Science Buddies: "Ask an Expert"

Hi

My daughter is looking to work on science fair project to compare the differences in calcium absorption with milk vs. milk with vitamin D. She was planning to use chicken bones and we were wondering if there would be a measurable weight change with the chicken bones. Would it be better to use hydroxyapatite and if so what form of hydroxyapatite should we procure for her. Her project is due 2nd week of January. I am not sure if she needs to get any special permission to use chicken bones.

Regards
Sram

Hi,

Welcome to Science Buddies! This is a very interesting idea for a science project. What type of experiment are you planning to do for your project? Normally, calcium is absorbed through the intestinal track and is deposited in bones by osteocytes. Vitamin D is required for the absorption process. Soaking chicken bones in milk would probably not reproduce the physiological process of calcium deposition.

http://www.vivo.colostate.edu/hbooks/pa ... erals.html

http://books.google.com/books?id=1R_a_D ... es&f=false


As you have learned chicken bones are composed of hydroxyapatite, or calcium phosphate. If you soaked chicken bones in milk, the hydroxyapatite could bind some calcium and will probably absorb some of the proteins in the milk, so this would be more of a chromatography experiment.

http://en.wikipedia.org/wiki/Hydroxylapatite

I think you are on the track for a great science project, and perhaps some more background reading will help you understand what to do. For a science project, you do need some way to measure your results. If you soak the chicken bones in milk, you would need to dry them and have a balance that would weigh to the closest .001 gram. Or, you would need to elute the adsorbed molecules from the chicken bones with a 500 mM phosphate buffer and measure the protein or calcium that was adsorbed from the sample.

I am not aware of any restrictions on using chicken bones in science fair projects; however, you do need to have the teacher's approval for any project before your start. You need to plan a big dinner and serve lots of legs and thighs so you will have a large quantity of good-sized bones to work with. You can boil the bones so all the meat comes off easily and then bleach the bones in 3% hydrogen peroxide overnight so they are nice and white to work with. Since the project is due the second week of January, I would try to finalize the experimental plan within the next week so you will have time to complete the project on time.

Donna Hardy

Donna

Thanks for the response. We were thinking of suspending part of the chicken bone in milk, milk+vitamin D, milk+turmeric solutions to determine if there is any weight change. If we suspend the bones in these liquids, it will be easy to remove and measure the weight. Would that work?

Do we need an access to a lab to use the 500 mM phosphate buffer? Could you direct us to links on how to use this and learn a little bit more about this?

Regards
Sram

Hi,

Yes, I think this will work. I would recommend soaking the bones in the various solutions for several hours or overnight. And plan to do the experiment in triplicate with separate containers for each bone if possible. You should do a turmeric only set of samples in addition to the milk and milk plus turmeric. You can dry the bones before you start and then dry them after they are soaked. You should rinse the bones with water after they are soaked to remove any protein or calcium that is not adsorbed specifically to the bones. You will need a definite protocol for drying the bones to make sure your results are consistent and reproducible, probably something like baking the bones in a single layer at lowest oven heat for 1-2 hours. To confirm the bones are dry, weigh them, and then rebake to confirm the weight has not changed. You will need a technique for identifying each bone so you can compare the weight before and after for the same bone. Be sure and include a control bone that is not soaked in anything. A positive control would be good to do also. Ask your daughter if she can think of something to use for a positive control.

It would be excellent if you could compare the weighing method vs. the elution method for this project. To elute bound proteins and calcium, you can make your own phosphate buffer. Reagent grade potassium phosphate is available from Carolina Biologicals, or you could use trisodium phosphate available from your local hardware store. Do you have a balance to use to weigh the powdered reagent? Let me know if you decide to do this option and I will explain how to make to 500 mM phosphate buffer.

http://www.carolina.com/product/potassi ... estMatches

http://shop.chemicalstore.com/navigatio ... ?id=TSP12T

If you elute the sample from the bones, you would need a method to measure the calcium and proteins eluted. There are test kits available for calcium and proteins can be quantitated using absorbance at 280 nm with a spectrophotometer or with a colorimetric protein assay. Does you daughter have access to a spectrophotometer at school? If not, I will try to suggest an alternative.

