If you take unwashed, washed, and boiled vegetables to see which has the most bacteria, then the unwashed spinach will have the most bacteria and will contain salmonella and E. coli. We believe that it will contain the most bacteria and will contain salmonella and E. coli because spinach is known to contain E. coli

Why do we need to wash our already healthy vegetables? Vegetables are sources of vitamins and minerals, and can provide health benefits. But why should you always wash your vegetables? you may ask. As most parents say, it is vital to always wash your vegetables because of the harmful bacteria that could be there. After hearing this many many times, my partner and I wondered what could be the real reason for washing your vegetables. This is the start of how my partner and I started researching and experimenting on the harmful bacteria that could be on different vegetables depending on how it was cleaned.

One part of our manipulated variable is the vegetables, spinach. Spinach is a nutrient rich vegetable that has many important vitamins and minerals. In other words, in an article about spinach, registered dietitian Kayla Kopp says, “Spinach is one of the most nutritious foods you can eat.”(Kopp, 2024)This is because spinach is rich in vitamins such as vitamin B and many more. Eating spinach can also protect against skin cancers and ultraviolet rays caused by the sun. While spinach is a vegetable loaded with minerals, it also is known to contain E Coli(Escherichia coli). For example, in an article about an E coli outbreak, the FDA warned people to avoid eating spinach. (Charatan, 2006)This was due to what they believed was spinach being contaminated through irrigation water. Spinach is a very healthy 

Our second manipulated variable is cabbage. Cabbage is filled with lots of vitamins C. It contains 60% Vitamin C, 5%Vitamin B6, 2% Iron, and 4% calcium. To add, in a study of cabbage, registered dietitian Julia Zumpano RD, LD, says “Cabbage is also a potassium-rich food, which can help lower high blood pressure. The more we learn about cabbage, the better it promises to be.” This vegetable can help you with your digestion and it can control your blood sugar. It can also lower your cholesterol and help maintain healthy bones and blood clotting.  Both of these leafy greens are high in vitamins. 

Our dependent variable is the amount of unhealthy bacteria and the type of bacteria. To measure the amount, we will swab the bacteria onto a petri dish to grow. Nutrient agar is dissolved in water and creates a jelly like substance, which will feed the bacteria in order for it to grow in the right temperatures, which is an excellent surface to grow agar. This is because bacteria use agar as a food source when growing. In fit temperatures, which is around 37 degrees celsius the bacteria can grow. Bacteria is a small and simple organism that some types can be harmful, or helpful, or also useless. Almost anywhere you go, whether or not it's in extreme conditions, you can find bacteria. Harmful bacteria can enter through your mouth and nose, and cause diseases. 

For our dependent variable, to identify the types of bacteria, we will identify it by bacteria colonies. We will use the different -characteristics of disease bacteria that is visible by the naked eye found in spinach and cabbage which will be E.coli and salmonella to compare it to the bacteria on the petri dishes. Some characteristics could be its shape, size, elevation, surface, colour, and structure. E. coli is known to be an off-white in colour and has a round, smooth shape. Its size is around 1-3 mm and it’s semi opaque and its elevation type is convex. Salmonella colonies look similar on nutrient agar. Just as the professor, Weidong Wang from the Department of Agriculture, Shanghai University, wrote in an article, “Colonies are large, round, 2-3 mm in diameter, slightly convex, smooth, and more translucent than colonies of E. coli.” (Wang, 2022). We can compare our petri dishes with these characteristics and identify the bacteria if they contain any of these two disease colonies.

Our scientific question is Spinach and cabbage are two leafy greens that offer large amounts of vitamins and minerals. Our problect is to inform people about  bacteria that can be on vegetables. This ensures people who think that since vegetables are already healthy, it does not need washing which is incorrect. So always remember to wash our vegetables to be healthy.

Our manipulated variables are the vegetables, being spinach and cabbage, and also how well each vegetable is washed. The responding variable is how much bacteria is grown, and identifying the differentd types of bacteria colonies. Our controlled variables are, the amount of heat used to grow the bacteria, the type of agar, the type and size of petri dishes, the size of the spinach and cabbage, the type of spinach, the type of cabbage, how long each boiled/washed vegetable is boiled/washed for.

