Date/Time

What we plan

See “Spring Lab Procedures” file for detailed information

Summary of what we did

See “Lab Report” file for more detailed information

Week 1: 4/24-26/2017

Monday 4/24/2017

4-6pm

• Inoculate E.coli

• Gathered items needed to be autoclaved

Tuesday 4/25/2017

12:30-1:00

3:00-5:30

• Prepare LB broth
• Autoclaved LB broth, pipet tips, and microtubes.
• E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking.
• Spectrophotometer testing with dead E.coli  

• Prepared 400mL LB broth
• Autoclaved LB broth, pipet tips, and microtubes.
• E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking.
• Spectrophotometer testing with dead E.coli to try to find a correlation between cell concentration and OD600 value (could not find a correlation)

Wednesday 4/26/2017

10am-5pm

• Prepare different concentration samples of methanol/ethanol. 10% intervals starting from 10% to 60%
• Place E.coli in different samples and perform Spectrophotometer testing to detect the initial and final concentration of E.coli every half an hour

• Prepared 800mL LB broth
• Prepared LB agar plates and LB/ampicillin agar plates
• Inoculate E.coli
• 20% ethanol survival test for 2 hours (15 min intervals for the first hour and 30 min intervals for the second hour)
• Correlation test for Bacterial Concentration and OD600- used alive E.coli (found no correlation again)

Week 2: 5/31/2017-6/2/2017

Wednesday

5/31/2017

2:00-4:00pm

Plan:

• Inoculate E.coli
• Spread glycerol stock E.coli on agar plates

Equipment: Sterile hood, spreader, shaker

Reagents: LB broth, agar plates

• Inoculated 6 tubes of E.coli. Started shaking incubator at 3:30pm.
• Revived glycerol stock by plating it onto 4 agar plates. Incubate overnight at 37C.

Thursday

6/1/2017

1:00-5:00pm

Plan:

• Spread inoculated E.coli on agar plates.
• If there is enough E.coli, conduct survival test in ethanol concentration at 10%, 20%, 30%, and 40%.

Equipment: Sterile Hood, spreader, spectrophotometer

Reagents: Ethanol, agar plates, LB broth

• Turn off shaking incubator and placed the tubes in 4C at 10:30am.
• 5 E.coli survival trials at 30% and 40% ethanol concentration (15 min intervals for the first hour and 30 min interval for the remaining two hours).
• Inoculated 4 tubes of E.coli. Start shaking incubator at 5pm

Friday

6/2/2017

9am-1pm

Plan:

• E.coli Survival test at 10% and 20% ethanol concentration.
• Try to find the maximum time at the optimal concentration.  

Equipment: Sterile Hood, spreader, spectrophotometer

Reagents: Ethanol, LB broth, agar plates

• (2) 5 E.coli survival trials at 10% ethanol concentration.
• First method: using ethanol as blank, get rid of LB broth
• Second method: made alcohol solution using cell culture LB broth rather than water
• Tested for 2 hours. 15 min intervals for the first hour and 30 min intervals for the second hour.
• Learned the procedures for trypan blue staining
• Competent DH5a E.coli cells arrived today

Week 5: 6/5-9/2017

Tuesday

6/6/2017

1:00-5:00pm

Plan:

• Inoculate E.coli
• Dead E.coli trypan blue staining under microscope

Equipment: Sterile Hood, spreader, microscope, counting grid

Reagents: LB broth, agar plates, trypan blue dye

• Begin development of ethanol/methanol resistant bacteria: Inoculate E. coli in 1% ethanol or methanol  

Wednesday

6/7/17

9am-5pm

Plan:

• E.coli Survival test at 10% and 5% ethanol concentration.
• Inoculate 4 tubes of E.coli

Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid

Reagents: Ethanol, agar plates, LB broth, trypan blue dye

• Trypan blue staining

• Could not distinguish E.coli cells from contaminants on the grid

• Ethanol and methanol resistant bacteria:

• Spread plates for 1% resistance; inoculate 2% resistance from the previous 1% inoculation
• Spread plates for 2% resistance; inoculate 3% resistance from the previous 2%

• Ordered fluorescence dye

Thursday

6/8/17

9am-5pm

Plan:

• Inoculate 1 colony each from the 1% ethanol/methanol plates
• Spread plates for 3% resistance; inoculate 4% resistance from the previous 3%
• If dye arrives, practice fluorescence microscope

Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid

Reagents: Ethanol, agar plates, LB broth, trypan blue dye

• Tried trypan blue staining using new microscope from genetics lab. Was not able to see the E.coli or the counting grids. Abandon using this technique.
• Inoculated E.coli in 4% methanol/ethanol solution for 6 hours. Spread onto agar plates to incubate.
• Spread 3% E.coli on agar plates  

Week 6: 6/19-23/2017

Monday

6/19/17

4-5pm

Plan:

• Inoculate E.coli

• Inoculated 2 tubes of regular LB broth 

Tuesday

6/20/17

9-5pm

Amy and Agnes

Plan:

• Prepare E.coli solution
• Prepare working solution for the dyes
• Learn to use the fluorescent microscope from Professor Wenshu. Perform initial testing together.
• Survival test 5% and 10% using the fluorescent microscope
• Inoculate E.coli

• Test different concentration of E.coli and dye under fluorescent microscope using the hoechst and propidium iodide dyes.
• Gene arrived. Stored in -20
• Inoculated 3 tubes of E.coli

Wednesday

6/21/17

9-5pm

Amy, Agnes, Julie, Simba

Plan:

• Survival test 5% and 10% using the fluorescent microscope
• Inoculate E.coli

Gene arrives:

• Prepare plasmid
• Transformation
• Grow overnight on agar plates

• Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution
• Cancelled plans for Gas Chromatography, initiated further research for methanol concentration testing

Thursday

6/22/17

9-5pm

• Survival test 5% and 10% using the fluorescent microscope
• Calibrating the OD600 measurement for DH5a.

• Inoculate one colony of transformed E.coli

• Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution
• Postponed OD600 calibration, continued Methanol Concentration testing research

Week 7: 6/26-30/2017

Monday

6/26/17

11am-5pm

Agnes and Eric

Plan:

• Autoclave and pour agar plates
• Inoculate E.coli

Materials and Instruments:

Sterile hood, autoclave

• Poured 40 10 mm agar plates and inoculated 3 tubes of E. coli

Tuesday

6/27/17

9-5pm

Agnes, Amy, Eric

• Fluorescence microscopy: E. coli ethanol survival testing

Materials and Instruments:

Sterile hood, fluorescence microscope

• Final Day of E.coli viability testing, increased concentration of dyes and found improved results
• Inoculated 2 tubes of E. coli

Wednesday

6/28/17

9-5pm

Agnes, Amy

• Fluorescence microscopy: E. coli ethanol survival testing

Materials and Instruments:

Sterile hood, fluorescence microscope

• Methanol survival testing: at 20 minutes of varying levels of methanol concentration (10%, 20%, 30%, 40%), we looked at the effect on fluorescence in our E. coli samples
• Began methanol clonogenic assay experiment, plated E. coli exposed to varying levels of methanol for 20 minutes (0%, 5%, 10%, 15%, 20%), at 10x dilution

Thursday

6/29/17

9-5pm

• Fluorescence microscopy: E. coli ethanol survival testing
• Methanol clonogenic assay

Materials and Instruments:

Sterile hood, fluorescence microscope

• Began OD600 Calibration, plated dilutions of 200x, 400x, 500x, 800x, and 1000x
• Methanol survival testing continued, varied concentration of dye and exposure to 20% methanol compared to the control

Friday

6/30/17

9-5pm

• Methanol clonogenic assay
• Fluorescence microscopy: E. coli ethanol survival testing

Materials and Instruments:

fluorescence microscope

• Visualized the results of previous day’s plating and updated lab reports

Week 8: 7/3-7/2017

Monday

7/3/2017

4-5pm

Agnes and Eric

Plan:

• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Inoculated  3 tubes

Tuesday

7/4/17

10-5pm

Julie, Agnes

Plan:

• Calibrating OD 600 value
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Calibration: Plated x200
• Inoculate 2 tubes

Wednesday

7/5/17

10-5pm

Julie, Simba, Agnes

Plan:

• Fluorescence Microscope testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room
• Fluorescence Microscope

• Calibration: Plated x200 and x500
• Inoculate 2 tubes
• Fluorescence microscope: varied time, switched water for PBS

Thursday

7/6/17

10-5pm

Julie, Agnes

Plan:

• Methanol clonogenic assay
• Inoculate E.coli

Materials and Instruments:

