Protocol for LIVE/DEAD^® BacLight^TM Bacterial Viability Assay

1. Introduction

The Turner BioSystems TD-700 Laboratory Fluorometer in combination with Molecular Probes' LIVE/DEAD^® BacLight^TM Bacterial Viability Kit provides a novel two-color fluorescence assay of bacterial viability that allows researchers to quantitatively distinguish live and dead bacteria in minutes, even in a mixed population containing a range of bacterial types. Conventional direct-count assays of bacterial viability are based on metabolic characteristics or membrane integrity. However, methods relying on metabolic characteristics often only work for a limited subset of bacterial groups,(1) and methods for assessing bacterial membrane integrity commonly have high levels of background fluorescence.(2) Both types of determinations suffer from being very sensitive to growth and staining conditions.(3,4)

The LIVE/DEAD BacLight Bacterial Viability assay utilizes mixtures of SYTO^® 9 green fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain labels bacteria with both intact and damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, competing with the SYTO 9 stain for nucleic acid binding sites when both dyes are present. When mixed in recommended proportions, SYTO 9 stain and propidium iodide produce green fluorescent staining of bacteria with intact cell membranes and red fluorescent staining of bacteria with damaged membranes. The background remains virtually nonfluorescent. Consequently, the ratio of green to red fluorescence intensities provides a quantitative index of bacterial viability (Figure 1).

2. Materials Required

• Turner BioSystems TD-700 Laboratory Fluorometer with standard PMT (P/N 7000-009)
• Quartz-halogen lamp (P/N 7000-930)
• 13 mm round test tube adapter (P/N 7000-981)
• Two pairs of filters are required: First pair includes: EX 486 nm filter (P/N 034-0486), EM 520 nm filter (P/N 034-0520). Second pair includes: EX 520 nm filter (P/N 034-0520), EM 325-700 nm (P/N 10-058R) and greater than 610 nm filter (P/N 10-054R).
• Filter o-rings (P/N 7000-949)
• 13 mm x 100 mm borosilicate glass test tubes (P/N 10-031)
• LIVE/DEAD® BacLight(TM) Bacterial Viability Kit (catalog number L-7012), supplied by Molecular Probes, Inc., Eugene, OR. This kit consists of three components: Component A, 300 µL of 3.34 mM SYTO 9 in anhydrous DMSO, Component B, 300 µL of 20 mM propidium iodide in anhydrous DMSO, and Component C, 10 mL of BacLight mounting oil (this component is not used in the fluorometric assay described here). One kit is sufficient for analysis of about 65 samples using this protocol. Handling, storage and the use of the reagents should be performed in accordance with the product information sheet supplied by Molecular Probes.
• Staphylococcus aureus suspension containing approximately 1 × 10-7 bacteria/mL. To adjust the concentration of the bacterial suspension, take a 3 mL sample in a 10 mm × 10 mm cuvette and measure the optical density at 670 nm (OD670) using a spectrophotometer. 1 × 10-7 bacteria/mL corresponds to OD670 ~0.15. Microcentrifuge (SUREspin(TM) Model 7040, Helena Labs, Beaumont, TX).
• Isopropyl alcohol.

Table 1. Preparation of isopropyl alcohol/water mixtures

3. Experimental Protocol

3.1. Disinfectant Treatment of Staphylococcus aureus with Isopropyl Alcohol

3.1.1 In a set of 5 labeled tubes, prepare 0, 10, 30, 70 and 100% isopropyl alcohol/water mixtures as outlined in Table 1.

3.1.2 Label a set of 5 microfuge tubes with the designation 0, 10, 30, 70, or 100%.

3.1.3 Transfer 1 mL of Staphylococcus aureus suspension to each of the labeled microfuge tubes^[A].

3.1.4 Centrifuge the microfuge tubes for 3 minutes at 10,000 rpm.

3.1.5 Remove and discard the supernatant from each microfuge tube using a glass Pasteur pipet.

3.1.6 Break up the bacterial pellets and resuspend each in 1 mL of 0, 10, 30, 70 or 100% isopropyl alcohol/water (step 3.1.1) according to the designation on the tube. For example, resuspend the pellet in the microfuge tube marked 10% in 1 mL of 10% isopropyl alcohol.

3.1.7 Incubate the bacterial suspensions in alcohol/water mixtures for 1 hour at room temperature.

3.1.8 Centrifuge the bacterial suspensions for 3 minutes at 10,000 rpm.

3.1.9 Break up bacterial pellets and resuspend each in 1 mL of sterile filtered water.

3.1.10 Centrifuge bacterial suspensions for 3 minutes at 10,000 rpm.

3.1.11 Break up bacterial pellets and resuspend each in 1.5 mL of sterile filtered water.^[A]

^[A] Although the experiment described here is based on a comparison of samples containing equal numbers of bacteria, it is also possible to compare viability of bacterial samples with different densities because the ratio of green to red fluorescence is relatively independent of the number of bacteria present.

