LIVE/DEAD® BacLight - Bacterial Viability Kit
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Kit Components
- Component A, DMAO: 2x100 µL, 5 mM in DMSO
- Component B, EthD-III: 2x150 µL, 2 mM in DMSO
Preparation of Live and Dead Bacterial Suspensions as Controls
- Grow 4 mL cultures of your bacteria to late log phase in nutrient broth.
- Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes.
- 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.
- 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.
- Incubate both samples at room temperature for 1 hour, mixing every 15 minutes.
- Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes.
- Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6.
- 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).
- Use live and dead bacteria at your desired concentration for staining experiments shown below.
Staining Bacteria in Suspension
- 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.
- For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture.
- Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
- Trap 5 µL of the stained bacterial suspension between a slide and an 18 mm square coverslip.
- Observe under a fluorescence microscope equipped with any of the filter sets as below.
Selection of Optical Filters
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
Bandpass filters for viewing DMAO alone
Omega Filters: XF22, XF23
Chroma Filters: 31001, 41001
Bandpass filters for viewing EthD-III alone
Omega Filters: XF32, XF43, XF102, XF108
Chroma Filters: 31002, 31004, 41002, 41004
Flow Cytometry
- 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.
- 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.
- 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.
Note: It may be desirable to prepare additional bacterial samples for staining with component A alone (stain both live and dead bacteria ) and with Component B alone (stain dead bacteria only). - Incubate at room temperature in the dark for 15 minutes.
- Analyze each bacterial sample by flow cytometry using the setting for fluorescein for DMAO positive cells and propidium iodide for EtD-III positive cells.
Table 1: Volume of Live- and dead-cell suspension to mix to achieve desired ratio of live:dead cell population
Ratio of Live:Dead Cells mL Live-Cell | Suspension mL Dead-Cell | Suspension |
0:100 | 0 | 1.0 |
10:90 | 0.1 | 0.9 |
20:80 | 0.2 | 0.8 |
30:70 | 0.3 | 0.7 |
40:60 | 0.4 | 0.6 |
50:50 | 0.5 | 0.5 |
60:40 | 0.6 | 0.4 |
70:30 | 0.7 | 0.3 |
80:20 | 0.8 | 0.2 |
90:10 | 0.9 | 0.1 |
100:0 | 1.0 | 0 |
Adapted from: http://www.openwetware.org/images/b/b9/Cell_Death_Assay.pdf