Safety Considerations:
Power Tools:
Wear goggles or safety glasses.
Do not work alone in the lab.
Dry your hands and clothing before working with electricity. Mop up all water spilled on the floor
Keep the lab clean.
Clamp down any objects that will be used.
Know of all switches and controls before using a power tool.
Keep hands, fingers, feet, and all objects not being used away from moving parts.
Use a vacuum system to keep debris away from cutting area.
UV-C light safety:
Limit UV exposure by placing the light source in a solid sealed case.
Wear UV protect goggles
Wear personal protection includes aprons and gloves
Never look directly into UV light source
When using UV-C, the light and reflection of light must be completely blocked before the unit is on.
Place a warning sign inn the lab area that a UV light source is being used.
Electrical Power Safety:
Limit the use of high power devices around water.
Never use frayed or cut wires when plugging in a device.
Never overload the outlet and use a surge protector.
All electrical devices should have an emergency cut off switch or ‘kill’switch’
Shut off and unplug all devices when not in use or when you leave the room unattended.
Chemical Safety:
During the part of the experiment that will be performed at the school, all chemicals will be used in accordance with the MSDS.
The use of all chemicals will be done wearing gloves, aprons, and goggles.
All chemicals that release fumes will be placed under the fume hood to prevent the release of fumes into the classroom.
A fire blanket, fume-hood, fire extinguisher, eyewash station, and shower are available for use
Upon leaving the lab, hands will be washed and all chemicals will be stored in a locked ventilated chemical storeroom.
Chemicals and Heat:
Goggles and aprons will be worn.
During heating, potholders (thermal gloves) will be used to handle hot surfaces and equipment coming off the hot plate.
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.
Robot Fabrication
Chassis:
1. Cut four pieces of aluminum with dimensions 5½ inches x 9 inches
2. Mark five drill holes ¼ inch from the 9 inch boarder on both sides of all pieces spacing each evenly along the length of the side
3. Clamp all four pieces together aligning the drill holes
4. Using a drill press, drill through all four pieces
5. Mark two lines parallel to the 9 inch length, the first 2½ inches from the 9 inch side, then the second mark 2½ inches from the previously marked line, creating a ½ inch gap between the second mark and the second side.
6. Repeat step five for all four pieces of aluminum
7. Once marked, bend each line exactly 135 degrees using a bending brake.
a.) Place the marked line horizontally along the bending line of the brake.
b.) Align the metal bar that runs across the length of the face with the line.
c.) Clamp the metal bar over the piece of aluminum onto the brake
d.) Pull up on the handles until they reach a pre-marked angle of 135 degrees.
e.) Remove the piece.
f.) Repeat steps a-e for the marked line on the piece of aluminum.
g.) Repeat process a-f for all four pieces of aluminum.
8. Align the five drill holes on each ½ inch side underneath the corresponding 5 drill holes on the 9 inch side of another piece.
9. Repeat until all holes are aligned and an octagonal prism is formed.
10. Once formed, both all pieces together through the drill holes.
11. Cut two more square pieces of aluminum 6 inches x 6 inches.
12. Mark the four corners of each piece to be bent along the inside walls of the octagonal prism.
13. Bend each marked corner 90 degrees
a.) Place the marked line horizontally along the bending line of the brake.
b.) Align the metal bar that runs across the length of the face with the line.
c.) Clamp the metal bar over the piece of aluminum onto the brake.
d.) Pull up on the handles until they reach a pre-marked angle of 90 degrees.
e.) Remove the piece.
f.) Repeat steps a-e for the marked line on the piece of aluminum.
g.) Repeat process a-f for both pieces of aluminum.
14. Place each face in the two open ends of the prism.
15. Drill through the bent lip of the each face and the corresponding side of the octagonal prism using a drill press.
16. Fasten each face to the prism using bolts.
17. Cut a hole 1 ¾ in the center of the first face using a hydraulic hole punch.
18. Using a die grinder, smooth the edges of the octagon.
19. Using a rotary rasp, remove the remaining aluminum necessary from the edge of the circle to house the fixture for the light.
20. Mark and cut seven ¼ inch holes for LED lights connected to the circuit boards to fit through on the left panel of the chassis.
