Chemical Safety:
1. During the part of the experiment that will be performed at the school, the chemicals of glucose, alanine, bean extract and sodium nitrate cocktail will be used in accordance with the MSDS.
2. The use of all chemicals will be done while wearing gloves, aprons, and goggles.
3. All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
4. A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use.
5. Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
1. During the part of the experiment that will be performed at the school, the chemicals of glucose, alanine, bean extract and sodium nitrate cocktail will be used in accordance with the MSDS.
2. The use of all chemicals will be done while wearing gloves, aprons, and goggles.
3. All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
4. A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use.
5. Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
Chemicals and Heat:
1. Goggles and aprons will be worn.
2. During heating, potholders (thermal gloves) will be used to handle hot surfaces and equipment coming off the hot plate.
3. No containers will be sealed when heated. In the event a closed container needs to be heated a pneumatic trough or pressure release value will be used.
Chemical Disposal:
1. According to the Flinn Catalogue the Nutrient Rick Cocktail containing alanine, glucose, bean extract and sodium nitrate must be disposed of down the drain and only if drains are connected to a sanitary sewer system.
2. These materials maybe disposed of in quantities that do not exceed 100 grams each day for each substance by rinsing them down the drain with a large excess of water.
3. Completely dissolve each substance in water in a separate container. Rinse this solution of a second substance down the drain with ten fold excess water. Repeat if necessary.
Aseptic Techniques:
1. Upon entering the lab, wash hands and arms up to the elbow with antibacterial soap. Before and during experimentation wear rubber gloves, apron, and goggles at all times. 2. Use 10 % bleach solution and wipe down the lab area and tabletop. The Transfer of any culture will be done with a mechanical pipette.
3. Place a biohazard sign in plain view for all to see near or at the testing site.
4. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use. Needles will be capped at all times.
4. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use. Needles will be capped at all times.
5. After experimentation, clean the lab counter with 10% bleach. Wash hands with 10% bleach.
Disposal:
1. Petri dishes and other disposable equipment will be placed in a plastic bag and autoclaved for 30 minutes at 20 psi, 120 degrees Celsius and disposed of.
2. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.
Electrical Power Safety:
1. Limit the use of high power hotplate around water.
2. Never use frayed or cut wires when plugging in a device.
3. Never overload the outlet and use a surge protector.
4. All electrical devices should have an emergency cut off switch or ‘kill’ switch’.
5. Shut off and unplug all devices when not in use or when you leave the room unattended.
1. Limit the use of high power hotplate around water.
2. Never use frayed or cut wires when plugging in a device.
3. Never overload the outlet and use a surge protector.
4. All electrical devices should have an emergency cut off switch or ‘kill’ switch’.
5. Shut off and unplug all devices when not in use or when you leave the room unattended.
Sharp Object Safety:
1. Use caution when using needles, scissors, and scalpels.
2. Restrict the use of "sharps" to a minimum.
3. Do not use bent or broken needles, knifes, or razor blades.
4. Cover or cap needle after every use.
5. Discard used syringes, needles, or scalpels into an approved sharps container.
6. Bring all needles in a sharps container to a local fire station for disposal.
1. Use caution when using needles, scissors, and scalpels.
2. Restrict the use of "sharps" to a minimum.
3. Do not use bent or broken needles, knifes, or razor blades.
4. Cover or cap needle after every use.
5. Discard used syringes, needles, or scalpels into an approved sharps container.
6. Bring all needles in a sharps container to a local fire station for disposal.
Safety Procedures for Sea Salt:
1. Using sea salt will be done with gloves, aprons, and goggles.
2. A fire blanket, fume hood fire extinguisher, shower and eyewash station are available for use.
3. According to the MSDS excess salt will rinsed down the sink with 10 fold excess water.
4. After using sea salt, hands will be washed with soap and water.
Safety Protocol for pH Buffer Calcium Carbonate
1. The use of Calcium carbonate will used in accordance with the MSDS
2. Using calcium carbonate will be done with gloves, aprons, and goggles.
3. A fire blanket, fume hood fire extinguisher, shower and eyewash station are available for use.
4. According to the Flinn catalogue, excess calcium carbonate will wrapped in newspaper, placed in a cardboard box, and disposed of in a landfill.