Your daughter needs to understand the science behind her project, so I would recommend doing Google searches for composition of milk, proteins in milk, composition of turmeric, and mechanism of hydroxyapatite chromatography. The experiment needs to be a carefully controlled experiment with quantitative results; the independent variable is the solution the bones will be soaked in. Let me know if your daughter has questions on any of the information.

Here is the Science Buddies website guide to doing a science project. After doing more background reading, your daughter should be able to write down the question she will be answering with her experiment before designing the experiment. Read through all of the steps so you can help her understand what she will be doing next.

https://www.sciencebuddies.org/science- ... ndex.shtml

Since this is an original project, I would recommend doing a pilot experiment using one control bone and one experimental bone. This will help your daughter gain the experience and solve experimental detail problems with a trial run. The definitive experiment will be more successful if you include this important step.


Donna Hardy

Donna

Thanks for the detailed explanation. She wants to set up a trial run as you suggest understand the experiment and then finalize the experiment.

We will enquire about the spectrophotometer in her school and see how to proceed on the elution part. Will keep you posted on the results.

Regards
Sram

Hi,

Yes, there are lots of details that need to be finalized, depending on availability of materials and equipment, but let me know what you will have available.

Donna Hardy

Donna

I have pinged their science teacher and will call them if I don't see a response today. But she is setting up the trial today. I have a kitchen balance at home that is accurate to 0.1gm, but checked online to see if I can procure one with higher accuracy. Looks like it is going to expensive to purchase one with the higher accuracy. Could you suggest a mentor/lab in the DFW area that we can have access to. My daughter who is in 6th grade, usually gets all the science fair experiment and boards completed during the winter break. If we need access to these equipment, she needs to complete as much as she can before winter break
!.

Regards
Sram

Hi Donna

I checked with their school and they have access to spectroscope but not a spectrophotometer. Let me know of any resources in the DFW area.

Regards
Sram

Hi,

The type of balance that you need to weigh to the closest milligram is too expensive to purchase for a science fair project. I don’t know of any mentors or labs in the DFW area. Ask the teacher, or call a local analytical lab, your local water company, or a university. I’m sure there is someone available to help weigh the bones. If your daughter has them all prepared and labeled, it will take just a few minutes to weigh.

Unfortunately, a spectroscope is not suitable for this analysis. I will think of something else.

Donna Hardy

Hi,

Here are suggestions for measuring the calcium and protein eluted from the chicken bones that will give you semi quantitative results for your project. The protein assay option would be a lot of work and there is not a lot of time left to get everything together, but it should make a really excellent science project you you can do it.

Protein can be measured using the Bradford Protein Assay:

http://en.wikipedia.org/wiki/Bradford_protein_assay

Here is a recipe for making the Bradford reagent using a blue dye called Coomassie Brilliant Blue G-250 (note you do not want R-250, which is used for a different application). The reagent requires methanol or ethanol to dissolve the dye and phosphoric acid. This protocol also includes using NaOH (sodium hydroxide), which interferes with the assay, so do not include this step). The protocol also calls for using a spectrophotometer to measure results, which you don’t have, but this assay turned bright blue, so you can make a series of different concentrations of protein and add the dye reagent and match the color of the unknown sample to the color of that closest standard. A range of standards from 0 to 2 mg/mL will give you a “standard curve.” that ranges from clear to very dark blue.

http://www.ruf.rice.edu/~bioslabs/metho ... dford.html

So this method would work if you can obtain Coomassie Brilliant Blue G-250, ethanol/methanol, and phosphoric acid, and a pure protein to use as a standard. You would also need some small test tubes, an accurate balance, and pipettes to measure volumes accurately.

Calcium can be measured with water hardness test kits that can be purchase on-line and possibly at a local home and garden store. There are lots of on-line options for these kits and they are usually titration kits that call for adding a reagent in one drop at a time until there is a color change. If you decide to pursue this option, make sure you get a kit that includes all of the ingredients so you will be able to explain the chemistry in the project write up. Let me know what kit you purchase if you need an explanation.

http://en.wikipedia.org/wiki/Hard_water

For this project, working with milk samples, it would be better to be able to measure the protein that binds to the chicken bones rather than the calcium. Protein should bind well to the bones so there should be a measurable result. I’m not sure how much calcium would bind to the chicken bones, and the test kits are not that sensitive.

I hope this helps. Let me know if you need more options.


Donna Hardy