Plant cancer

[hanzhongxuan2]

Hi! My name is Tony and I am currently working on a science fair project with my partner. It is about finding a possible preventive anti-carcinogen for plant cancer caused by the Agrobacterium Tumefaciens bacteria (crown gall). I will first expose healthy bean plants to an anti-carcinogen extract (turmeric, oregano, and Vitamin C) and then give it the cancer. I will see the percent of plants that get prevented from the cancer. However, I have some questions about my experiment. I have searched up on where to buy the bacteria, and I found a science supply store (Boreal) that sells them. However, there are two products that both contain the bacteria, and I don’t know which to buy. Also, once I buy the bacteria, how will I be able to transfer it to the plant to give it cancer? Thank you very much!

[SciB]

Hi Tony,

You certainly picked a fascinating subject to do a science project on! I hope you let us know by a later post what your results are, because I am really curious to know if the anticancer extract works on plants.

As to where to get the AT bacterial culture and how to use it on the plants, Carolina Biological sells a kit that will allow you to do just that: http://www.carolina.com/bacteria/caroli ... obacterium
They also sell the bacterial culture by itself. I think you would learn more and feel like a real scientist if you did some googling and found out how to inoculate a plant with AT, how long it takes for the ‘tumor’ to grow and how to measure the tumor accurately so you can compare your treated plants to controls. We can help you when you have questions.

Of course, you can buy the AT culture from the company you mentioned, Boreal Scientific Supply, as it is the same as what Carolina sells. The one thing I noticed, however, is that both companies require that you fill out a USDA permit application to obtain the bacteria because they are pathogenic to many plants and the USDA regulates interstate transport of such pathogens. I downloaded the form and attached it for you to look at. How soon do you need to get started? They said it could take a month to get the permit. You could try asking around at a college, university or agricultural extension station to see if anyone local has the bacteria and could supply you with a culture.

How did you plan on doing the experiments? You could do two types of treatment. One is called prophylactic and is like the flu shot that you take before you get exposed to the virus. You could water your plants or spray them with a solution of the anticancer extract or just water as a control, then a week later inoculate them with AT. The other type of experiment is known as therapeutic because you treat the plants after they develop a tumor. You could apply the anticancer treatment to the tumor itself or you could spray the whole plant.

Let us know what you plan to do and we will help you with the details.

Good luck!

Sybee

[hanzhongxuan2]

Hi, Sybee!
Thanks for the reply!
I live in Ontario in Canada, so I don't think there is a permit requirement.
http://www.inspection.gc.ca/plants/plan ... 315732659/

The AT bacteria can be imported without any permits.
Also, that is the reason why I need to buy the AT from Boreal, because it is an Ontario company.

I tried doing some research about how to "give" the plants bacteria, but I found that there weren't any information, because people want to prevent the bacteria, and not grow it. So, that is the main area where I will need help in. I have done some research, and I found some people give the plants the AT via a needle prick.

I am having trouble on deciding the control for the project, and the availability of science equipment decides the control variable. One one hand, I want to do a therapeutic experiment, and the control would be the percentage of the decrease in size of the tumor. But this method requires high-tech equipment! I have heard some people used mass spectrometers, but I don't know what they are for. In addition, it is difficult to obtain the exact size of the tumor on day 1 and day 10 (you can't just take a ruler and measure it!).

So currently, I am leaning more towards the prophylactic approach (first anti-carc. then AT), and calculating on the percentage of the plants that get protected from the AT. But this approach takes a lot of tests! I would have to grow a lot of bean plants! (Because if I grow 10 bean plants and 8 get prevented, I can't really prove that the anti-carc. is really effective).

I have tried contacting a professor at the University of Toronto, but I haven't heard from him yet. Also, I don't think any people would lend students high-tech equipment!

Another problem is handling the bacteria. I need to give them agar to keep them alive. I found this guide and I will follow after I order my bacteria.

http://www.carolina.com/teacher-resourc ... tr10478.tr

Thank you so much for helping me!

Tony

[SciB]

Hi again Tony,

I am happy to help a young scientist do a good project!

Let me answer your last question first because that one has to do with keeping your bacteria alive. When you buy the bacterial culture it comes in a sterile tube growing on a nutrient agar ‘slant’. I have worked with other bacteria, but not AT, so I don’t know how fast it grows on agar or how dense the culture is. What I am thinking is that you might be able use the slant tube culture as your bacterial source for inoculation of the plants and not bother with agar in a petri dish.

You will be inoculating the plants only once with AT, so I would think that the slant culture will provide you with enough bacteria to infect many plants. I looked up how to work with AT and it seems pretty simple:
http://link.springer.com/protocol/10.13 ... -130-4%3A3
http://www.cals.ncsu.edu/course/pp728/A ... rofile.htm
http://www.protocol-online.org/biology- ... /9567.html
http://cshprotocols.cshlp.org/content/2 ... t5428.full

You are correct that the best way to inoculate the plants with AT is to prick the stem with a needle. You need to make an actual wound in the stem for the AT to be able to enter and transform the plant cells into tumor cells. Do you have or can you borrow an inoculating needle and loop from someone? If not, you can buy them from Carolina Biological: http://www.carolina.com/catalog/search- ... SearchForm

The problem I see is how you will inoculate each plant with exactly the same amount of AT bacteria so that you can get an accurate comparison of the treated plants with the untreated controls. You could simply wound each plant by pricking the stem with a needle that you sterilize by holding in a candle flame then scrape up a bit of the AT culture from the agar slant and apply it to the wound. But I am concerned that you will not be able to scrape up the same amount of bacterial culture each time and some plants may get more AT than others and maybe grow tumors faster. I am assuming that the more AT you inject into the plant, the quicker the tumor will form, but I do not know that this is true since I have never worked with AT. Maybe one of the other experts has experience with this method and can help.