21. Mark and cut a rectangle for the motor on the robotic arm and circle for the test tube on the right side of the chassis.
22. Mark and cut holes for the 3 switches to be mounted through on the top of the robot.
23. Mark and cut holes for the individual sensors to be mounted through on the side of the chassis.
24. Mark and drill holes for the eight legs to be bolted through on each diagonal panel of the chassis.
25. Disassemble an 18v drill and socket.
26. Cut a hole large enough for the neck of the socket to fit through on the back face of the chassis.
27. Screw the socket into place.
Light:
1.Wire the 18v battery to a 15v reducer..
2. Wire the 15v to the 120v inverter.
3. Wire the 120v inverter to power the ballast. .
4. Attach light to base and screw onto ballast connector.
Legs:
1. Cut eight 2 ½ inch long segments of a 1 inch diameter aluminum rod.
2. Cut eight 2 inch long segments of ½ inch diameter PVC.
3. Mark two lines down the length of each of the PVC segments 180 degrees apart and use a hack saw to cut grooves along the lines.
4. Cut a ½ inch wooden dowel into eight segments and a 1 inch wooden dowel into eight segments.
5. Drill holes through all dowels.
6. Attach one of each size dowel to each side of the eight compression springs.
7. Push the dowels into place in the aluminum and PVC segments.
8. Screw the spring through both dowels using a washer and sheet metal screws.
9. Align the grooves on the PVC with the grooves inside the aluminum rods.
10. Bolt the eight legs through the eight holes drilled in the chassis.
Circuitry:
1. Cut the socket of a drill or similar tool that runs using an 18 volt battery and wire directly into the socket.
2. Wire the 18v to a 15v and 5v reducer.
3. Wire the 5v to the logic board and 15v to the timing circuit board.
4. Using the wire wrap method, attach the various components of the circuit board in the correct sequence.
5. Solder.
6. Connect the 120v inverter to the 15v coming from the timer on the circuit board.
7. Connect the light ballast to the 120v inverter the power the UV-C.
Arm:
1. Cut two aluminum strips, ½ inch by 2 inches.
2. Mount the first servo motor flush with the side of the chassis by cutting a hole in the chassis and a hole for the test tube.
3. Connect the second and third servo motors with a piece of aluminum 5” x 2” with two rectangular holes to place the servos through and screw to the aluminum.
4. Attach the third servo motor to a test tube stopper using on of the two strips of aluminum.
5. Attach the second servo to the first using the second strip of aluminum.
6. Mount the first servo through the hole, and feed the test tube through the pre-cut hole along the chassis.
7. Feed the stopper into the test tube.
Microbiological Experimentation
BSL 1 Aseptic Techniques
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 bio hazard 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.
Autoclave:
1. Always wear goggles and aprons when using the autoclave.
2. The bottom of the autoclave should have about 2 inches of cleaned distilled water before operation.
3. Check the pressure release value prior to use (it should be clear)
4. Use potholders when opening or handling hot surfaces.
5. Opening the autoclave can be done only after the sterilizer after it has cooled (gauge should read zero.) and all the steam has been allowed to escape.
6. Place the control knob in the straight up position. This allows the unit to operate at 16-21 psi.
Preparation of Agar:
1. Measure out 39 grams of Potato Dextrose agar dehydrated media.
2. Measure out 1.0 L of distilled water in a graduated cylinder. Place in a 2 L Erlenmeyer flask.
3. Put flask with water onto hot plate and bring to a boil.
4. Once boiling, dissolve 39 grams of agar into the water. Boil for one minute to dissolve.
5. Pour liquid agar into reagent bottles.
6. Autoclave for 30 minutes at 20psi, 120 degrees C.
7. Store in refrigerator when cooled down after autoclaving.
Culturing:
1. On the underside of each petri plate label with each plate with a wax pencil.
2. Heat the potato dextrose agar in a microwave or water bath to 50 degrees 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 degrees C.
5. After the medium agar has solidified, streak the plate with a continuous streak.
6. Once the plates have been streaked, invert and close the petri dish, tape the edges if you wish and incubate them at 37 degrees C for 24-48 hours unless otherwise noted.
