IGEM:IMPERIAL/2009/M3/Assays/Membrane staining

Membrane Staining Assay

Cell Membrane Staining Assay: This assay uses a kit with certain stains to determine whether cells are alive or dead. The assay stains those cells with intact cell membranes green (the 'alive' cells), and those with damaged cell membranes red (the 'dead' cells). This is a quantitative measure of cell death. However, as we are using restriction enzymes, we will not directly affect the cell membrane, so this assay may not work. Natural degredation of the membrane without maintenance from the cell may occur, in which case this would be a useful guide. In either case, it would be useful as an indicator of the viability of the cells with disrupted genetic material. Another issue is the fact that our cells are to be encapsulated, so whether this staining technique can work through the colanic acid capsule is unclear.

Viability/Cytotoxicity Assay Kit for Bacteria Live & Dead Cell Staining Kit (30027) provides two-color fluorescence staining on both live (green) and dead (red) bacteria using two probes, DMAO and EtD-III.

• DMAO is a green-fluorescent nucleic acid dye that stains both live and dead bacteria with intact and damaged cell membranes.
• EtD-III is a red-fluorescent nucleic acid dye that stains only dead bacteria with damaged cell membranes.
• With an appropriate mixture of DMAO and EtD-III, bacteria with intact cell membranes is stained fluorescent green, whereas bacteria with damaged cell membranes is stained fluorescent red.
• The kit is suitable for use with fluorescence microscopes and flow cytometers. The assay principles are general and applicable to most bacteria types.

Membrane Staining Protocol

Things we need:

• 0.85% NaCl solution
• Slides and 18 mm square coverslips

Preparation of Live and Dead Bacterial Suspensions as Controls

• 1.1 Grow 4 mL cultures of your bacteria to late log phase in nutrient broth.

• 1.2 Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes.

• 1.3 Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl.

• 1.4 Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria.

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

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

• 1.7 Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6.

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

• 1.9 Use live and dead bacteria at your desired concentration for staining experiments shown below.

Fluorescence Microscopy Protocol

NB: Care must be taken to remove traces of growth medium before staining bacteria with these kit reagents. A single wash step is usually sufficient to remove significant traces of interfering media components from the bacterial suspension.

Staining Bacteria in Suspension

• 2.1 Combine one volume of Component A and two volumes of Component B in a microcentrifuge tube, mix thoroughly and add 8 volumes of 0.85% NaCl solution to derive 100X dye solution.

• 2.2 For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture.

• 2.3 Mix thoroughly and incubate at room temperature in the dark for 15 minutes.

• 2.4 Trap 5 μL of the stained bacterial suspension between a slide and an 18 mm square coverslip.

• 2.5 Observe under a fluorescence microscope equipped with any of the filter sets as below.

Filter We Will Use

• Longpass and dual emission filters useful for simultaneous viewing of DMAO and EthD-III stains.

Omega Filters: XF25, XF26, XF115 Chroma Filters: 11001, 41012, 71010

Flow Cytometry

• 3.1 Adjust the E. coli suspensions (live and killed) to 1 × 108 bacteria/mL (~0.03 OD670), then dilute them 1:100 in filter-sterilized dH2O to reach a final density of 1 × 106 bacteria/mL if needed.

• 3.2 Mix 11 different proportions of E. coli in 16 × 125 mm borosilicate glass tubes according to Table 1. The volume of each of the 11 samples will be 1 mL.

• 3.3 Mix 12 μL of Component A with 24 μL of Component B in a microcentrifuge tube. Add 3 μL of the combined reagent mixture to each of the 11 samples and mix thoroughly by pipetting up and down several times.

• 3.4 Incubate at room temperature in the dark for 15 minutes.

• 3.5 Analyze each bacterial sample by flow cytometry using the setting for fluorescein for DMAO positive cells and propidium iodide for EtD-III positive cells.