        Materials:(materials stated are for 3 trials)

• petri dishes               Vegetables (cabbage and spinach)               Agar (16 grams)               cardboard box                     heater pads                Water (750 ml+a seperate amount of water for boiling+tap water to wash)               2 pots               food thermometer and                               Double-sided tape               Measuring cup            18 swabs               Fridge               Mask                Towel               Gloves               18 petri dishes (100 mm in diameter)               {(9 spinach leaves           9 cabbage leaves) around the same size}         stove             tape               marker               spoon(for stiring)               soap(cleaning)

Steps: 

Making the petri dishes 

1. Put on gloves and mask for sanitization
2. Place a pot on the stove on high heat with 750 ml of water until it boils
3. Once boiling, add the 16 grams of agar into the water
4. Stir for around 15 minutes, waiting for the agar solution to turn into a slightly thick mixture(keep at 100 degrees)
5. Cool the solution until it becomes around 50-55 degrees
6. Pour 1/8 cup agar solution into each petri dish (there will be extra)
7. Leave the dishes to cool for 30 minutes
8. After 30 minutes, place the petri dishes into the fridge upside down for atleast an hour before use (with the cap/top)
9. Wash all equipment used is washed thoroughly with warm soap and water

Spinach group

1. Label each petri dish with S, for the type of vegetable, and U, W, or B depending on how it is washed (3 of each combination). 
2. Take 9 roughly equal sized leaves 
3. Take 3 unwashed leaves and use sterile, unopened swabs and swab each leaf
4. For each swab, use 1 of the S.U labelled petri dishes and across the agar jelly, go in a zig zag pattern, rotating the petri dish (use a different swab for different petri dishes)
5. Take another 3 leaves, but this time washing them with cold tap water
6. Use 3 swabs and swab leaf using the different swabs
7. Now use 1 swab for each S.W labbeled petri dish, going in a zig zag pattern from top to bottom, rotating the petri dish.
8. Take 3 leaves, and and place each in boiled water for 10 seconds
9. Swab each leaf and in swab the S.B petri dish on the agar in a zig zag pattern from top to bottom and rotate the petri dish. 

Cabbage group

1. Label each petri dish with C, for the type of vegetable, and U, W, or B depending on how it is washed (3 of each combination). 
2. Take 9 roughly equal sized cabbage leaves
3. Take 3 unwashed leaves and use sterile, unopened swabs and swab each leaf
4. For each swab, use 1 of the C.U labelled petri dishes and across the agar jelly, go in a zig zag pattern, rotating the petri dish (use a different swab for different petri dishes)
5. Take another 3 leaves, but this time washing them with cold tap water
6. Use 3 swabs and swab leaf using the different swabs
7. Now use 1 swab for each C.W labbeled petri dish, going in a zig zag pattern from top to bottom, rotating the petri dish.
8. Take 3 leaves, and and place each in boiled water for 10 seconds
9. Swab each leaf and in swab the C.B labelled petri dish on the agar in a zig zag pattern from top to bottom and rotate the petri dish. 

Setting up 

1. Take heating pads  (around 12 watts of electricity depending on the size of the box) and place them on the walls of the box (making sure everywhere is heated and also make sure to plug them in) 
2. Place all 18 petri dishes right side up into the box evenly making sure they do not over lap 
3. Keep a thermometer with a wired probe inside the box to make sure during the growth of the bacteria is stays around 35 degrees
4. Take pictures and record data every morning and night for 7 days
5. Record data and make obserobservation
6. To dispose safely, use 20% bleach solution to kill bacteria and dispose in the regular trash                                                                  OR                                                             
7. Dispose using 70% isopropyl alcohol to kill bacteria, and then dispose in the regular trash.

On the first morning, there were a few small light dot-looking colonies on some of the petri dishes while others were still clear. The unwashed spinach petri dishes had very few white colonies spread out. The washed spinach had even less colonies of bacteria, one being completely clear as if it was brand new. The boiled spinach has no bacteria colonies. Surprisingly, the unwashed cabbage barely has any noticeable bacteria colonies and was similar to the unwashed spinach. Surprisingly, the washed cabbage was close to clear with small bumpy areas. With the boiled cabbage 1 of the 3 trials had small pecks of white, while the rest are blank. The morning of day 1 had mostly empty or very little amounts of bacteria colonies

On the second day not much had changed, but almost all the petri dishes had a cloudy film on the agar. Some white little spots sparkly spots that may be bacteria formed on the unwashed cabbage. On the top of one of the unwashed cabbage petri dishes had some bubble looking shape. The washed cabbage looked very similar to the unwashed cabbage but the colonies are slightly larger. The boiled cabbage had small spots everywhere that was very hard to see. Having a lot more translucent white spots, the unwashed spinach had a crowded surface with tiny specs. The washed spinach was much less foggy and was less crowded, but instead had slightly larger spots. The petri dishes with the least amount of bacteria was the boiled spinach. Although it had some larger colonies compared to the other petri dishes, it had by far the least. Compared to day one, lots of tiny colonies grew.

