IGEM:IMPERIAL/2009/M3/Assays/Staining

=Staining Cells (determining rate of killing)=

Aims

 * We want to determine the rate of killing of the restriction enzymes after induction.


 * By measuring the fluorescent staining of live and dead cells, this gives a quantitative analysis of how number of cells killed varies with time.

Staining Assay
The two stains involved are: 1) green-fluorescent nucleic acid stain SYTO® 9

The SYTO 9 stain generally labels all bacteria in a population — those with intact membranes and those with damaged membranes.

2) red-fluorescent nucleic acid stain propidium iodide.

Propidium iodide penetrates only bacteria with damaged membranes.

Overall, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red.

The excitation/emission maxima for these dyes are about 480/500nm for SYTO 9 stain and 490/635nm for propidium iodide.

2) Fluorescence Spectroscopy

 * Fluorescence spectrophotometer

Kit Contents for Viability Kit, L7012
DMSO
 * SYTO 9 dye, 3.34 mM (Component A), 300 μL solution in

in DMSO
 * Propidium iodide, 20 mM (Component B), 300 μL solution

1) Calibrating standard curve of percentage live/dead
1. Grow 30 mL cultures of Escherichia coli to late log phase in nutrient broth.

2. Concentrate 25 mL of the bacterial culture by centrifugation at 10,000 × g for 10–15 minutes.

3. Remove the supernatant and resuspend the pellet in 2 mL of 0.85% NaCl or appropriate buffer.

4. Add 1 mL of this suspension to each of two 30–40 mL centrifuge tubes containing either 20 mL of 0.85% NaCl or appropriate buffer (for live bacteria) or 20 mL of 70% isopropyl alcohol (for killed bacteria).

5. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes.

6. Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes.

7. Resuspend the pellets in 20 mL of 0.85% NaCl or appropriate buffer and centrifuge again as in step 6.

8. Resuspend both pellets in separate tubes with 10 mL of 0.85% NaCl or appropriate buffer each.

9. Determine the optical density at 670 nm (OD670) of a 3 mL aliquot of the bacterial suspensions in glass or acrylic absorption cuvettes (1 cm pathlength).

10. Adjust the E. coli suspensions (live and killed) to 1 × 108 bacteria/mL (~0.03 OD670). Mix 5 different proportions of the bacterial suspensions to get a range of percent live bacteria.



11. Use the protocol for either the spectroscopy (protocol 2) or fluorescence microplate reader (protocol 3)

12. Plot the ratio of integrated green fluorescence to integrated red fluorescence (RG/R) versus percentage of live cells in the E. coli suspension  for either Protocol 2 (fluorescence spectroscopy) or Protocol 3 (fluorescence microplate reader)



2) Fluorescence spectroscopy
1. Grow 30 mL cultures of Escherichia coli  to late log phase in nutrient broth.

2. Measure the OD of the culture.

3. Adding 1mM Arabinose solution and mix well.

4. At time point 0 and 20 minute intervals thereafter, withdraw 9 μL of sample into 1 cm acrylic, glass or quartz fluorescence cuvettes. The total volume of each of the 5 samples will be 3 mL.

5. Prepare a combined reagent mixture in a microfuge tube by adding 30 μL of Component A to 30 μL of Component B.

6. Add 9 μL of the combined reagent mixture to each of the 5 samples (5 samples × 9 μL = 45 μL total) and mix thoroughly by pipetting up and down several times.

7. Incubate at room temperature in the dark for 15 minutes.

8. Measure the fluorescence emission spectrum (excitation 470 nm, emission 490–700 nm) of each cell suspension (Fcell) in a fluorescence spectrophotometer.



9. Calculate the ratio of the integrated intensity of the portion of each spectrum between 510–540 nm (em1; green) to that between 620–650 (em2; red) for each bacterial suspension.



10. Plot the RatioG/R versus time in the E. coli suspension.

3) Fluorescence Microplate Readers
1. Grow 30 mL cultures of Escherichia coli  to late log phase in nutrient broth.

2. Measure the OD of the culture.

3. Adding 1mM Arabinose solution and mix well.

4. At time point 0 and 20 minute intervals thereafter, pipet 100 μL of each of the bacterial cell suspension mixtures into separate wells of a 96-well flat-bottom microplate.  It is recommended that you prepare samples in triplicate. The outside wells (rows A and H and columns 1 and 12) are usually kept empty to avoid spurious readings.

5. Mix 6 μL of Component A with 6 μL of Component B in a microfuge tube.

6. Prepare a 2X stain solution by adding the entire 12 μL of the above mixture to 2.0 mL of filter-sterilized dH2O in a 16 × 125 mm borosilicate glass culture tube and mix well.

7. Using a new tip for each well, pipet 100 μL of the 2X staining solution (from step 4) to each well and mix thoroughly by pipetting up and down several times.

8. Incubate at room temperature in the dark for 15 minutes.

9. With the excitation wavelength centered at about 485 nm, measure the fluorescence intensity at a wavelength centered at about 530 nm (emission 1; green) for each well of the entire plate.

10. Analyse the data by dividing the fluorescence intensity of the stained bacterial suspensions (Fcell) at emission 1 by the fluorescence intensity at emission 2.



11. Plot the RatioG/R versus time in the E. coli suspension.