February+2012+Z

__‍Wednesday February 1st ‍__ Today, I used 7 day old zebrafish embryos and 200mg Walgreens caffeine tablets to test the effect of caffeine on zebrafish. Whereas caffeine heightens the heart rate of humans, from the paper I read, caffeine is supposed to continuously lower the heart rate of zebrafish until eventually the heart stops.

The first step in my experiment was to make a 80mM stock solution of caffeine knowing the molar weight of caffeine is 194.19 g/mol.
 * 1) To do this, I dissolved 4 caffeine tablets in water.
 * 2) Then I filtered the dissolved caffeine tablets to get rid of the fillers (the yellow color remains after filtering)
 * 3) The 4 tablets dissolved and diluted to 50mL, gives a 80mM solution.
 * 4) Next, I made solutions of different concentrations to use to test if the effects of the caffeine are dose-dependent
 * 5) Using the stock solution and egg water, I made solutions of concentrations 5mM, 10mM, 15mM, 20mM, and 25mM.

The next step was to set up the experiment.
 * 1) I set up petri dishes with quadrants.
 * 2) Egg water (the embryos started here)
 * 3) Tricaine
 * 4) 5mM caffeine solution
 * 5) 25mM caffeine solution
 * 6) Wash out in egg water
 * 7) I used a microscope to count the beats of the zebrafish heart in a minute, to measure heart rate.
 * 8) In order to measure the heart rate, it was necessary to first anesthitize the fish so that they were still (this allowed me to know that the movements I was counting within the fish were because of the heart pumping). Therefore, the fish were first put in tricaine methane sulphonate and I counted their heart rate.
 * 9) Next, I moved the embryo to the solution of caffeine concentraion that I wanted to test (for now, I tested 2 embryos in 5mM and 2 in 25 mM to see the extremes). After the embryo sat in the solution for a minute, I picked up a bubble of solution, with the embryo in it, and put it on a slide under the microscope so that I could remeasure the heart rate with the effect of the caffeine.
 * 10) After I had remeasured the heart rate, I put the embryo in the wash out to allow the embryo to recover.

__Results__
 * = Fish Number ||= Concentration Used (mM) ||= Starting Heart Rate (beats/min) ||= Caffeine Heart Rate (beats/min) ||
 * = 1 ||= 5 ||= 127 ||= Inconclusive ||
 * = 2 ||= 5 ||= 123 ||= 119 ||
 * = 3 ||= 25 ||= 115 ||= 68 ||
 * = 4 ||= 25 ||= 117 ||= 62 ||

__Observations__ The 5mM solution with only a one minute waiting period did not prove to show much of a change in heart rate. However, on the first trial, I was having difficulty establishing an efficient technique and the embryo was left in the caffeine for a few minutes. As a result, the embryo began to convulse. However, I am unsure if this is a result of an overdose of caffeine or the tricaine wearing off. The 25mM solution worked very well in this experiment. In the one minute wait period, the heart rate dropped significantly. This would be a good test for the high school students to run to test the effects of caffeine on the heart rate of the zebrafish.

__Next Step__ Next Saturday, I will be further testing for heart rate and I will be doing research this week to find another aspect from heart rate to test the effects of caffeine.

__Saturday, February 11th__ Aprell and I have decided that we are going to focus on the same test that we ran before. We will use some variations, but we want to collect good data to analyze this one aspect. Today, I ran the same test as before. However, I had to make new solutions because the caffeine does not have a long shelf life when left unrefrigerated. Therefore, I made a new 100mM stock solution and trial solutions of concentrations 5mM, 10mM, and 15mM. From this data, I was able to see that the 15mM solution has a great enough effect that there is no need to use a higher concentration. However, I would like to test the gaps in between the concentrations I have already tested.
 * Resting HR || Concentration Tested || Experimental HR ||
 * 116 || 5mM || 108 ||
 * 120 || 5mM || 112 ||
 * 122 || 5mM || 115 ||
 * 119 || 10mM || 45 ||
 * 121 || 10mM || 47 ||
 * 114 || 10mM || 51 ||
 * 124 || 15mM || 39 ||
 * 123 || 15mM || 53 ||
 * 120 || 15mM || 38 ||

__Tuesday, February 14th__ Today, I am running another of the same test to collect more data for our graphs and information to be presented at Jordan Hall. I had to make another new set of stock solution and experimental concentration solutions. This time I am still using 5mM and 10mM, but I am also testing 7mM to see what the readings will be in the gap between 5mM and 10 mM. It is important to note that this time, I am using 5 days old embryos; if there is a big difference in data, this could be the reason.

The data today was interesting. The 7mM concentration of caffeine seems to have the same amount of effect as the 10mM solution. Future tests will have to show if this is a consistent result.
 * Resting HR || Concentration Tested || Experiemental HR ||
 * 117 || 5mM || 111 ||
 * 112 || 5mM || 115 ||
 * 124 || 5mM || 118 ||
 * 108 || 7mM || 57 ||
 * 111 || 7mM || 60 ||
 * 109 || 7mM || 54 ||
 * 122 || 10mM || 52 ||
 * 119 || 10mM || 40 ||
 * 113 || 10mM || 48 ||

__Thursday, February 16th__ Today I am using energy/caffeine supplements to test to see if they have the same effect as pure caffeine. Over the next few lab days, I will be testing NXT energy blend, NOS energy drink, and Red Bull Sugar Free/ Lightly Carbonated energy drink. Today, I am using NXT. There is no way to know exact concentrations of caffeine, therefore, I will be using dilutions. 5x, 10x, and 20x.

This data has the same general trend as pure caffeine. Higher amounts of caffeine drop the HR more. The fish were starting to move more during these test, so the data could have some error, but I am confident in the results. However, I may need to make new tricaine MS22 next week.
 * Starting HR || Dilution Tested || Experimental HR ||
 * 132 || 5x || 59 ||
 * 137 || 5x || 63 ||
 * 128 || 5x || 41 ||
 * 126 || 10x || 83 ||
 * 135 || 10x || 82 ||
 * 133 || 10x || 85 ||
 * 138 || 20x || 112 ||
 * 121 || 20x || 107 ||
 * 117 || 20x || 112 ||

__Tuesday February 21st__ Today, I am running the same test that I did on the 16th but using Red Bull and NOS. As I had mentioned before, I had to made new tricaine today so that the fish stay still while I count the HR.

Red Bull Data:
 * Starting HR || Dilution Tested || Experimental HR ||
 * 117 || 5x || 119 ||
 * 119 || 5x || 117 ||
 * 118 || 10x || 111 ||
 * 120 || 10x || 113 ||
 * 121 || 20x || 117 ||
 * 116 || 20x || 110 ||

NOS Data: Tested || Experimental HR || The results that I found today with the energy drinks, was very interesting. There seemed to be a slight drop in HR, but nothing significant. And there was no difference in the HRs between the three different dilutions tested.
 * Starting HR || Dilution
 * 119 || 5x || 114 ||
 * 120 || 5x || 118 ||
 * 123 || 5x || 114 ||
 * 121 || 10x || 114 ||
 * 120 || 10x || 123 ||
 * 119 || 10x || 121 ||
 * 121 || 20x || 113 ||
 * 127 || 20x || 112 ||
 * 123 || 20x || 115 ||

Results overall: The caffeine that we used proved to be dose-dependent and lowered the HR of the embryos. The more caffeine, the larger the drop in HR. Eventually, the heart rate will completely stop.