Surprisingly on the third day, the cloudy film disappeared. As it did the day before, one of the unwashed cabbage was still crowded but looked like it had an oily-looking film on it. The crowded areas in the other two unwashed cabbage dishes seemed to disappear and instead was replaced with larger different coloured bacteria colonies that were slightly large. In the washed cabbage dishes, there were lots of orange bacteria colonies. On the other hand, the boiled cabbage had many small whiteish bacteria colonies all over the agar. Looking almost identical to the boiled cabbage, the unwashed spinach had tiny and slight bigger yellow and white spots. With around the most bacteria for the third day, the washed spinach had varying sizes of yellow and white dots. Following the pattern, the boiled spinach had close together whiteish yellow coloured spots. The third morning had lots of small colonies of bacteria.

It seemed that the bacteria had grown so much on the fourth day. The unwashed cabbage had one petri dish that had lots of crowded and small colonies of yellow and white bacteria, while the other two had larger spread out colonies that ranged in oranges, yellows and whites. All three of the washed cabbage had lots of small white spots of bacteria with a few larger yellow bacteria colonies. While the washed cabbage had large yellow bacteria colonies, the boiled cabbage had much smaller and crowded spots  that were white, grey, and yellow. To our surprise, the unwashed spinach had not as much bacteria as we had expected, being that it had grown lots of little spot like bacteria colonies in greyish yellow and orange. What was even more surprising was that the washed spinach had lots of large orange bacteria colonies. The boiled spinach has lots of translucent grey spots and even more yellow dot like colonies.

On day 5, there weren't any new colonies growing but the colonies that had already formed had began to grow larger. The unwasehd cabbage has many colonies but they were all small. There is on petri dish with lots of large colonies and bacteria. The washed cabbage on the other hand, there are many small colonies but there were a few yellow colonies which are larger.  Boiled cabbage has many small yellow colonies and a few yellow colonies which were a little larger than white colonies. The unwashed spinach has many small light yellow dots all over the petri dish but some has only white small dots as colonies all over the petri dish. Washed spinach has yellow dots just like the spinach-unwashed but the washed spinach has less than the spinach-unwashed. Unsurprisingly, the noiled spinach has the least colonies and has a few larger yellow colonies. 

On day 6, lots of progress was made as the unwashed cabbage has lots more large colonies with different colors, some are white, some are yellow and some are orange. The washed cabbage looks like it has more white than yellow colonies but the yellow colonies are larger than the white colonies. Our boiled cabbage has lots of small colonies which are yellow but not as much as the washed cabbage or unwashed cabbage. The unwashed spinach on the other hand has lots of small white colonies spread all over the petri dishes. The washed spinach has many small colonies all over the petri dish which are different shades of yellow. The boiled spinach has some white but a lot of yellow which are small. There are few large colonies on day six and quiet alot of small colonies.

On day 7, the unwashed cabbage had grown lots and has huge colonies which are mostly in the center of the petri dish. The washed cabbage has a lot more colonies but most are quite small. 1/3 looks different than the others. The boiled cabbage has less colonies than the washed cabbage and the colonies are a lot smaller. The unwashed spinach has many small white coloured colonies. The washed spinach on the other hand, has less colonies but the colonies are larger and yellow. The boiled spinach doesn't have as many colonies as the washed and unwashed spinach with a few larger colonies.

On the final night, all the petri dishes has lots of bacteria growing on them. The petri dish with the most bacteria is ours 2nd trial, unwashed cabbage. This is very surprising as when we did our research, we thought that the spinach unwashed would have the most bacteria. Some other petri dishes with lots of bacteria were the unwashed spinach and another unwashed cabbage petri dish. The petri dishes with the least bacteria was the boiled cabbage and the boiled spinach, which we had expected as boiling/cooking vegetables is what kills bacteria.On the last day of growing the bacteria, we discovered that there were little amounts of E coli growing in the unwashed spinach. The unwashed cabbage was alike to the unwashed spinach, having colonies of E. Coli growing in different places. All of the petri dishes either had none, or very little amounts of bacteria colonies that looked like E. coli. What was shocking, was that most of the cabbage dishes had salmonella on them, especially the unwashed ones. 

