Positive and negative controls

[Sareena Avadhany]

Hi Experts,

I have started my project this year, and I am testing the effects of curcumin on an organism (what organism, I haven't decided. However this is not relevant to my first question). My teacher was talking about what I could do, and she mentioned positive and negative controls. I have tried reading up on what positive and negative controls are, but haven't gotten a clear answer. It would also be really great if an example could be given as well.

For my second question, this regards the organism I have to choose. I am most definitely conducting my experiment at school, so I cannot use any harmful micro organisms. I began this year thinking about testing the effect of curcumin on paramecium, but my teacher said it would be really hard to acquire 100 +/- samples of this protist because it is so hard to 'catch' if that is the right word. The reason I initially chose paramecium is because it can be used as a model in my experiment. If curcumin inhibits growth of paramecium, then I can predict it can for other closely related protists. She suggested brine shrimp and E.Coli as other possible organisms to conduct the test on. I have many choices, but I am confused on what to choose. If I could be provided with suggestions or information or websites that can help me make my decision, I would be most grateful.

My third question is to do with isolating curcumin, which comes from curcuma longa. Curcumin is the active ingredient in curcuma longa, but from what I have research, there are other components of the plant that also show some healing properties. I have researched on paper thin chromatography, and it seems like the best way to go in terms of isolating curcumin. When the results show the separated substances in a certain order from let's say substances 1-4, if I conduct the same test, will substance 2, for example, always be shown in the same location? Would I be able to control the order in which the substances separate?

Thank you very much!

Sincerely,
Sareena

[hhemken]

Sareena,

As to how to design your experiment, please read some posts I have made in the "Plant Nutrition" thread here in the Life, Earth, and Social Sciences forum. It sounds as if your experiment may be as simple as one test group with curcumin, and one without. The one without is to be treated with the same materials as that of curcumin, exept without curcumin. This means that if you needed to dissolve the curcumin in 70% alcohol in water and applied 5 ml of that solution, you would apply 5 ml of 70% alcohol in water without the curcumin to the control group. Yoyu may even have an additional control group to which you add 5 ml of pure water to control for the effects of the alcohol.

I assume you have already seen:
http://en.wikipedia.org/wiki/Experiment
http://en.wikipedia.org/wiki/Negative_control
http://en.wikipedia.org/wiki/Positive_control

I don't think the terminology of positive or negative control is important in your case. The purpose of the controls is to tease out the effects of all of the treatments you are using, and leave no doubt as to what component is causing what effect.

As to purifying curcumin, you cannot do it on a useful scale with paper chromatography, which is a method to detect the presence of substances in a mixture. The amounts used are miniscule, and will not be useful to purify any useful amount to use as a treatment. If you purify curcumin yourself, you will use paper chromatography or thin layer chromatography (TLC) to see how pure it is, usually by comparing to a standard of known purity.

Curcumin is obtained by crystallization. It is very soluble in ethanol, apparently less so in water. It would be laborious and distracting for you to purify it yourself. You would be better off purchasing it. Search for these terms on google:

curcumin crystalline buy

For example, here is one source (I know nothing about them, and this does not constitute a recommendation to buy from them):

http://www.naturalpharmacy.com/suppleme ... y/Curcumin

There may be other sources. Try visiting nutritional supplement stores in your locality.

You will probably want to test several doses on your model organism. You may want to look into using baker's dry yeast, available at many supermarkets. If you do, buy several packages and make sure they are all from the same manufacturing batch.

You need to think what will be your dependent variable. What will you measure, and how will you measure it? Growth rate? Carbon dioxide production (especially with yeast)?

[Sareena Avadhany]

Thank you Heinz,

I talked to my mentor about the isolation of curcumin from turmeric, and she said paper chromatography. From what you said and from what I've read, paper chromatography is not an effective method to test its effects on micro organisms. Since the samples are extremely small, different concentrations does not seem feasible.
When I re-read my notes on curcumin, I realized that curcumin is the active ingredient in the root curcuma longa and turmeric. There are other components that show remedial properties, but curcumin is the only component that shows microbial activity. Doesn't that mean that even if I don't isolate curcumin from turmeric and test turmeric's effect on microorganisms - I've chosen E.Coli - then I can safely assume that curcumin is the property that is e.coli is trying to resist to, right?
If I can just test turmeric and not have to isolate curcumin, how is this possible? I can find turmeric in powder form. I would I set it up in petri dishes when I inoculate it with e.coli? Is there any website or text that shows ways to test concentrations of substances in a powder form?

Thanks!

Sareena

[carolinethorn]

Hi Sareena,

Good to hear that things are progressing so well with your ideas.

In my opinion i think it would be appropraite to use the tumeric for your experiments and then in your report to state your assumptions about the active component of it being curcumin based on your background research. I think that is preferable than trying to purify the curcumin as you may not end up with enough to do the number of repeats of the experiments. I would be especially carefull that all the turmeric comes from the same batch as mentioned before by Heinze, as each packet may have a slightly different amount of active ingredient/curcumin in it. Maybe even work out how many packets you might use and thn blend them all together at the beginning of your experiment just to make sure it is homogenous.