Streaking Plates:
1. Sterilize work area and safety equipment
2. Take out plates from refrigerator and allow to warm to room temperature.
3. Take out Aspergillus niger and Rhizopus stolonifer cultures 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. Streak 30 plates with Aspergillus niger and 30 plates with Rhizopus stolonifer.
9. When done streaking plates, and experimenting put into incubator upside down. Depending upon the microorganism, growth can appear as soon as 24 hours later.
10. Dispose of materials.
Cleaning up:
1. After each day of experimentation, clean the lab counter with isopropyl.
2. Wash hands with isopropyl.
3. Clean with isopropyl all glassware and related items. Autoclave new equipment as it becomes 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:
1. Items of biological hazard 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 autoclave bag. Put bag in autoclave.
5. Autoclave for thirty minutes at 20 psi, 120 degrees C.
6. When able, remove from autoclave and transport to dumpster.
Non-pulsating UV-C radiation:
1. Prepare 80 petri plates inoculated with Aspergillus niger
2. Place 20 plates on the left, bottom, right, and top wall respectively on the inside of the ductwork.
3. Run the robot inside the ductwork, irradiating the mold with non-pulsating UV-C radiation for 0 minutes.
4. Remove the plates and place them in an incubator for 24 hours.
5.) After the incubation period, take the plates out of the incubator and count the number of living spores on each plate.
6. Record the number of each plate.
7. Repeat steps 1-6 irradiating the mold for 4, 6, 8, and 10 minutes respectively.
8. Repeat the entire experiment two more times, resulting in 3 trials of 0, 4, 6, 8, and 10 minutes each.
9. Repeat steps 1-8 using Rhizopus stolonifer in place of Aspergillus niger.
Pulsating radiation:
1. Prepare 80 petri plates inoculated with Aspergillus niger.
2. Place 20 plates on the left, bottom, right, and top wall respectively on the inside of the ductwork.
3. Run the robot inside the ductwork, irradiating the mold with pulsating UV-C radiation for 0 minutes.
4. Remove the plates and place them in an incubator for 24 hours.
5. After the incubation period, take the plates out of the incubator and count the number of living spores on each plate.
6. Record the number of each plate.
7. Repeat steps 1-6 irradiating the mold for 4, 6, 8, and 10 minutes respectively.
8. Repeat the entire experiment two more times, resulting in 3 trials of 0, 4, 6, 8, and 10 minutes each.
9. Repeat steps 1-8 using Rhizopus stolonifer in place of Aspergillus niger.
Data Collection
Non-pulsating:
1. Group together plates of Aspergillus niger irradiated for 0 minutes using non-pulsating radiation (control)
2. Record the average number of living spores on all of the plates combined
3. Graph.
4. Separate the plates by the wall they were each irradiated on, resulting in 20 plates on the left, right, bottom, and top wall resulting in the total of 80 plates.
5. Record the average number of spores on the 20 plates for each wall.
6. Create a graph comparing the average number of spores on the plates irradiated on the different walls against each other.
7. Use the data to create and ANOVA or analysis of variance to determine whether the IV had a statistical effect.
8. Repeat steps 1-7 using the data collected from the experiments that irradiated Aspergillus niger with non-pulsating UV-C radiation for 4, 6, 8, and 10 minutes respectively.
9. Repeat steps 1-8 using the data collected from Rhizopus stolonifer in place of Aspergillus niger.
Pulsating:
1. Group together plates of Aspergillus niger irradiated for 0 minutes using pulsating radiation (control)
2. Record the average number of living spores on all of the plates combined
3. Graph.
4. Separate the plates by the wall they were each irradiated on, resulting in 20 plates on the left, right, bottom, and top wall resulting in the total of 80 plates.
5. Record the average number of spores on the 20 plates for each wall.
6. Create a graph comparing the average number of spores on the plates irradiated on the different walls against each other.
7. Use the data to create and ANOVA or analysis of variance to determine whether the IV had a statistical effect.
8. Repeat steps 1-7 using the data collected from the experiments that irradiated Aspergillus niger with pulsating UV-C radiation for 4, 6, 8, and 10 minutes respectively.
9. Repeat steps 1-8 using the data collected from Rhizopus stolonifer in place of Aspergillus niger.