What I would do to ensure that each plant got the same amount of AT would be to make a liquid suspension of the bacteria. You will need some sterile saline, which you can get at a drugstore. Just make sure that it does NOT contain a preservative as this could kill the bacteria. Just to be safe you could tell the pharmacist what you need it for and have them suggest a brand for you.

You want to have your plants all grown and ready for the inoculation before you make the saline culture. The control plants will be wounded with the needle and you will apply a loop full of saline, but no AT.

To make the bacterial suspension you will have to carefully add about 1 milliliter of saline to the AT culture tube. The volume does not have to be accurately measured. You just want to have enough to make a good suspension. Take the cap off and hold it between your fingers without touching the inside so it stays sterile. After you add the saline, put the cap back on tight and shake the tube gently for some time until you see that the saline is cloudy with a suspension of the bacteria.

Now sterilize your needle in the flame, let it cool for a few seconds then make a small puncture in the stem of a plant. Swirl the AT suspension a bit to make sure the bacteria are uniformly distributed and using the disposable sterile loops, get a loop full of the suspension and apply it to the puncture wound. Repeat these steps for each plant (except adding saline only for the controls) plant until you have inoculated all of them.

This is a lot of work and maybe you want to just try scraping off some of the bacteria from the slant and applying that to the wound. That’s OK if you want to do it that way. I just think the liquid suspension would insure a more uniform application of bacteria to each plant.

From what I have read, you should see some changes in the plant stem after 7-10 days and a tumor should form in 3-4 weeks. As to how to quantitate the difference in tumor size between your controls and treated plants, you will definitely want to take pictures at successive days to document the changes. Try to include a ruler in each photo for scale. From the google images photos of the tumors (‘galls’) that form on plant stems, it looks like you might be able to cut off the tumor and weigh it. I think the shape is variable so measuring the size would be a problem; but if you can remove all the tumor tissue, you should be able to quantitate the growth by weight.

If you have a microscope and some slides and a way to make sections of the galls it would be really cool to compare them microscopically. If you have a biologist friend in a lab with a microscope that has a camera mounted on it, you could get some nice photos after staining the sections.
Let us know what you plan to do. You don’t have a lot of time before your January deadline, so get started right away. We’ll be here to help with the details.

Best wishes,

Sybee

[SciB]

Hi Tony,

I forgot--there was a file I wanted to send you with some instructions on AT inoculation, so here it is attached. Maybe you have seen it already.

Good luck!

Sybee

[hanzhongxuan2]

Thanks for the information on how to evenly apply the AT to the plant! It sounds slightly confusing and kind of difficult to do, having to purchase more supplies. Also, I have discovered that I would not be able to use the inoculating needle because it can't be shipped to my school before the holidays. I was wondering if I could simply combine, say 10 mL of AT (bacteria is being shipped in tube form) and 200 mL of water. This way, the bacteria is kind of diluted and the difference won't be so great when I apply one drop of the solution to the plant. But I am unsure on if the water itself or the temperature of the water might somehow kill the bacteria. Also, to save money, is it alright to sterilize equipment in rubbing alcohol (70% ethanol) instead of under a flame?

I think that dissecting the tumor from the plant and weighing it is a great idea! I am going to change my prophylactic experiment into a therapeutic experiment. At school, my teacher has a gram scale going up to the tenths (i.e. 1.2 g). Is that accurate enough, or do I need a scale going up the hundredths?

To be more specific, I am going to conduct the experiment 3 times. Each experiment will involve testing 60 plants in total. Of these 60 plants, 20 will be grown in turmeric, oregano, and Vitamin C respectively. I have included (part of) my report as an attachment.

I have asked my science teacher to order the bacteria today, and the company says it will arrive in 2 days. When the bacteria arrives, I'm afraid it will die off from the cold. Also, another urgent problem is will the the bacteria be able to survive for two weeks? (I am just beginning to grow the bacteria today, and the beans take about 10 days to sprout.

Thanks,
Tony

[deleted-71625]

Hello Tony,

Your experiment sounds really neat! I know that SciB is helping you with your questions, but I thought I would just answer a few things and SciB can add to it later

Diluting the bacteria down is usually the procedure, using a serial dilution, which dilutes the bacteria to manageable numbers. You can use sterile deionized water to dilute. I'm not sure if you need to dilute the bacteria since you are only using it to infect the plant, and not using it for counting purposes. Although, having a standardized bacterial solution (the same amount of solution applied to each plant wound) is ideal. Maybe SciB can elaborate.

You asked about using alcohol to sterilize your materials? Yes, you can do this. Usually, in a lab, we dip our instruments in isopropyl alcohol and sterilize it by using a bunsen burner. The alcohol basically dries out the bacteria, so they shrivel up and die, and the flame just an extra precaution to kill them. You can use a flame from a candle, making sure that you dip your loop (or inoculator) into the alcohol, then into the flame, and just let the alcohol burn off. No need to shake it or anything. Use safety precautions. Of course, you need to wait for the inoculating loop to cool before dipping it into the bacterial solution, or you could singe and kill the bacteria. If you decide to use only the alcohol, make sure that you leave your materials sitting in it for a couple of minutes, then take out and let dry completely on several clean paper towels.

As far as the bacterium goes, Carolina biologicals had this article you could check out:
http://www.carolina.com/bacteria/agroba ... en%2C+tube

I found some information from Boreal Sciences that I have attached about what temperature to keep them at. Hopefully, when you get them, it will come with an instruction sheet. You want to keep them at 30 degrees Celsius.[attachment=0]Bacteria_&_Fungi.pdf[/attachment] . Also read about how to inoculate in the second article, pdf "working with bacteria and fungi" (I can only include 1 attachment).

