Shakson Isaac Research

Procedures/Methods

 

Experimental Setup: Design of a MFC (Testing if metabolic activity combined with substrates influence electrical output)

 

Procedure:

Pre-setup: Made empty MFC jars containing electrodes and wires

1. Add 100g of soil to 3 jars.

2. Seal the jars and add silicone to seal the hole.

3. Put the MFCs to autoclave at 121 degrees Celsius at 15 min at 15 psi to eliminate 99.9% of the bacteria present

4. Take out the MFCs from the autoclave (label Autoclaved Dirt 1 through 3)

5. Add 100g of soil to 9 other jars

6. 3 of the jars are sealed and labeled (Soil 1 through 3)

7. Add 2% of glucose (2 mL of 20% aqueous solution of glucose/ 100 g of soil) to 3 jars

8. Seal the 3 jars and label them (soil w/ glucose 1 through 3)

9. Add 2% of peptone (2 mL of 20% aqueous solution of peptone/ 100 g of soil) to 3 jars

10. Seal the 3 jars and label them (soil w/ peptone 1 through 3)

11. Check time zero measurement of the MFC electrical output

12. Check the electrical output for 40 days and come back to the lab to add substrates again (steps 7-10) and add distilled water the jar labeled soil 1-3

13. End the measurement of electrical output on day 65 and provide standard deviation for each average of 3 jars.

14. Measure how many MFC’s it takes to charge an LED for all 65 days

Note: each experimental variable jars and the control were done in triplicates

 

 

Culturing and isolating bacteria

Procedure:  

1. Get a clean, sterile cotton swab and dip it into a test tube of sterile water.

2. Swab the anode and cathode separately and apply directly to the agar for the bacteria to grow.

3. Put the bacteria in room temperature since the MFC’s where in room temperature and higher temperatures might kill the bacteria.

4. On the LB agar plate (bacteria from the electrodes) select three or more different colonies of bacteria to isolate.

5. Use an inoculating loop and put under a flame from a Bunsen burner to sterilize

6. Obtain the bacteria selected and streak across one quadrant of the agar plate

7. Sterilize the loop ended inoculating wire

8. Swipe the inoculating loop across quadrant one to quadrant two.

9. Repeat steps 3-5 for quadrant 3 and 4

10. Repeat steps 1-6 for the next bacterial colony selected on the agar plate.

11. Put all the petri dishes into an incubator but set at room temperature since working with environmental bacteria.

12. Pick 3-4 bacteria to freeze to use for later

 

Creating MFCs with isolated bacteria

Procedure:

Pre-setup: Made empty MFC jars containing electrodes and wires

1. Add 100g of soil to 15 jars

2. Autoclave the jars 121 degrees Celsius at 15 min at 15 psi

3. Take out the jars a label the jars accordingly (control (that had broth), sga21, sgc11, spa11, and spc1F).

4. Add the broth with no bacteria that was used to cultivate the other bacteria in the control jar

5. Add 5 mL of labelled isolated bacteria contain the same broth as the control to the appropriate jar (ex. 5 mL of sga21 isolated bacteria added to sga21 jar).

6. Check time zero measurement of the MFC electrical output

7. Check the electrical output for 40 days

8. End the measurement of electrical output on day 40 and provide standard deviation for each average of 3 jars.

9. Measure how many MFC’s it takes to charge an LED for all 40 days

Note: each experimental variable jars and the control were done in triplicates

Second Note: same procedure for the addition of bacteria with known metabolism (Escherichia coli and Staphylococcus aureus) same amount of bacteria just different labels and also done in triplicates

 

Sequencing of Bacteria Isolates

Procedure:

1. DNA Extraction Procedure

• Pick colony using toothpick

• Swirl into 500 µl water

• Boil for 5 minutes

2. PCR Reaction mix

• 2X Short mix (Bioline)

• Forward Primer (F-341 U1)

• Reverse Primer (R1346)

PCR Cycle conditions

• 5:00 at 95°C

• 34 cycles of:

• 00:30 at 95°C

• 00:30 at 55°C

• 1:00 at 72°C

• 10:00 at 72°C

3. Gel electrophoresis conditions

• 0.7% agarose gel

• 45 minutes at 100V

4. PCR purification

• QIAquick Spin Kit (Qiagen)

5. DNA quantified using Nanovue spectrophotometer

6. Sequencing (sanger sequencing)

• 200 ng of DNA amplified by PCR

• 3.2 p moles forward primer or reverse primer (as used in PCR)

• Sequencing reaction performed by ASGPB at UH-Manoa