Procedures:
Chemical Safety:
1. During the part of the experiment that will be performed at the school, Alisma and Potassium Hydroxide pellets 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.
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.
Electrical Power Safety
1. Limit the use of dissolve oxygen meter and filters 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.
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 a sharp object.
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.
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 Protocol for pH Buffer Calcium Carbonate
- The uses 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.
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
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:
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.
Treatment
Creating Alisma Solution:
- Take 2 tablet of 600 mg of Alisma .
- Crush with pestle and mortar till a fine powder.
- Add powder with 100 mL of distilled water.
Creating Sugar Water Solution:
- Measure 180.2 g of glucose in a graduated cylinder
- Add water until the total volume reaches 1 liter
- Dilute this solution to needed concentrations
Injection:
- Fill syringe with .5 cc of Alisma solution.
- Inject crayfish in soft tissue between arm and Thorax with syringe and inject the solution.
- Wait 1 week.
- Repeat with 9 crayfish per group.
- For control inject with 1 cc of distilled water.
Ammonia Levels:
- Place 100 mL of freshwater in a bucket.
- Place crayfish in bucket and let sit for 5 minutes.
- Run ammonia test strip through water for 10 seconds.
- Compare test strip color to container for a reading of ideal, stress, and harmful.
Oxygen:
- Drill a hole through a size 6 rubber stopper.
- Cover the hole with soap and push pipette through.
- Put wet paper towels in a 250 mL Eryrlmeyer flask.
- Place a test tube through the hole in the top filled with 20 potassium hydroxide pellets.
- Insert crayfish and place rubber stopper in flask.
- Insert green food coloring into the end of pipette.
- Slightly elevate pipette and let sit for 1 hour.
- Take reading from end of the green dye.
*Take crayfish out of the flask if given a reading of 1.64. This is a 20% drop of oxygen in the air and stresses the organism.
Blood Sugar Levels:
1. Place each crayfish in a culture dish filled half way with fresh water.
2. Draw blood with syringe.
3. Mix 3 drops of blood with 1 drop of sugar water solution.
4. Test with blood meter.
Cleaning up:
1. After each day of experimentation, clean the lab counter with 10% bleach.
2.
3. Clean with 10% bleach all glassware and related items. Auto clave new equipment as needed.
4. 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.
5. All related glassware and related equipment would be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.
Disposal/ Blood Samples/ Equipment:
1. Items of crayfish blood must be disposed of after autoclaving.
2. Put on protective equipment (goggles, gloves, aprons).
3. Gather biological items to be autoclaved prior to disposal.
4. Put items into autoclavable bag. Put bag in autoclave.
5. Autoclave for thirty minutes at 20psi, 121 degrees C.
6. When able, remove from autoclave and transport to dumpster.
Glucose Disposal:
- Dilute glucose by mixing with 100ml of water.
- Pour solution down drain with running water.
Analysis:
- Comparison of food vs. Glucose Level of Crayfish
- Comparison of food vs. Ammonia Levels
- Comparison of food vs. Dissolved Oxygen Level
Apparatus used to measure the intake of oxygen by crayfish.
These are the strips used to measure how stressed the crayfish got with the injection of the solution.
This is the injection of the crayfish. Injected with .2 CC of Alisma solution