As far as the galls go for weight, I'm not sure if measuring 10ths of grams will be accurate enough, especially given the time period in which you have to measure them; having a scale that measures the 1/100th of a gram would be ideal, since the weight of your galls may only have a small difference in weight.

Your sample size is great. The more the merrier and will definitely help solidify your data when it comes time to analyze it. It kind of depends on when your project is due and how many variables you have, a sample size of 10 might be good enough if you don't have the time to do a full 20 of each. But I say try for 20, as you planned.

Hope that helps. I look forward to seeing how your project comes along.

[hanzhongxuan2]

Hi Sarah!
Thank you for helping me with my project! (I never knew my experiment would attract so much attention!)
In the end, me and my partner decided to grow 180 plants in total (60 each in turmeric, oregano, Vitamin C). The experiment will be done three times, so 20 plants in each of the three variables will be experimented upon in each experiment.
What we plan to do is to grow each plant in 250 g. of soil (is that enough?). We will water each plant with 1 tbsp. of water (15 mL) until the plants are 20 cm tall. Then, we will inject the AT bacteria. We will continue watering for two weeks, and then we will switch to the anti carcinogen water. We will repeat this for 2 weeks, and record the weight difference before and after tumor to calculate the decrease in weight of the tumor.

We will grow the plants over the holidays from now until January 6, and then we will order the bacteria and administer the bacteria. Since we do not have an inoculating loop, we will dilute the bacteria in tap water and "dab" it onto the wound in the plant. The dilution is to ensure that equal amounts of bacteria are being transferred to the plant. Is regular tap water okay? I want to minimize my budget as much as possible.

As for the scale, I do not have access to the hundredth-gram scale, but only a tenth-gram scale. I will try to ask my teachers and other people if they could lend us the scale.

Finally, the biggest area of help I need in is proving that the anti-carcinogens are effective in humans. If my data shows that, say, turmeric can eliminate plant cancer by 90%, then how might I prove that it also works with humans? Our main argument is that since plants are more simple organisms than humans, and their immune system is not as evolved as humans, then humans have a better chance to fight the bacteria. We fear that this argument is very far-fetched, unsupported by proof, and is un-scientific. Adding on to that, plant cancer does not spread to other parts of the plant! It remains immobile, which differs from cancer in humans. We need some help backing our argument up. So what are the differences in plant tumors and human tumors? Are the tumors different somehow?

I have attached my materials below and the procedure as an attachment.

Materials:
 200 soy beans (Glycine max)
 45 kg soil
 180 Separate Small Growing Containers
 1.8 kg Turmeric
 1.8 kg Oregano
 45 Ascorbic Acid (Vitamin C) Pills (500 mg.)
 Crown Gall bacteria (Agrobacterium Tumefaciens)
 3.3 g Nutrient Broth Powder
 Prepared Agar Petri Dishes or 7 g Nutrient Agar Powder and Petri Dishes
 Measuring Cup and Spoon (tbsp. and tsp.)
 Bowl
 Rolling Pin and Mat (for crushing Vitamin C pill)
 Watering Can
 Sewing Needle
 Clear and Masking Tape
 Recording Utensils
 Sterile Test Tubes
 50 Wooden skewers (40 cm)
 Yarn
 Scissors
 Scale (preferably up to hundredths of a gram)
 Inoculating Loop (nichrome) (optional but preferred)
 Pairs of Latex Gloves
 Rubbing Alcohol (70%+ ethanol) (for sterilization)
 Disposable Sterile Cotton Wipes
 Household Bleach
 Water


Thanks for helping us!

[SciB]

Hi Tony,

I read through the procedure that you attached and it seems to be an awful lot of work that may not be necessary. Are you following that procedure exactly? I think there is some confusion in the details of how to prepare the bacterial culture for applying to the plant. As I have said, and also Sarah, it is very important to use the same amount of bacteria to inoculate each plant so that the tumors that form are all about the same size. If not then comparing them by weighing will be meaningless.

I think you may not understand in what form the bacteria that you receive from Boreal will be. As I understood it from their product description, the AT is growing on the surface of solid nutrient agar in a sterile tube. That means the layer of bacteria is in solid form—not liquid. That is why in my first post I suggested adding sterile saline to the tube and shaking it to make a liquid suspension of the bacteria. Bacterial cells can grow equally well as solid colonies on agar or as dividing cells in nutrient broth. To apply an identical amount of bacteria to each plant wound, the culture has to be liquid.

Also, you said you were worried about cold harming your bacteria. It will not kill them. All it does is slow down their growth and cell division. You could store them in the refrigerator for weeks and they would remain healthy—just not dividing. From my reading, I see that their ideal growing temperature is 25-30C. Unless you can find someone who has worked with AT and knows exactly how to treat it, I would suggest making a liquid culture of the bacteria with sterile saline and leaving it in the refrigerator until your plants are ready to be inoculated.

You said you wanted to do therapeutic rather than prophylactic treatment of the plants. Cancers are easier to prevent than to treat. Once a large tumor forms, it is usually removed surgically and the patient is treated with chemotherapeutic drugs or radiation to kill any leftover cancer cells, and this can take a year or longer. I am afraid that once the crown galls form on your bean plants, making them shrink and go away by treatment with turmeric or the other compounds will take a LONG time—longer than your deadline. So, I would suggest you try prevention rather than cure. Water the plants with the chemical solutions (or just water for the controls) for a week or so to get them well treated, THEN try to induce tumors by inoculating with the AT bacteria. You can take photos of some of the plants every couple of days to monitor growth at the wound site and keep applying the anticancer agents. This is what humans do when they take turmeric every day as a supplement, or vitamin C; they are hoping to STOP cancer before it starts.

