Procedure

Procedure


Safety Procedures for Sea Salt:1. The use of sea salt will used in accordance with the MSDS2. Using sea salt 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 MSDS excess salt will rinsed down the sink with 10 fold excess water.5. 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 MSDS2.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 a tank that can hold at least 800 gallons.2. Fill the 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. Check the pre-filter and clean it once a week.6. Change and remove the carbon filter every month.7. Every month change 10% of the water with clean fresh DO water.8. 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 testingAdjusting 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.



Making Agar:1. Agar is to be prepared by following the proportions of dehydrated media to distilled water and then autoclaved.2. Store sterile agar in refrigerator.3. Excess sterile agar will be wrapped in news paper and disposed of by placing in a landfill.



Experimentation:

1. Take shark out of tank with a medium sized net.

2. Put beaker underneath the shark’s snout.

3. Press down on the top of the shark’s snout with your thumbs. Make sure you wear gloves.

4. Wait for gel to gather in the beaker below.

5. When gel has been collected put the shark back in the tank.

6. Dilute the gel with water for testing, 25% shark gel and 75% water.

7. Soak 1 cm. disks of paper in the diluted shark gel.

8. Place 4 disks on each petri dish.

9. Check each petri dish every 2 days and measure the zone of inhibition.

10. Run experiment one more time.

Culturing:1. On the underside of each petri plate label with each plate with a wax pencil.2. Heat the nutrient agar in a microwave or water bath to 50º C.3. Pour a thin layer of agar on the bottom of each plate and cover immediately.4. Let agar plate solidify. Liquid agar will solidify at about 42 º C.5. After the medium agar has solidified, streak the plate. 6. Once the plates have been streaked, invert and close the petri dish, tape the edges if you wish and incubate them at 25º C for 24-48 hours unless other wise noted.



Streaking Plates:1. Sterilize work area and don safety equipment2. Take out plates from refrigerator and allow to warm up to room temperature.3. Take out bacterial culture to be streaked onto plates.4. Take inoculating loop and scrape off growth from bacterial culture.5. Place inoculating loop into sterile broth of distilled water and agitate.6. Dip sterile swab into wash.7. Lift up lid of plate and streak wet swab onto plate being sure to cover the entire area. Plate can be streaked multiple times in a sweeping motion to ensure plate overage.8. When done streaking plates, put into incubator upside down. Depending upon the bacteria, growth can appear as soon as 24 hours later.9. Dispose of materials.

Animal Safety:

1. Feed shark everyday, once per day.

2. Use cinder blocks to separate shark from protein skimmer.

3. Cover tank to keep shark from jumping out of the tank.

4. Temperature should be at 72-78oF.

5. pH should be at 8.1-8.4.

6. Salinity should be at 1.020-1.025.

7. Regularly check salinity, pH, water temperature, and water level.

8. Clean out the collection dish in the protein skimmer every 1-2 days.

9. Coat the bottom of the tank with smooth gravel or sand.

Cleaning up:1. After each day of experimentation, clean the lab counter with 10% bleach.2. Wash hands with 10% bleach.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.



Aseptic Techniques:1. Upon entering the lab, wash hands and arms up to the elbow with antibacterial soap.2. Before and during experimentation wear rubber gloves, apron, and goggles at all times.3. All glassware and material should be autoclaved for 30 minutes at 20 psi, 120 degrees Celsius.4. Any open flask, container, or beaker should be covered with aluminum foil when placed in the autoclave. 5. Use 10 % bleach solution and wipe down the lab area and tabletop.6. Transfer of any culture will be done with a mechanical pipette.7. Place a biohazard sign in plain view for all to see.8. Loops and Needles used to transfer the culture will be sterilized by flame heating until bright red prior to use. When not in use needles, will always be capped or covered. When not in use, place loops face up in a beaker. Never place loops on the counter.9. The top of the culture-tube will be flame heated before inserting a needle or loop for culture transfer.



Electrical Power Safety:1. Limit the use of high power devices 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



Descriptive statistics:1. Data will first be organized in a data table based on groups or variables.2. The mean or average for each group or variable tested will be calculated.3. Using an excel spread sheet the standard deviation for each group of variable will be calculated. Correlations:A relationship between 2 variables identified.1. The independent variable will be identified and be known as ‘x2. The dependent variable will be identified and know as ‘y3. x2 will be calculated for each test.4. y2 will be calculated for each test.5. xy will also be calculated and used to find the correlation coefficient (r)6. Using the formula for the correlation coefficient, (r) is calculated7. If the r value is close to zero, there is no correlation.8. If the r value is close to positive 1, there is a positive correlation. As one goes up the other goes up.9. If the r value close to negative 1, there is a negative correlation. As one goes up the other goes down.