I would recommend that you make a solution of the tumeric for treating the organisms with. That way you can measure it very accurately to add it at low concentrations and for doing a range of concentrations. It should be water soluble. If you are making your agar plates yourself you could add hte solution to the medium when it has cooled slightly and then pour them out, but if you have pre-poured plates or are making ones with several concentrations it might be better to add the tumeric solution after by putting a set volume on and spreading it over the plate with a cell spreader and then letting it soak in. Another way people test antibiotic solutions on agar plates is to spread the organisms first and then put a small circle of filter paper (only 5mm across) in the center and pipette the test antibiotic onto that. I will see if i can find a website that describes that.

Best of luck!
-Caroline

[Sareena Avadhany]

Thanks Dr. Thorn,

I was so confused as to how to test the effects of turmeric when it is in powder form; this makes much more sense.

The experimental set-up is due November 25th for the Synopsys Science Fair, so I have just started writing my procedures. I plan to finish soon, but I never knew there were so many things to consider when studying microbial cultures.

Thanks for your help.

Sincerely,
Sareena

[carolinethorn]

Hi Sareena,

I just realized we spent all of the thread talking about how to prepare the test compound and didn't really talk about your question on positive and negative controls. Now that you have an idea about dissolving the tumeric, what do you think would be your negative controls? and do you have plan for a positive control? (maybe a little more tricky)

-Caroline

[Sareena Avadhany]

Hi Dr. Thorn,

I am sorry for not getting back to you sooner. There are many updates on my project. As for now, my positive and negative controls are a disk saturated in iodine and just the disk, respectively. As for the concentrations of curcumin, I am going to have 10%, 20%, 50% and 100%, and two trials, so 8 petri dishes for my experimental set-up will be used.

I talked to my teacher about dissolving curcumin in water; she recommends alcohol and sodium sulphide. What would be a better solvent? Since curcumin is an organic substance, would it dissolve better in alcohol and sodium sulphide?

Here is a rough sketch of my experimental set-up:

I am inoculating petri-dishes with e.coli and placing a disk saturated with curcumin in the dish, and then measuring the zone of inhibition 24 hours later after incubation. After waiting a period of time to notice a decrease in size of the zone, I will take a sample from a colony grown in the zone and plate the e.coli in a new petri dish of a nutrient medium with curcumin, to see if e.coli has grown resistant to it.

When I am conducting the second part of the experiment, inoculating plates to test the resistance of e.coli, do I need to use the same solution as I did with the first part? If I do, since alcohol evaporates, won't that change the concentration of curcumin?

There was another question that popped into my mind: what if the colonies that grow within the zone are not because of resistance, rather, it is because the curcumin molecule broke down? So I thought about how we would prove that it is the e.coli that developed the resistance and not the molecule break down. In my second part of the experiment, I am inoculating plates of a nutrient medium containing curcumin with the e.coli from the zone of inhibition, and I am also going to need to control. I told my mentor that my control would be the plate of nutrient medium with curcumin, but with e.coli not from the zone of inhibition. She told me that it we would need to take e.coli from the zone of inhibition and plate it on a dish not containing curcumin.

I think my control was correct, because if e.coli from the zone grows on the new plate with curcumin, and the e.coli not from the zone doesn't grow on the new plate with curcumin, we know for sure that it is the e.coli, not the curcumin. My mentor told me to think about the suggested the control to be, but I cannot understant how the control would tell me that it is the e.coli, not the curcumin. Her control would tell me that the e.coli is viable, so should I have two controls? One with broth containing curcumin, and one without, both plated with e.coli?

Thanks Dr. Thorn.

Sincerely,
Sareena

[carolinethorn]

Hi Sareena,

I can see you have been thinking about a lot of things and got some good plans.

To decide which solvent to use I would try a quick pilot experiment of attempting to dissolve the curcumin in the different solvents you have and seeing which works best. I would start with a set volume of solvents and at a low concentrations and keep adding a set mass of solute (the curcumin) until it becomes saturated - ie. no more will dissolve and you can see it starting to settle at the bottom of the tube instead. Remember to keep track in your notebook about this.

Your set up for the first experiment sounds great. Remember - you will need some solvent alone, rather than a blank disc, to use for the negative control so that you can demonstrate that it was the solute (the curcumin) that was inhibiting any bacteria.

I think you will probably want to repeat the first experiment at least once to se if you get a good "dose-response" - that the ones with more concentration curcumin have larger zones of inhibition.

I am not a microbiologist really so I think the second part will be interesting as I have no idea how long it takes bacteria to become resistant to an inhibiting agent. I think having both controls is an excellent idea. After bacteria have been growing on a plate for a while it starts to use up all of the nutrients and also may exctrete waste products into the plate so i think both controls will show you important information.