You asked a lot of questions about human cancer vs plant cancer. I know a lot about human cancer, but little about plant cancer. If you have read the Wikipedia entry on Agrobacterium, then you know that it is not the bacteria itself that causes the galls, it is a small piece of DNA called a plasmid. Many different types of bacteria have plasmids and they can be transferred from one bacterium to another or in the case of AT, transferred into plant cells. So, what does this Ti (tumor-inducing) plasmid actually do? The plasmid is a circular piece of DNA that contains several genes, some of which can stimulate local growth of plant tissue—that’s what makes the tumor form. Human cancers can occur in a similar way but usually start because of mutations in a cell’s DNA that allow it to grow and divide when it is not supposed to. This is a very complicated subject. If I were talking to you in person with a blackboard where I could explain things with pictures and answer your questions as you thought of them, it would be much easier to help you understand. You’ll just have to repost with more questions.

For now, you should get your beans planted and work out all the details of the experiment ahead of time. Where are you planning on growing the bean plants in December? They need to have a temperature of at least 25C and 6-8 hours of sun a day. If it is too cold, the bacteria may not be able to form galls very quickly.

You said you could not order the inoculating loops, but you can make one easily from a piece of wire: http://en.wikipedia.org/wiki/Inoculation_loop
As for sterilizing the loop, you certainly must have a candle. All you have to do is dip the loop into alcohol then put it in the candle flame for a couple of seconds and let it cool for another 5-10 seconds before putting it into the liquid culture of bacteria. This is how bacteriologists have been doing it since the time of Louis Pasteur and it is still the best way.

OK. I think I’ve gone on long enough! Repost with more questions and we will help you get this great project going.

Best wishes for the holidays,

Sybee

[hanzhongxuan2]

Hi Sybee! Merry Christmas!

Sorry I couldn't post back... There was a power outage for a few days because of the ice storm in Toronto.
What you said in your last post was really beginning to clear things up! I totally understand the bacteria suspension now!
Also, I never knew that the bacteria was being shipped in tubes and grown on top of solid agar - now I know what you meant! I can't just simply pour out the bacteria because it is not in liquid form!

I've done some more research today about experiments similar to mine, and indeed, I found that the time frames were WAY larger than mine! You're right - not only is preventing the cancer much more easier it also takes less time. If I do have extra time, I would have definitely done the other way.

I have 2 options for what to compare in the observations:
Option A:

For each independent variable including control:
(Average weight AFTER bacteria - Average weight BEFORE bacteria) / Average weight AFTER bacteria

Option B:

For each independent variable control:
Plants with tumor / Plants WITHOUT tumor

Personally, I think B is less accurate, but A has some technical errors: the plants will grow - thus increasing its mass - and this will affect the weight of the tumor (i.e. if the plant was 20 cm before the tumor and after the tumor it was 30 cm, we cannot calculate the tumor size accurately by subtraction).

Do you think there are any other options on what to measure?

Happy Holidays!

Tony
My partner started growing the beans on the 20th, so that means I have 3 weeks of time (roughly) for them to grow until I get back to school.

[SciB]

Hi and a Happy Christmas to you too, and a great New Year!

Sorry to hear that the big storm knocked out your power for so long. I hope everything is ok now.

I'm glad you understand now about the way bacteria are grown and handled. It's not something they would teach you in high school biology!

So, you only have about one month to complete the project. Did you start treating the bean plants with vitamin C and the other anticancer agents? I would do that and continue with the treatment even after you inoculate the plants with AT. I think that may be the only way you can see an affect of the treatment in so short a time--assuming that the treatment does reduce the tumorigenicity of AT. I don't know the answer to that, but it really is a cool question.

Did you order the bacteria yet? I don't know how long it takes for the Ti plasmid from the bacteria to start modifying the plant tissue but the sooner you start, the more time you'll have and the better your chances of seeing some effects. Since you are in Canada, I assume your bean plants are being grown indoors under lights. Are you using wide-spectrum fluorescent lights? Are all the plants getting the same amount of light? Is the temperature of the room where you are growing them warm enough and the temp fairly constant--say 18-24C?

So, if I were doing this, what would I measure? First, I would take lots of photos. Label all the plants so you can tell in the photo which is which. Try to take the pictures from the same relative location and distance to make it easier to compare the growth of the plants over time.

I understand your two options but really, shouldn’t you be comparing plants inoculated with AT with and without anticancer prophylaxis [let’s call this ACP—scientists love to use acronyms for everything!]? However, in order to do that, you have to prove that ACP itself does not affect the growth of the plants. That means you need two more groups—one without ACP or AT and one with ACP but without AT. Do you have enough plants to do these 4 groups: (1) no ACP and no AT, (2) ACP but no AT, (3) no ACP + AT, and (4) ACP +AT

You definitely want to compare the weights of the plants at the start of the experiment to their weights at the end. This will tell you what effect ACP by itself or AT by itself had on the plants' growth. I have no idea how long it will take for AT to cause a measurable change in your bean plants, but if you find no statistical difference between the weight gain in group 1 compared to group 3, then you have to conclude that the bacteria did not cause a measurable change in growth rate over your experimental time period. Then you have to compare group 1 to group 2 to see if the ACP stimulated or retarded growth. Finally, you compare group 3 to group 4 to see whether the ACP altered growth.

But is growth the only thing you can measure? Certainly not! What I would really like to see is a microscopic comparison of the plant tissues. Since the initial changes caused by the Ti plasmid occur at a cellular level, that’s where you should look. Do you have access to a research microscope with a digital camera? This would be a lot of work but it would give you a really great project. You would have to make sections through the stems of the bean plants at the location of the AT inoculation, stain them and take pictures through the microscope. You would have to do some reading of scientific papers to understand what to look for or measure, and if you could find a plant researcher to help you it would be a lot easier. You would need to take photos of a normal bean plant stem section for comparison, then one after the AT inoculation with and without ACP. I don’t know what you would see in terms of differences—more cell division, abnormal looking cells, tumor tissue? It would certainly be worth taking a look if you can find a person in the biology department of some university who would be willing to let you use their microscope and camera for a few hours. It would be a great learning experience for you too.