3.2 Bacterial Staining

3.2.1 Prepare a 2X BacLight staining solution from the kit reagents by mixing 3 uL of component A and 3 uL of component B per 1 mL of sterile water. To prepare sufficient staining solution for the five samples and one reagent blank used in the experiment described here, add 27 uL of BacLight component A and 27 uL of BacLight component B to 9 mL of sterile water.

3.2.2 Label six 13 mm glass test tubes with reagent blank, 0, 10, 30, 70, and 100%.

3.2.3 Transfer 1.5 mL of 2X BacLight (prepared in 3.2.1) to each labeled test tube.

3.2.4 Transfer 1.5 mL of sterile water to the reagent blank tube, making up a total of 3 mL in this test tube.

3.2.5 Transfer 1.5 mL of the isopropyl alcohol-exposed Staphylococcus aureus from the microfuge tubes to the correspondingly labeled glass test tubes, making up a total of 3 mL in each test tube.

3.3 Fluorescence Measurements (Green Fluorescence)

3.3.1 Install the 486 nm filter in A-EX and 520 nm filter in A-EM.

3.3.2 Rotate the TD-700 filter cylinder to place the 485 nm excitation and 530 nm emission filters in the optical path.

3.3.3 You will be performing a simple mode calibration (refer to your manual if need be). Press "enter" from the keypad. Select [1] on the keypad to enter "setup" then select [1] to enter "mode".

3.3.4 Using the arrow key to choose the mode, select "simple, then [ESC] twice to return to the setup/calibration screen. Select [2], then press [ENTER].

3.3.5 The TD-700 will prompt you to insert a typical sample. Insert the sample containing the largest number of live bacteria; this sample should have the highest intensity of green fluorescence among those to be measured. In the experiment described here, this will be the sample containing 0% isopropyl alcohol.

3.3.6 Press [ENTER]. The instrument detection sensitivity will be automatically optimized to match the fluorescence of the sample. When the screen indicates that the sample is 500, press [ENTER]. Your TD-700 is now calibrated.

3.3.7 Press [ENTER] again and read the remaining samples in the order of reagent blank followed by the bacterial samples in order of increasing isopropyl alcohol concentration.

3.3.8 For each sample, record the fluorescence intensity value shown on the fluorometer display screen. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. Correct data for the dye background by subtracting the reagent blank fluorescence value from each bacterial sample fluorescence reading.

3.4 Fluorescence Measurements (Red Fluorescence)

3.4.1 Rotate the TD-700 filter cylinder to place the 520 nm excitation and 325-700 nm and greater than 610 nm emission filters in the optical path ("B" on the left hand side lined up with silver dot).

3.4.2 Press [ENTER] on the TD-700 key pad.

3.4.3 Select [2] CALIBRATION from the displayed options.

3.4.4 Insert the sample containing the largest number of dead bacteria; this sample should have the highest intensity of red fluorescence among those to be measured. In the experiment described here, this will be the sample containing 100% isopropyl alcohol.

3.4.5 Press ENTER. The instrument detection sensitivity will be automatically optimized to match the fluorescence of the sample.

3.4.6 Press ENTER again and read the remaining samples in the order of reagent blank followed by the bacterial samples in order of increasing isopropyl alcohol concentration.

3.4.7 For each sample, record the fluorescence intensity value shown on the fluorometer display screen. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. Correct data for the dye background by subtracting the reagent blank fluorescence value from each bacterial sample fluorescence reading.

3.5 Data Analysis and Interpretation

3.5.1 For each sample, divide the background-corrected green fluorescence intensity by the background-corrected red fluorescence intensity.

3.5.2 Plot the calculated green/red fluorescence intensity ratio (Y-axis) as a function of isopropyl alcohol concentration (X-axis).

3.5.3 Plotted data should show decreasing green/red fluorescence intensity ratios as the number of dead bacteria increases (i.e. as the isopropyl alcohol concentration increases; Figure 1).

Figure 1. Analysis of Staphylococcus aureus viability following one hour exposure to varying concentrations of isopropyl alcohol. The TD-700 Laboratory Fluorometer was used to separately quantitate the green and red fluorescence emission produced by staining with SYTO 9 and propidium iodide respectively. The ratio of green/red fluorescence intensity was calculated for each sample and plotted against isopropyl alcohol concentration.

4. References

1. J Appl Bacteriol 72, 410 (1992)
2. Lett Appl Microbiol 13, 58 (1991)
3. Curr Microbiol 4, 321 (1980)
4. J Microbiol Meth 13, 87 (1991)

5. Patent and Trademark Information

LIVE/DEAD and SYTO are registered trademarks of Molecular Probes, Inc. BacLight is a trademark of Molecular Probes, Inc. SUREspin is a trademark of Helena Laboratories. The LIVE/DEAD BacLight Bacterial Viability assay is covered by current or pending U.S. and foreign patents.