Your question about the concentration for the second experiment is a good one. It shouldn't matter if some of the alcohol evaporates after the curcumin has been distributed through the agar plate but you would need to think about how much to add to have the concentration be the same as experiment 1 given the difference in the area of the plate where the curcumin would be effecting the bacteria.

Best of luck,
Caroline

[Sareena Avadhany]

Hi Dr. Thorn,

I am sorry I have not gotten back to you in a while.

I have again made many changes in my project the last week. I looked over my experiment and realized it was actually two science projects. Seeing whether E.coli has the ability to grow resistant to curcumin is to me a continuation project. I had to think about why I wanted to do this particular experiment and its applicability.

I then went back to what aroused my interest in curcumin; it was mainly because I discovered Ayurveda and noticed that we used turmeric powder in practically all of our Indian cooking. So instead of seeing whether E.Coli has the ability to grow resistant to curcumin, I decided to compare the effects of turmeric and curcumin on E.Coli, and see if there is a recognizable difference in the zone of inhibition. I know that curcumin is a component of turmeric, and I know that curcumin shows antibacterial property. What I don't know is that if it inhibits the growth of E.Coli and if turmeric is composed of other properties besides curcumin that show anti-bacterial properties. This is relevant because most people who use turmeric powder use the powder form, not curcumin. I am not exactly sure whether my indecisiveness about choosing a set project and sticking with it is bad; I have changed my project many times in a short time period. The SRC application date is nearing as well.

I am keen on pursuing the 2nd part of my initial experiment. I find it extremely interesting; I expressed my interest in conducting the experiment to my advisor, and she said that if there is time after the fair, I would be able to do it.

We also discussed the isolation of curcumin. It does not seem that we have the facilities to isolate curcumin and acquire the concentrations I would need. I explored the possibility of paper chromatography and posted a question about it on a previous message, but have concluded that it is insufficient for the quantity I need. Mr. Hemken had provided websites to purchase curcumin tablets, but I have no idea whether what I'll be purchasing has additives that help produce the desired results or make the curcumin powder dissolvable. This is a big stumbling block for me. I talked to my teacher about dissolving it in alcohol and in water. In terms of knowing which solvent would work best, how would I be able to recognize? Would it be which solvent can dissolve the solute best?

The outlined of my planned procedure is:

Three set-ups will be used. One dish of curcumin, another of turmeric powder store-bought, and the last is the ground root. The dish will be divided into four quadrants - one quadrant for the positive control (C+), C- (just the disk) and another C-(the solvent) and the substance (Either curcumin, turmeric powder store-bought or ground root). A lawn application will be used, and the nutrient medium will be LB broth. I will incubate each disk for 24 hours and record my results the next day.

Thank you for all your help Dr. Thorn. I have settled on this experiment, and am definitely sticking to it - the SRC date is too close to make any major changes.

Sincerely,
Sareena

[carolinethorn]

Hi Sareena,

My apologies that I did not see your message right away.

Do not worry that you changed your mind many times before you decided on your project. As a graduate student I changed my end goal many times! I think you have shown maturity about recognising that what you had initially planned was really 2 projects. I have had graduate students who were not so mature and tried to do big overly complicated experiments and then forgot to include basic controls and so in general its often better to keep it simple so that it answers the question. I think your experimental procedure design looks good.

If the websites for purchasing the tablets are biomedical suppliers they should be able to tell you the approximate purity of the curcumin. If they are health food places, probably not. One of the most commonly used suppliers of biomedical chemicals is Sigma Aldrich (signmaaldrich.com). They have a couple of different grades or purity of curcumin available. The purer it is the more expensive it is and the less you get! I would say that the 70% purity is probably fine as its going to be a big enough difference from the raw ground root, and i doubt it is worth spending a lot of money to get the 94% pure stuff. It lists how pure it is in the"properties" section of the compound description under assay. It also mentions there what the solubility limit is and how well is dissolves in different solvents so that should help answer your question. Most biomedical suppliers should give that information. You can tell if it has dissolved when there is no particulate matter in there and the solution is clear. Most people just do this by eye and shaking or vortexing the tube to check that no specks of powder are visible.

Hope that helps. I am going to be away over thanksgiving but am sure other people will help out if anything more comes up.
Best of luck,
Caroline

[hhemken]

Sareena,

I think you are too nervous. Your experiment is well designed, and you will learn several important lessons. It is not important whether the purchased curcumin is absolutely pure. You would be testing several concentrations in which the curcumin would be by far the most concentrated solute.

Also, I suggest you use neither alcohol nor sodium sulphide (nor hydrogen sulphide, if that's what your teacher meant). Water is most likely the best choice. Try dissolving some today, if possible, to show yourself if it will be OK. If the store-bought curcumin dissolves slowly, you can let it sit in a cool dark place for a few hours with occasional stirring.

In any case, be calm and collected, your experiment will work. I do suggest you have 3 or more, preferably 5 or more, replicates for each treatment. This way you will have good quality averages and standard deviations which can be used in a Student's t Test (research this). With that statistical calculation, you will be able to confidently state whether curcumin or turmeric have statistically significant effects.

Good luck!