• Sterile room
• Fluorescence Microscope

• Calibration: Plated x2000

Friday

7/7/17

10-11am

Plan:

• Check on agar plates

• Checked on agar plates

Week 9: 7/10-14/2017

Note: Methanol concentration testing will proceed if all chemicals arrive by Tuesday 7/11;

Update: Chemicals have not arrived by Tuesday 7/11

Monday

7/10/2017

(10)4-5pm

Agnes, Eric

Plan:

• Inoculate E.coli
• (Prepare Magenta Sulfite Colorimetric solutions)

Materials and Instruments:

• Sterile room

• Inoculate 2 tubes E. coli

Tuesday

7/11/17

10-5pm

Plan:

• Fluorescence testing
• OD600 Calibration
• Inoculate E.coli

Materials and Instruments:

• Sterile room
• Fluorescence Microscope

• Fluorescence: Varied exposure time (1 min, 5 min, 10 min) to methanol at a concentration of 15% methanol; controls: no exposure, 50% methanol at 10 minutes
• OD600 Calibration

Wednesday

7/12/17

10-5pm

Plan:

• Pour ampicillin plates
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Methanol Clonogenic assay using 5% and 15% methanol at different incubation times (5, 10, 15, 20)

Thursday

7/13/17

10-5pm

Plan:

• Methanol clonogenic assay
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Methanol clonogenic assay with 15% and 10% methanol at different time intervals (5, 10, 15, 20)

Friday

7/14/17

10-5pm

Plan:

• Check on agar plates

• Checked on agar plates and took picture

Week 10: 7/17-21/2017

Note: Magenta Sulfite Colorimetric testing will proceed if all chemicals arrive by Tue 7/18

Monday

7/17/2017

3-5pm

Agnes, Eric

Plan:

• Inoculate E.coli
• Spread new E.coli on LB and LB/amp

• 50+500, 10/25/50/75/100

Materials and Instruments:

• Sterile room

• Inoculated E.coli
• Plated competent E.coli on agar plates to find suitable plating amount.
• Plated normal E.coli on LB/amp plates to check if the antibiotic works

Tuesday

7/18/17

10-5pm

Plan:

• Transformation
• Methanol clonogenic assay
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Methanol clonogenic assay with different concentrations of methanol 20%-50%

Wednesday

7/19/17

10-5pm

Plan:

• Prepare Magenta Sulfite Colorimetric solutions (must be autoclaved)
• Methanol clonogenic assay
• Inoculate E.coli

Materials and Instruments:

• Sterile room, Autoclave

• Clonogenic assay with different concentrations of ethanol. 10%-40%
• Filming

Thursday

7/20/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Filming
• Fluorescent Microscopy with 30% ethanol and 50% methanol

Friday

7/21/17

10-115pm

Note: Due to the fact that the chemicals for the Magenta Sulfite Colorimetric testing will be arriving today, we will only go to lab in the morning

• Took the plates out of the incubator. Took pictures
• Filmed scenes for video
• Chemicals arrived!

Week 11: 7/24-28/2017

Monday

7/24/2017

1:30-5pm

Agnes, Eric

Plan:

• Inoculate E.coli
• Transformation

Materials and Instruments:

• Sterile room

• Inoculated 5 tubes of transformed E.coli
• Prepared chemical solutions

Tuesday

7/25/17

1-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room, Microplate reader

• Prepared chemical solutions
• Inoculated 2 tubes of transformed E.coli
• Reviewed Interlab protocols. Just found out that our school’s plate reader cannot measure fluorescence  

Wednesday

7/26/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Methanol clonogenic assay
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Magenta Sulfite Colorimetric

Thursday

7/27/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room

• Magenta Sulfite Colorimetric

Week 11: 7/31-8/4/2017

We will NOT be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week.

Week 11: 8/7-8/11/2017

We will not be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week.

Week 11: 8/14-18/2017

Monday

8/14/2017

1:30-5pm

Plan:

• Inoculate E.coli
• Transformation

Materials and Instruments:

• Sterile room

Tuesday

8/15/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room

Same as planed

Wednesday

8/16/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing
• Inoculate E.coli

Materials and Instruments:

• Sterile room

Same as planned

Thursday

8/17/17

10-5pm

Plan:

• Magenta Sulfite Colorimetric testing

Materials and Instruments:

Same as planned