5. After using calcium carbonate, hands will be washed with soap and water.
Tank Set Up:
1. Obtain 4 ten-gallon tanks.
2. Fill each tank with tap water and let sit for one week to allow the chlorine to evaporate.
3. Add 1 inch of gravel to the bottom of each tank.
4. Place a water filter on each tank and check that the spinner does not clog or stop on a daily basis.
5. Add two shrimp to the tank to help the nitrogen cycle.
6. Check the pre-filter and clean it once a week.
7. Change and remove the carbon filter every month.
8. Every month change 10% of the water with clean fresh DO water.
9. Check the pH, salt level, and temperature every week with the Hanna pH EC/TDS HI98130 probe.
* a. Press and hold the mode button to turn on.
* b. Select the item to be tested with the set/hold button.
* c. Submerge the probe into the tank to be tested.
* d. When the stability clock disappears the measurement can be taken.
* e. Be sure to calibrate the probe before each test.
* f. Turn off the probe when done testing
Adjusting the Tanks:
1. Identify the water level to be about 2-3 cm below the black plastic lip of the tank.
2. Add reversed Osmosis water to replace evaporated water.
3. To adjust the pH small amounts of vinegar will be used to adjust for acidic environment and small amounts of baking soda (sodium bicarbonate) will be used to create a basic environment. (pH-up and pH down can also be used.)
4. In the event that salinity levels change more water will be used to dilute the levels and more marine salt will be added to increase salinity.
Inserting Living Organisms:
1. Place the container or bag in which organisms came in into the tank for 15 to 30 minutes. During this time they can be acclimated to the water temperature.
2. Add half of the tank water to the bag and reinsert the bag into the tank for another 15 to 30 minutes.
3. Release the organism and water into the tank.
4. Do not feed the organism for a day allowing it to get used to the environment.
1. Using sea salt will be done with gloves, aprons, and goggles.
2. A fire blanket, fume hood fire extinguisher, shower and eyewash station are available for use.
3. According to the MSDS excess salt will rinsed down the sink with 10 fold excess water.
4. After using sea salt, hands will be washed with soap and water.
Safety Protocol for pH Buffer Calcium Carbonate
1. The use of Calcium carbonate will used in accordance with the MSDS
2. Using calcium carbonate will be done with gloves, aprons, and goggles.
3. A fire blanket, fume hood fire extinguisher, shower and eyewash station are available for use.
4. According to the Flinn catalogue, excess calcium carbonate will wrapped in newspaper, placed in a cardboard box, and disposed of in a landfill.
5. After using calcium carbonate, hands will be washed with soap and water.
Tank Set Up:
1. Obtain 4 ten-gallon tanks.
2. Fill each tank with tap water and let sit for one week to allow the chlorine to evaporate.
3. Add 1 inch of gravel to the bottom of each tank.
4. Place a water filter on each tank and check that the spinner does not clog or stop on a daily basis.
5. Add two shrimp to the tank to help the nitrogen cycle.
6. Check the pre-filter and clean it once a week.
7. Change and remove the carbon filter every month.
8. Every month change 10% of the water with clean fresh DO water.
9. Check the pH, salt level, and temperature every week with the Hanna pH EC/TDS HI98130 probe.
* a. Press and hold the mode button to turn on.
* b. Select the item to be tested with the set/hold button.
* c. Submerge the probe into the tank to be tested.
* d. When the stability clock disappears the measurement can be taken.
* e. Be sure to calibrate the probe before each test.
* f. Turn off the probe when done testing
Adjusting the Tanks:
1. Identify the water level to be about 2-3 cm below the black plastic lip of the tank.
2. Add reversed Osmosis water to replace evaporated water.
3. To adjust the pH small amounts of vinegar will be used to adjust for acidic environment and small amounts of baking soda (sodium bicarbonate) will be used to create a basic environment. (pH-up and pH down can also be used.)
4. In the event that salinity levels change more water will be used to dilute the levels and more marine salt will be added to increase salinity.
Inserting Living Organisms:
1. Place the container or bag in which organisms came in into the tank for 15 to 30 minutes. During this time they can be acclimated to the water temperature.