Let us know what you plan to do and whatever you decide, we’ll be here to help you complete the project and write up the results.

Have a great New Year!

Sybee

[hanzhongxuan2]

Thanks!

I probably can't get the microscope, but at least I can take some photos of the tumors.
I am going to ask my science teacher for an electronic force probe (measures up to hundredths of a newton) to weigh the plants. As for the microscope I am going to try to ask someone for it.

I am not really sure how much anti-carcinogen to put in the solution. They have to be the same amount, and right now I'm settling on 250 mg. of Anti-carcinogen. Is that enough?

The plants will be watered with the AC for 3 weeks. After the first week, I will apply the bacteria and continue to water AC for another two weeks. I have time to do this.

Currently, my plan is to mix 350 mL water and 250 mg. anti-carcinogen and water the plants with 50 mL solution (lasts 7 days). I have to make a total of 3 batches of solution for one plant. I think that will be enough.

Tony

[deleted-71625]

Hi Tony,
Happy New Year!

Thanks for keeping us updated on your project. Here are my ideas. I'm not sure how effective weighing the entire plant will be, since each plant size will vary as well as the weight of the plant will change, due to the growth between the time of inoculation and the end of the experiment.

In thinking of how to set up your experiment, keeping track of data, and the validity of your results, I would highly recommend only choosing 1 anticarcinogen (i.e. Vitamin C OR oregano). This will help simplify. Scientists only test one variable at a time (in your case ONE anticarcinogen) to keep other factors from interfering, and also having a larger sample size will better solidify your results. You can choose to do it how you want, but I think given your time frame, this would dramatically help the ease of your project. In science, more is not necessarily better, but consistency is. Just something to think about.

My suggestion are 3 things:
1. first take a photo of each plant before inoculation, weekly, and then also at the end when there is bacterium growth (the galls). Take close up photos of each gall as well. In your conclusions you can compare/contrast size, texture, color of each type of gall. Also take note of any plant physical changes (did the area of inoculation change color, become dry or 'sick' looking, etc) You can pick 3-4 photos of each scenario (i.e. each anti-carcinogen used) to use on your display board (pictures speak a thousand words and help clarify your project).

2. Take the weight of each plant will the gall still attached (just in case you need it later). When you do your weights, make sure to use a scale that has 1/100th grams, and record all decimal places. The weights of the galls will most likely (I'm assuming) be similar to one another.

3. Do this step last, since you will no longer have your plant connected. Cut off the gall of each plant and weigh the gall, rather than the plant. I think if you weigh the plant, there will be too many variables (size of each plant in comparison, amount of soil left on roots after weighing, etc.) Also, I'm not sure how you concluded using Newtons as a weight, because that is for energy/caloric measurements: http://en.wikipedia.org/wiki/Newton_scale

As far as the concentration of the carcinogen to water goes, that sounds like an awfully high ratio-almost 70%! I don't really know exactly at what concentration you would need. Here are a couple articles I found that could help:
http://www.theepochtimes.com/n2/health/ ... -2807.html

Also, will you be using liquid or solid oregano, vitamin C, etc? They should all be water soluble or you will run into problems. Vitamin C is water soluble, but I don't know what concentration you would use; some research would be helpful here. Is there an oregano extract? That would be ideal, and it should tell you the concentration of it on the bottle. To find the saturation level, you can start with your 350 mL of water (needs to be sterile deionized, so no contaminants) and slowly add increments of Vitamin C and mix with a spoon (for example, add 1 mg at a time and mix, then add increments, making sure to keep track of all your weights). You will do this part before your experiment, so you need extra water and extra Vitamin C to make mistakes with. You might have to do several trials to find the point of saturation. This is why I say it is easier if you already have an idea of what concentration of Vitamin C (and other anticarcinogens) to use.

Let's say your research shows that 10% vitamin C shows an increase in cancer fighting (you'll have to read some articles or studies about this: try a Google Scholar search), then if you used a 10% solution, you would need 10mL of vitamin C liquid drops for every 90mL of liquid (for a total of 100mL). Look at Solution 2 on this site to help explain this: http://www.sciencecompany.com/Preparing ... spx#volume
I think that buying a premade Vitamin C solution is easiest, like at a health food store it will be no problem finding it. Just remember that if you have a 10% solution of it, DO not dilute it with water, or you will change the concentration.

To obtain a 10% solution, how much of it do you need if your total volume of water is 350mL?
10% = 0.10mL Vitamin C
percentage Vit. C x total volume = 0.10mL x 350mL = 35mL Vitamin C
350mL total volume - 35mL Vit. C = 300mL deionized water
So, 35mL Vit. C + 300mL water = 350mL total volume

Hope that helps. Please let us know if this is confusing or when you have further questions.

[hanzhongxuan2]

Thanks, Sarah, for your contribution! You're right. I shouldn't have that many variables. I have finalized my decision to only Vitamin C and turmeric. This helps simplify everything greatly!

The experiement is well underway, and the plants are mature and ready to be tested. We have taken many photos of the plants

Meanwhile, I have a question:

Is there a chemical I can add to turmeric (the antioxidant curcumin) to make it water soluble? It is poorly soluble in water, and soluble in oil.
I can't water the plants with oil or any lipids, or it will die.
I looked it up online and I found adding carboxymethyl cellulose ("sodium salt") can make it soluble in water. Also, I found that it is soluble in alkali water. Is this true?
If it is true, would methyl cellulose also work?
Also, does changing the acidity of the water affect the effect of solubility? (i.e. curcumin in acid water vs curcumin in alkaline water)

Thanks!

