important. The purpose of my experiment was to simply show the huge difference in the amount of bacteria in pre-sliced packaged apples compared to fresh apples.










Bacteria Colonies on Agar Plates After Rubbing with Apple Slices

Apple Type

Number of Colonies

Organic Fuji Unwashed

5

Fuji Washed

1

Organic Pink Lady Unwashed

8

Pink Lady Washed

2

Organic Honeycrisp Unwashed

3

Honeycrisp Unwashed

2

Red delicious washed

1

Golden delicious washed

0

Organic Golden Delicious Unwashed

8

Crunch Pak Slices

1000

Crunch Pak Foodles

1000

Crunch Pak Dipperz

1000

Marketside Apples

1000

Walmart Apple Rings

1000

McDonalds Apple Slices

1000

Organic Crunch Pak Slices

1000

Trader Joe’s Organic Slices

1000

Positive Control

656

Negative Control

2






Bacteria on fresh-sliced apples after 1 week in the fridge

I wondered if the huge amount of bacteria in the pre-sliced apples, was simply because the peel was no longer protecting the flesh of the apple. So, I decided to see if I could make fresh-sliced apples that germy. I washed and sliced Ambrosia, Gala, Pink Lady, Red Delicious, and Golden Delicious apples and put them in Ziplock sandwich bags. Then I put the bags of apples in the fridge for a week. The fridge was 3 degrees Celsius. All of the apples that were refrigerated for a week produced very few colonies with the exception of the Red Delicious apple. The Red Delicious turned mushier and browner after a week in the fridge than the rest of the apples did.






Bacteria on fresh-sliced apples after 9 days in the fridge

After 9 days in the fridge, the Gala and Ambrosia had a few colonies. The Red Delicious had a lot of colonies, similar to the pre-sliced apples. However, the colonies on the Red Delicious were a different color (yellow) than the colonies on the pre-sliced apple plates, so they are likely a different type of bacteria.



Bacteria on fresh-sliced apples left out at room temperature

In another attempt to make fresh-sliced apples as germy as the pre-sliced apples, I left freshly sliced Honeycrisp apple slices in a Ziplock bag on the countertop for 24 hours and 3 days. The Honeycrisp did not grow any colonies after 24 hours, but had a significant number of colonies after 3 days.


Brown Spot on Apple

I even tested a brown spot on an unwashed apple to see if it would grow much bacteria. It didn’t.



Rotten Apple

I also tested the bacteria on an unwashed rotten Red Delicious apple that I’d been saving for 2 months. The apple was mushy. It just grew a little bacteria.







NatureSeal Experiments

    After seeing how germy the pre-sliced apples were and how relatively difficult it is to grow that much bacteria on a fresh apple slice, I decided to do some tests with NatureSeal. I wanted to determine if NatureSeal itself contains bacteria or if it helps bacteria grow on an apple. I ordered NatureSeal here on Amazon. I noticed right away that the box says that the product does not inhibit microbial growth. I mixed up the NatureSeal according to the instructions by mixing one packet of NatureSeal with 8 oz of bottled water in a red plastic cup. To compare, I put 8 oz of water in a red plastic cut without NatureSeal. I swabbed the bottled water and the NatureSeal in the red cups to make sure that they didn’t contain any bacteria. They didn’t.


Next, I washed and sliced up a Fuji, Ambrosia, Golden Delicious, and Red Delicious Apple. I soaked 4 slices of each apple in the NatureSeal for 5 minutes. (The instructions say to soak the fruit for 1-5 minutes). As a control, I soaked 4 slices of each apple in the plain water cup for 5 minutes. Then I put the apple slices in labeled Ziplock bags and put them in the fridge. After 5 days and 11 days, I looked at the apples. The apples soaked in NatureSeal looked good. The apples soaked in water were brown, with the exception of the Ambrosia apple which still looked surprisingly good. I pressed 2 apple slices of each onto agar plates and incubated them for 48 hours.




Apple Slices After 5 Days in Fridge











Apples After 11 Days in Fridge










The results show that none of the apples had any significant amount of bacteria at the 5 day mark. There was a little bacteria at the 11-day mark, but it was far less than the bacteria on the packaged pre-sliced apples that I tested previously which were about 11 days old. The NatureSeal did not seem to increase bacterial growth at the 5 day or 11 day mark, and it does a nice job keeping apples from browning. Interestingly, the Red Delicious apple did not grow as much bacteria this time compared to the first time I tested Red Delicious apple slices for a week in the fridge. Also, Ambrosia seems to turn the least brown of all the apples that I tested without NatureSeal.



Analysis and Conclusion

From my experiments, I have shown that pre-sliced packaged apples have far more bacteria than freshly sliced apples. While the Natureseal calcium ascorbate preservative is very good at keeping sliced apples from browning, it doesn’t prevent bacteria from growing. Since it was not easy for me to grow that much bacteria on a fresh apple slice, I wondered if the bacteria in the pre-sliced apples was a special strain that was put there on purpose to help preserve the apple. I called the Crunch Pak company on January 5, 2018, and they said that they do not add any bacteria to their apples and don’t know why any would be there. So it seems that the bacteria in the pre-sliced apples is likely there unintentionally. The apples could be contaminated with bacteria and/or held at too warm of a temperature at some point during the manufacturing process. Most of this bacteria is probably harmless because people eat these apples all the time and aren't usually sick. However, sometimes these apples are recalled due to the presence of dangerous germs, so some of the bacteria in the pre-sliced apples could be harmful. My hypothesis that the pre-sliced apples would have more bacteria than freshly sliced apples is correct.

Implications for Further Research

There is plenty more work to be done on this project. I would like to know what specific kinds of bacteria are in the pre-sliced apples and if they are harmless or pathogenic. These experiments would require staining and selective growth media, which I don’t have the equipment or training to do. It is also important to figure out if the pre-sliced apples are getting contaminated with bacteria during the manufacturing process or if are they are being held at the wrong temperature at some point which is allowing bacteria to grow. This could be done by testing for bacteria after each step in the manufacturing process.





Acknowledgements:

I would like to thank Mr. Craig Reinhart for teaching me to use a microscope and for helping me look at the bacteria in my apple samples. I would also like to thank my parents for helping me with this project, my dog Luke for tolerating his drool being swabbed, and Mrs. C. St. Pierre for being a great science teacher.




Well, that was my son's awesome science fair project! He got a 100%! If you would like to read any of MY experiments, please check out this list. I am constantly doing more experiments and adding them to my website. If you would like to be informed when new experiments are posted, suggest an experiment, or suggest a product for testing, please like or post on my 
facebook page. You can also email me directly phd.annie@gmail.com. Thank you!

--Annie Pryor, Ph.D.