Our hypothesis was partially correct. We were correct that the unwashed spinach would contain E. Colli, but incorrect that it would have the most amount of bacteria on the last day. When you add all the points together and find the average, the result comes out to be boiled cabbage. It was also shown in the tables that cabbage overall had more bacteria colonies compared to spinach. This may be because we have either accidentally washed and boiled the spinach for more than we intended, which could have changed our answers. With this data, we can conclude that that while washing your vegetables is a key component, the type of vegetable could be a key part for different amounts of bacteria. 

Our experiment will benefit people in their daily lifes, with their health every time they eat a meal. Even though most people know not washing your vegetables is a health hazard, people can understand better now. By a couple years ago, it was said in a food safety article by the World Health Organization, "An estimated 600 million – almost 1 in 10 people in the world – fall ill after eating contaminated food and 420 000 die every year, resulting in the loss of 33 million healthy life years" With this study we wish we prove to people that washing your vegetables can not only save from a stomach ache but many foodborne illnesses too.

During or project, we could've added wrong amounts of agar as when we were adding the agar into the petri dishes, there were traces of agar in the measuring cup which has solidified which could've changed the amount agar placed into the petri dish. Another mistake we had man that could've affected our results, was that we didn't have an incubator so we had to use heaters instead which might also change our results.

medicinenet.com https://www.medicinenet.com/is_eating_spinach_every_day_good_for_you/article.htm  Karthik Kumar, MediciNet

microbiologysociety.org /https://microbiologysociety.org/why-microbiology-matters/what-is-microbiology/bacteria/observing-bacteria-in-a-petri-dish.html#:~:text=Different%20types%20of%20bacteria%20will,to%20describe%20common%20colony%20types. Microbiology Society 

health.clevelandclinic.org https://health.clevelandclinic.org/benefits-of-spinach Cleveland Clinic

World Book. (2014), Gary J. Olsen, Bacteria. Pages 19, 20, 21

genome.gov https://www.genome.gov/genetics-glossary/Bacteria National Human Genome Research Institute  

wardsi.com https://www.wardsci.com/assetsvc/asset/en_US/id/16920402/contents Ward’s Science

cdc.gov https://www.cdc.gov/foodsafety/communication/leafy-greens.html#:~:text=Harmful%20germs%20sometimes%20found%20on,Salmonella%2C%20Listeria%2C%20and%20Cyclospora.Centers for Disease Control and Prevention

webmd.com https://www.webmd.com/food-recipes/ss/slideshow-cabbage-benefits Paul Frysh, WebMD

fdc.nal.usda.gov/https://fdc.nal.usda.gov/fdc-app.html#/food-details/169975/nutrients U.S. Department Of Agriculture

health.clevelandclinic.org https://health.clevelandclinic.org/benefits-of-cabbage  Cleveland Clinic

https://milnepublishing.geneseo.edu/https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/bacteriological-culture-methods/#:~:text=Agar%2C%20which%20is%20a%20polysaccharide,that%20we%20call%20colonies%20form.
https://www.revsci.com/pages/bacterial-growth#:~:text=Petri%20plates%20are%20filled%20with,colony%20forming%20units%20(CFU's) Milne Library

https://sciencing.com/https://sciencing.com/colony-characteristics-ecoli-8507841.html Sciencing

ncbi.nlm.nih.gov/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006063/#:~:text=After%20streaking%20and%20incubating%20the,off%2Dwhite%20in%20color Curr Protec, National Library of Medicine

ntechopen.com/https://www.intechopen.com/chapters/84764 M. Basavaraju and B.S. Gunashree,Chaptermatrixoverview 

walshmedicalmedia.com/https://www.walshmedicalmedia.com/open-access/salmonella-typhi-characteristics-and-cultural-methods-117685.html#:~:text=Colonies%20are%20large%2C%20round%2C%202,convex%20surfaces%20and%20complete%20margins. Weidong Wang, Walsh Medical Media

Throughout the time we had to work on the project, Mr. Baillie motivated us to work on the project and taught us what to do. Our science teacher was there throughout our journey to get into CYSF to answer our questions when we had any. Mr. Baillie walked us through every step of our project with lots of assistance. Another person who helped us a lot with our project is our parents. While we worked on the presentation and the trifold, our parents were there to help us in case we needed them