[hanzhongxuan2]

Nevermind, I found the answer to my own question! Increasing the temperature of water can increase the solubility of turmeric "12-fold". But now I have a new question: How do I measure solubility? What unit do I measure it in?

(If I were to find out the solubility of turmeric, what would I do?)

[SciB]

Hi Tony,

How is your experiment going? Did you get the Agrobacterium? Did you find someone with a microscope and camera that you can use to take microphotos of the plant tissue at the site where you inoculate with AT? I would really like to see you do that because that would make your project really interesting. I am concerned that you might get very little gall growth in only two weeks. By the way—I want you to be clear on what is happening to the plants after you inoculate them with AT. The bacteria contain a plasmid called the Ti plasmid (‘Ti’ for tumor induction) and it is this little bit of DNA that causes the gall, NOT bacterial growth. The gall that forms after bacterial inoculation is made up of plant cells just as a tumor in your body is made up of your body cells.

In your previous post you said you would make the vitamin C solution by dissolving 250 mg of ascorbic acid in 350 ml of water. Did you choose this amount of C because you have a 250 mg tablet? That’s OK. I really have no idea how much vit C you would need to use to have an anticancer effect. Vit C has a solubility in water of 17.6 grams per 100 ml at 20 degrees C so dissolving a 250 mg tablet in 350 ml of water is no problem.

Curcumin from turmeric is poorly soluble in water as you said. Be careful here because turmeric and curcumin are not the same thing. Turmeric is an extract from the root of a plant and may contain 3-5% curcuminoids which are the active compounds. Instead of using turmeric, I would get a curcumin supplement that tells you the amount of curcumin on the label. I think the only way to increase the water solubility of the curcumin molecule is by chemically modifying it, so adding methyl cellulose to the solution won’t help. There are some water soluble curcumins that are being tested in the lab, and there are some that you can buy that claim to be more water soluble. Google ‘water-soluble curcumin’ and you will find several examples. You could go to a health-food store and see if you could find a curcumin capsule that is water soluble and use a solution of that for watering your plants. You said that heating the water increases the solubility of curcumin and that is true except that when it cools off it may not stay dissolved. You also asked about the units. Try to find a curcumin supplement with enhanced water solubility that lists the amount in milligrams or micrograms per capsule and mix one capsule with 350 ml of warm water. Swirl it around and let it sit for half an hour or so with occasional swirling. When you water the plants with the curcumin ‘solution’, swirl it around before you measure the 50 ml volume so that you get a uniform suspension. Even if the curcumin is not dissolved the plants’ roots may be able to absorb some of it from the tiny curcumin particles that are in suspension in the water

Again let me emphasize the importance of a microscopic examination of the plant tissues at the end of the experiment. You don’t need a big fancy microscope. One that will give you a clear magnification of 200X would be fine. You don’t even have to have a camera attached to the scope. I have taken photos through the eyepiece with the camera of my cell phone. You have to hold it real steady and adjust the light and focus carefully but it can be done. Making a section through the plant stem to examine under the scope is a bit of a challenge but you can do it with some practice. Here’s some information on how plant sections are made: http://www.hometrainingtools.com/micros ... ts/a/1239/
Try to find someone in the biology lab to help you with this. This is a really interesting project and I am sure someone would be glad to help you to see if there is a difference at the cellular level in the control plants (water only) compared to those watered with vit C or curcumin.

I’ve given you a lot of technical information here, so please if there is something you don’t understand, don’t be afraid to ask. We’re here to teach you and help you do the best science project you can.

Best wishes,

Sybee

[deleted-71625]

Hello Tony,
I am so happy to see that you are so proactive in your search for your own answers-good job!

I'm not sure why you need to "measure the solubility" of the mixture or what you mean by that; maybe you mean concentration? If you make up a solution of turmeric in hot water (see the article, http://www.medindia.net/alternativemedi ... powder.asp), you can calculate the concentration of solid to water and use that in your results. You can do the thing I suggested in the previous post about adding a little solid to the water at a time to the hot water, while keeping track of how much you put in until you can't dissolve any more (aka, saturation point).

Also, from all the research I've done on turmeric, it is water soluble--it is used primarily in teas and foods, so I'm not sure what resource said that it was water insoluble. However, if you are adding salts to your solution, you will be changing the alkalinity of the soil of the plant. Some plants do well in acidic soils and some do well in basic or neutral soils, so it depends on the needs of the plant. Changing the alkalinity of the food given to the plant, will obviously need to be in your results section, as this factor could cause your plant to die if not given the correct conditions. No problem with doing that, but just make sure it goes in your "Discussion" section of your report.

I did find some scientific articles on the water insolubility of curcumin: http://www.ncbi.nlm.nih.gov/pubmed/17116380, and http://www.ncbi.nlm.nih.gov/pubmed/17767425. Was this the article you found? I also read (from some researchers doing in vivo studies) that you can use acetone as the solvent with curcumin, you can read more here, as this person asked the same question: http://www.researchgate.net/post/Is_wat ... _curcumin2
Also, here's another article that explains the process of getting the powdery form, turmeric:
https://www.google.com/patents/WO201002 ... CE0Q6AEwAg; in simpler terms, when we buy turmeric powder at the store for cooking, it has already been prepared by harvesting the rhizome, crushed and stripped of the oily residue (via acetone treatments), and then dried in order to become the water soluble powder. So, given that, I would use the over the counter turmeric and use water to dissolve.

