Team:NYU Shanghai/Experiments

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Protocols

Double Staining Fluorescence Microscopy:

Procedure:
Remove 1mL of overnight bacterial suspension into a microcentrifuge tube
Centrifuge at 12,000rpm for 3 minutes
Remove supernatant
Ethanol/Methanol treatment for 30 mins
Centrifuge at 12,000rpm for 3 minutes
Remove ethanol/methanol solution
Resuspense E. coli with PBS (Dilute as much as necessary)
Use 200uL of the diluted E. coli suspension
Add 100uL of Hoechst dye (working concentration: 5ug/mL)
Add 100uL of PI dye (working concentration: 5ug/mL)
Incubate at room temperature under dark conditions for 15-20 minutes.
Clean microscope slides and coverslip and label
Pipett 10uL of bacterial/staining dye solution onto a microscope slide and cover
Observe under brightfield, DAPI, and Texas Red channel

Clonogenic Assay:

Procedure:
Take the OD600 value of the overnight culture
Take 1mL of the overnight culture and place it into a microcentrifuge tube
Centrifuge at 12,000rpm for 5 mins
Remove the supernatant
Add different concentrations of methanol/ethanol for a certain period of time
Centrifuge at 12,000rpm for 3 mins
Remove the methanol/ethanol supernatant
Dilute 5000x -10000x with LB
Plate the diluted, treated sample onto agar plates
Incubate the plates at 37°C at 220 rpm for 16-18 hours

Magenta Sulfite Colorimetric:

Procedure:

Preparation of solutions
1. potassium permanganate-phosphoric acid solution
Weigh 15g of potassium permanganate and add into 75 ml 85% phosphoric acid and 350ml of water. After the potassium permanganate has dissolved, control the volume to 500ml. Store the solution in brown bottle.
2.oxalic acid-sulfuric acid  
weigh 5g of oxalic acid and dissolve in 1:1 cold sulfuric acid and add water until the 100 ml mark, mix well and store in brown bottle.
3. aniline red- sulfinic acid
weigh 0.1g of aniline red and dissolve with 60ml 80 degree C water. After cooling add 1g sodium sulfite, 1ml of concentrated hydrochloric acid and add water until the 100 ml mark, mix well, filter and store in brown bottle. Let solution stand overnight. if the solution has color, use small amounts of activated charcoal to filter. if the solution turns red it should be discarded.
4. methanol standard solution 1(10mg/ml)
Measure 1.27ml of methanol and place in a 100ml volume controller with a small amount of distilled water, add water up to then 100ml mark, mix well. Store in low temperature
5.methanol standard solution 2 (1mg/ml)
Take 10 ml of the methanol standard solution1 and place in 100 ml volume controller and add water to the 100 ml mark, mix well.
6. 10% sodium sulfite solution

Experimental Procedure
drawing the standard curve (absorbance at 590nm vs. mass of methanol)
Take 0, 0.06, 0.12, 0.18,0.24 and 0.30 ml of methanol (containing 0, 0.06mg, 0.12mg, 0.18mg, 0.24mg and 0.30 mg of methanol) standard solution 2 and place in 2ml microcentrifuge tube. Add water to each microcentrifuge tube up to 0.5ml, and then 0.2 ml of potassium permanganate-phosphoric acid solution, mix well and let the solution sit for 10 min. Add 0.2ml of oxalic acid-sulfinic acid solution, mix well and let the solution sit until the color fades. Then add 0.5ml of aniline red-sulfinic acid solution, mix well and let the solution sit for 0.5 hours (at temperature over 20 degrees centigrade). Transfer 1ml of the solution in each microcentrifuge tube into a cuvette and test the absorbance at 590nm and plot the standard curve of absorbance at 590nm Vs. mass of methanol

Testing
a) methanol concentration 5g/100ml (needs to be diluted 100 times)
Take 0.631ml (0.5 g of methanol) place into test tube and add water up to 10ml. Add 9

transformed E. coli. After 15, 30, 45, and 60 minutes, take 1 ml of solution and centrifuge. Then take 0.005ml/ 0.0025ml of the upper clear solution place in another centrifuge tube, add water until 0.5 ml (diluted 100 times) and then 0.2 ml of potassium permanganate-phosphoric acid solution, mix well and let the solution sit for 10 min. Add 0.2ml of oxalic acid-sulfuric acid solution, mix well and let the solution sit until the color fades. Then add 0.5ml of aniline red-sulfinic acid solution, mix well and let the solution sit for 0.5 hours (at temperature over 20 degrees centigrade). Transfer 1ml of the solution in each microcentrifuge tube into a cuvette and test the absorbance at 590nm. Then plot the values onto the standard curve to find the concentration (needs to be times 200).