2. Add half of the tank water to the bag and reinsert the bag into the tank for another 15 to 30 minutes.
3. Release the organism and water into the tank.
4. Do not feed the organism for a day allowing it to get used to the environment.
Bean Extract:
1. Using a measuring cup, measure out a cup of beans.
2. Use a large beaker and fill 3/4 full of water.
3. Place beans in beaker, if beaker is over flowing with water, spill about an inch of water on to drain from beaker.
4. Prepare hotplate at 34 degrees Celsius.
5. Place beaker on to hotplate and let boil for half an hour.
6. Remove beaker and turn off hotplate.
7. Prepare blender.
8. Place beans in blender and blend beans until turned into a very liquid mix.
9. Use a new clean beaker and place filter paper over beaker.
10. Use bean mix from blender and pour onto filter.
11. You have now prepared bean extract.
Cocktail Procedures:
1. Get glucose, alanine, sodium nitrate, beans, and scale ready.
2. Make sure scale is set to 0, and there is no residue on weighing plate of scale. If there is residue remove it by using a wet wipe. Then once its dry you may begin to weigh objects.
3. Measure out the following: .6 grams of glucose, .2 grams of sodium nitrate, .7 grams of protein, and .6 grams of alanine.
3. Get a beaker that measures one liter. Place measured glucose, alanine, protein, and sodium nitrate in beaker.
4. Fill beaker with water until it reaches 1 full Liter.
5. Stir solution.
6. It is now ready for use. Store at room temperature, away from edges from where it could spill or fall and away from sun light. Place paper on top of beaker to prevent any loose particles from getting in.
Injection Methods:
1. Take one syringe from where it has been stored properly.
2. Remove cap carefully and place on a sterile surface.
3. Pull the plunger up to suck a little bit off air. The tip of plunger should never be in contact with any fluid while measuring.
4. Insert the syringe needle into the solution and suck up an amount great than 1 cc.
5. Push out the excess until the syringe reads 1 cc.
6. You have now properly measured 1 cc of desired fluid.
7. Grab one anemone and insert about quarter of an inch of needle onto the upper column close to the oral disk and below the tentacle row. If done properly the needle should not hit the actinopharynx of the anemone.
8. Gently begin to inject anemone. When solution has been dispensed, gently remove needle away from anemone.
9. With alcohol and cotton ball clean needle. Cap needle when done.
10. Clean syringe barrel by removing syringe plunger and placing it under running water. Rinse both barrel and plunger.
11. Wait until syringe barrel and plunger are dry and assemble syringe again.
12. Store syringe away to be reused or just throw away syringe in trash and needle in sharps box.
13. Repeat procedures every time when injecting an anemone.
14. Select anemones will be injected once a week with Nutrient Rich Cocktail for a month.
Data Collection of Zooxanthellae:
1. For each anemone, perform a Zooxanthellae count
2. Label test tubes, 1,2,3,4,5,6,7,8,9,10,11, and 12.
3. Have test tube stand ready and close by, away from any edges where it could fall.
4. Be sure not confuse which test tubes belong to which tanks. You should have three test tubes per tank, 12 test tubes total.
5. Clip about 4 centimeters of tentacle off all 12 anemones.
6. Add each tentacle piece to its labeled test tube with 3 ml of salt water. Then place test tube on test tube stand.
7. Set up hotplate and heart 250 ml of water in beaker to 34 degrees Celsius.
8. Place the twelve test tubes in beaker of heated water for five minutes.
9. The place test tubes in Centrifuge 2000 RPM for two minutes. Make sure the test tubes are place in an equal weight distribution inside the Centrifuge.
10. Once done with Centrifuge, stir the solution in each test tube.
11. Remove two drops from test tube labeled 1 and dispose of pipette. All other test tubes are to be held in test tube stand till needed. Dispose of pipette when done.
12. Place the two drops into a wet mount slide.
13. View under high power on a Compound Light Microscope.
14. Count the number of Zooxanthellae cells in one field.
15. Count the number of Zooxanthellae cells in four more random views.
16. Take an average.
17. Repeat this for each anemone tentacle in all the remaining test tubes.
18. This is to be repeated twice a week, at the beginning of the week and towards the end of the week.
19. Test tubes and clipping scissors will be autoclaved to be reused. See aseptic technique disposal above.
Descriptive statistics:
1. Data will be first entered in first by date and then by group. This is raw data to be displayed on the board.
1. Data will be first entered in first by date and then by group. This is raw data to be displayed on the board.
2. The averages for each group on each date will be calculated.
3. The averages will be organized by group and then by date.
4. The averages will be used to create a graph for each group.
5. The data will further be analyzed by performing a z test on each group using the control as the standard of comparison.
6. In order to perform the z test, the standard deviation for each group on the final date of measurement will be calculated.
7. The formula for z test is as follows:
where
8. In order to accept the null, the z-value must fall within the range of -1.96 to 1.96.