What do you think? Let us know if you have further questions. And I'm glad to hear that you simplified your experiment to using just 2 antioxidants. I look forward to hearing from you

[SciB]

Hi Tony,

I was just looking for some microscope photos of crown galls online and found several links that might be useful to you. Here are some good sites with general information about plant galls:
http://www.treesforlife.org.uk/forest/e ... galls.html
http://www.s-cool.co.uk/a-level/biology ... rs-and-pcr

If you want to see what a section through a crown gall looks like, here is one from Carolina Biologicals which sells a slide with a stained section through a crown gall: http://www.carolina.com/plant-microscop ... /294432.pr

Here’s a paper about the anticancer activity of curcumin against melanoma in mice. Feeding the mice a diet containing 4% curcumin reduced tumors significantly and had an effect on the levels of certain micro-RNAs which regulate a number of genes. http://www-ncbi-nlm-nih-gov.ezproxy.hsc ... d/24349037

Let us know if you need more information.

[hanzhongxuan2]

Thank you for all your help!

I have watered the plants with the AC, and next week I am going to start inoculating them. There is enough time.



My teacher has a microscope (school grade) and camera that I can use. In your microscope link, what is the difference between a microscope slide and slip cover? And do I need to dye/stain my cross-section before using?

Tony

[SciB]

Hi Tony,

A slide is what you put the specimen, the stem section in this case, ON--the thin glass coverslip is smaller than the slide and goes on top of the section to protect it and to keep the lens of the microscope from touching it: http://en.wikipedia.org/wiki/Microscope_slide
Here is a method for cutting plant sections that works well but requires double-edged razor blades that are pretty hard to find: http://www.microscopy-uk.org.uk/mag/ind ... icera.html

Hopefully your teacher can help you with cutting the sections. You don't have to stain the sections, but it does help to show certain details. Staining can be pretty complicated and require special chemicals that can be hazardous. You can experiment with simple stains like food coloring, ink or iodine. Ask your teacher. The most important thing is to look for consistent differences between the control plants and the ones treated with the vit C or curcumin. I have never looked at plants infected with AT so i have no idea what the plant sections would look like.

If you want to improve the power of your data you could do the slide photography 'blinded' [http://en.wikipedia.org/wiki/Blind_experiment]. This means that you don't know which plant the section is from so the photographs you take are not biased. Your teacher could code the slides so that she knows what they are but you won't until she gives you the code. You don't have to do this, but it really impresses the judges if you are presenting your results at a science fair.

I am really looking forward to hearing about the results, so be sure to post back after the experiment is complete. Very few people do this and so we never know how the experiments turned out. You can be one of the star students who do let us know the results!

All the best,

Sybee

[hanzhongxuan2]

Sybee, the "blind testing" seems really interesting!

Just to make it clear, do I cut the sections, and my teacher labels it and I don't know what group of plant it came from?
I'm not quite sure how it works. So there are two independent variables: T, and VC. The six groups are: normal, normal bact., t, t bact, vc, vc bact.
Let's say I cut two cross sections from each plant (12 in ttl). I cut the cross sections, and put the 12 into a plastic bag (each bag corresponds to the plant above it). Then, I ask someone (not doing our experiment), to label the sections from 1 to 12 in a RANDOM ORDER. That person then records the sections on a seperate piece of paper (1 = vc #2, 3 = nb #1 etc). The paper is placed in an sealed envelope which we will not look at.
What will I do when I take the pictures through the microscope? Also, I can easily distinguish the normal plant (no bacteria OR act) from the rest because it has no tumors. What is the point of the blind test? What data and conclusions do we get from it? Will we ever open the envelope in the end?
Do I try and figure out which one is which?

[SciB]

Hi Tony,

I am glads you are thinking about this and asking questions. In human drug testing research we actually do a DOUBLE blind in which the person administering the drug or placebo does not know which it is and the people running tests to determine the effects of the drug do not know which patient got placebo and which got drug. In scientific language this is called a 'double-blind, placebo-controlled drug trial'.

In your case it does not have to be DOUBLE blind because you cannot influence the plants' responses by knowing which treatment you are giving. You could be biased in looking at the sections, however. That's why i want you to examine the slides and take the pictures and record your evaluations without knowing which is which. OK, you may be able to tell the difference between a section from a plant that received no AT and one that did, but you probably won't be able to tell the difference between the groups that have AT and one of the treatments or no treatment.

What I want you to do is cut the THINNEST possible sections through the inoculation site that you can using the double razor blade cutter if you were able to make one. The sections have to be very thin because you are looking though them under the microscope. When we make sections in the lab we use a machine that has a special knife and holder so that the sections are less than one cell in thickness.

You should cut 4 sections from each plant and DO NOT put them into a bag! The stem sections should be small enough so that all 4 will fit on one slide and you need to put them on the slide immediately. If the sections are too large, then use more slides. Here's the info about doing this again: http://www.hometrainingtools.com/micros ... aredSlides

If you are going to try staining a couple of the sections you should do it as soon as you cut them. Add the stain for a couple of minutes then, using a dropper, carefully rinse the sections taking care not to wash them off the slide. If you have a helper, then they can be doing this while you continue to cut sections.

For the unstained sections, add a drop of water and cover them with a cover slip so they do not dry out. You can seal the edges of the coverslip with vaseline or clear nail polish. This is going to take some time so don't start it until you have a couple of hours to spend in completing it. You really should take the photos the same day because once you cut the sections, the cells will start to break down and the sections will dry out unless you seal the edges of the cover slip. I know this sounds like a lot of work and it is but I think I remember you said you were working as a team so you should have everyone there to help.

Your teacher or a helper should make a random code number for each plant and write this number on the slide containing its sections and record which plant and group the number corresponds to in the code book. Write the number on the slide using a Sharpie marking pen so that it won't come off. Your helper keeps the code in a secret place so that you cannot see it. You will not know which number goes with which group until the code is broken after the slides are examined. The purpose of this is so you do not unconsciously select parts of the section to photograph that look like what you expect them to. Do you understand now how this works?

I do not know how you will 'score' the results based on the slide pictures. You should take photos at low magnification, say 40X, then some at 100X or 200X. What should you look for? I really don't know. The TI plasmid from the bacteria stimulates plant cell growth so there should be extra tissue at the inoculation site, but I don't know if the 'tumor' tissue will look different from normal tissue. If the vit C or curcumin treatment prevented the tumor from forming then there should be less of the new tissue and this is really the main thing that you are looking for. Maybe you could estimate the size of the growth as a percentage of the control without AT.

How do the AT-inoculated plants look? Is there some growth of plant tissue at or near the inoculation site? Are you taking lots of photos and keeping a careful record of which photo corresponds to which plant? Also record your own visual observations and impressions in your lab book.

I've given you a lot of detailed information here so please read my post carefully and if you don't understand something then please ASK! It would be a lot easier if I could be there to show you how to do this, but we'll just have to make it work virtually.

Good luck!

Sybee

[hanzhongxuan2]

Hi Sybee!

Everything's going great: there were noticible brown-blackish "lumps" at the inoculation sight. We've been recording everything and taking photos.
I don't have to worry about anything more, except for maybe finishing the board and memorizing the script.

I am going to aim towards using the sections ASAP (the best i can do is a day). I will seal the edges with vaseline to prevent the section drying out. Other than that, is there a way to secure the section, slide, and slip together into one "bundle" for easier transport? Also, how can I preserve the sections more?

As for the blind test, I will do that next week. I will cut the sections, examine them, and THEN look at the code sheet to prevent bias.
Everything's on track and well.

I really appreciate all the help you gave me, Sybee!
On my "Acknowledgements" section, I need the full name of the people that helped me. Can I have your last name? And Sarah's too, if you can find her's.

Thanks a lot!

[SciB]

Hi Tony,

I'm glad to hear that the plant tumors are growing. Do you see any difference yet between the controls and the plants treated with turmeric or vit C? It's probably too early to tell. You are still treating them with T and VC, right?

You are certainly welcome to any help I have given you. I am happy that you have been successful and are learning about how science really works. I hope you decide on a scientific career. It's great fun, exciting and satisfying to know that you have advanced the knowledge of a subject and maybe found something to prevent disease or make a better computer chip. You can just acknowledge the Experts at Science Buddies. We do this because we love to help young scientists-to-be, not for any recognition.

Does your school have a website where science fair projects are posted? We would really like to see your poster. Most of the time we help people all the way through a project then never get to see the results. We're curious too!

I'm sure you will have some more questions before everything is done, so we will be here when you need us.

Good luck!

Sybee

[SciB]

Hi Tony,

I haven't heard from you since the last post and I was wondering how your plant sections looked under the microscope. Were you able to cut thin sections using the razor blade method? Did you use a stain on the sections? Did the vit C or turmeric treatment prevent or slow down the growth of the AT tumors? I'd love to see some photos of the plants with the tumors and of your microscope sections. Does your school have a website that you will be posting them to?

I really would like to know how the project I helped you with turned out. This may help me to learn better ways to help the next person. Your feedback is important and if you have suggestions, I'd like to hear them.

Best wishes,

Sybee

[hanzhongxuan2]

Hi Sybee!
The plants are doing "great" (some of them have tumors; so it's "great" for me)!
From what I've heard from my partner, the plants are developing small black-brown discolorations and "lumps".
All of the plants that were inoculated have them.
My partner hasn't emailed the pictures to me yet, but I do have some pictures of the plants before the inoculation, if that will help.

I think it is still too early to compare the results between the control plants and the AC plants. But hopefully, after a few days, my hypothesis will be supported!

The tumors still have 10 days left to develop.
I was wondering if there was any way to possibly accelerate the growth of the tumors? Maybe increasing humidity, temperature?

Unfortunately, my school does not have a website for science fair.
And also, some picture files are too large (>2MB)
I'll do what I can to upload them!

Tony

[SciB]

Hi Tony,

Thanks for letting me know the status of your project. I was curious to know if you could see a difference in tumor growth yet between the control plants and the ones that got vit C or turmeric. You are wise to wait till the end of the experiment to comment on the results!

How warm is the place where you have the plants? I assume since you are in Canada that they are inside your house, but if you could increase the local warmth with a couple of incandescent lamps, that should speed up the growth. What kind of light are they getting and for how long? You can promote growth of the whole plant and presumably also the tumors by increasing either the light intensity or the duration. I have lettuce and herbs growing inside under broad-spectrum fluorescent lights and I keep them on for 14 hours a day and the plants grow great.

You can always decrease image file size by putting the photos in a powerpoint and saving it as a pdf. You probably already knew that and also that if you make any changes to the image files that you should save them with a different name so your original files remain unchanged.

Good luck!

Sybee

[hanzhongxuan2]

Hi Sybee! There was a problem...

The plants did not develop tumors, however those that were inoculated died, except for all of the Vitamin C treated ones, and four of the turmeric treated ones. I plan to just say that the plants treated with VC have prevented the cancer more effectively than tumor. That's ok, right?

I've sent you my final file for you to look at, with all the data.

[hanzhongxuan2]

Sorry, the previous file was the science fair. This is the file that has all the data.

[SciB]

I'm totally confused! Your data for the AT-inoculated plants has a Y in the column for tumor which i assume means YES there was a tumor; but you just said the plants did NOT develop tumors. Which plants are you talking about?

Where are your photographs of the inoculation area and of the whole plant? I'd like to see some of those, especially ones of the vit C and turmeric-treated plants inoculated with AT compared to plants that had been inoculated but only given water.

So, did you make sections through the stem in the inoculation area as I asked you to? That was a really important part of your study--to examine the plants at the cellular level. That is the kind of data a scientist is looking for.

WHEN did the inoculated plants die? How did they look. Can you send photos? WHY do you think they died? I thought AT formed galls on a plant but did not kill it.

Please get back to me about your project. I think you are leaving out too much without explaining anything.

Sybee