Accurate Assessment of Microbial Viability by Flow Cytometry

One of the most fundamental applications in microbiology is the accurate assessment of culture viability and cell count. The Molecular Probes® LIVE/DEAD® BacLight™ Bacterial Viability Kit provides a novel two-color fluorescence assay that allows easy and reliable quantification of live and dead bacteria, even in a mixed population containing a range of bacterial types. When used with the new Attune® Acoustic Focusing Cytometer, the LIVE/DEAD® BacLight™ kit enables rapid, sensitive viability detection. This kit can be used with multiple instrument platforms including fluorescence imaging, fluorescence spectroscopy, and flow cytometry.

Increase the Speed of Your Microbial Viability Measurement

The traditional approach to microbial viability assessment is based on plate counting, which involves dilution, spread-plating, and manual colony counting. This method is time consuming (requiring at least 24 hours before results may be interpreted) and labor intensive, and does not account for viable but unculturable microorganisms. Attempts have been made to develop other bacterial viability assays based on metabolic characteristics or membrane integrity, but often these methods only work for a limited subset of bacterial groups [1], have high levels of background fluorescence [2], or are sensitive to growth and staining conditions [3]. In contrast, the LIVE/DEAD® BacLight™ Bacterial Viability Kit allows researchers to reliably distinguish and quantitate live and dead bacteria by flow cytometry within minutes.

Bright Staining of Live and Dead Bacterial Cells

The LIVE/DEAD® BacLight™ Bacterial Viability Kit contains two nucleic acid–binding dyes, SYTO® 9 and propidium iodide (PI), which are used to differentiate live from dead cells. SYTO® 9 is a cell-permeant dye that generally labels all bacteria within a population regardless of membrane integrity. In contrast, PI is a cell-impermeant stain that only crosses compromised or damaged cell membranes. When both stains are present in cells with compromised membrane, SYTO® 9 fluorescence is reduced by displacement of the stain by PI. With an appropriate mixture of the two stains, bacteria with intact cell membranes stain green; those with damaged membranes stain red.

The excitation/emission maxima for the dyes are approximately 480/500 nm (SYTO® 9) and 490/635 nm (PI), and the background remains virtually nonfluorescent (Figure 1). The two dye components in the LIVE/DEAD® BacLight™ Bacterial Viability Kit have been balanced so that a 1:1 mixture provides live/dead discrimination in most applications but occasionally, the dye ratio must be adjusted for optimal discrimination. To thoroughly optimize staining, we recommend experimenting with a range of dye concentrations.

The Attune® Acoustic Focusing Cytometer (see sidebar, next page) offers higher sensitivity with shorter acquisition times, which is especially valuable when samples are limited. When used with the Attune® cytometer, the LIVE/DEAD® BacLight™ Bacterial Viability Kit enables microbial viability to be determined quickly and accurately.

LIVE/DEAD® BacLight™ Bacterial Viability Kit
Figure 1. Bright staining of E. coli cells with the LIVE/DEAD® BacLight™ Bacterial Viability Kit. A culture of E. coli cells was washed and diluted to ~1 x 106 CFU/mL in 0.85% sodium chloride buffer prior to staining with SYTO® 9 and propidium iodide (PI) provided in the LIVE/DEAD® BacLight™ Bacterial Viability Kit. Cells were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser for SYTO® 9 and PI fluorescence excitation. Data were acquired using the standard 25 µL/min collection rate. SYTO® 9 fluorescence emission was detected in the BL1 channel (530/30 bandpass filter) and PI fluorescence emission was detected in the BL3 channel (640 longpass filter). Histogram overlays indicate the increase in fluorescence upon staining with either (left) SYTO® 9 or (right) PI. U = unstained cells; S = stained cells.

Quick and Easy Viability, No Matter What Species

The LIVE/DEAD® BacLight™ Bacterial Viability Kit has been thoroughly tested with a variety of organisms under several different conditions. Species tested include:

Bacillus cereus Klebsiella pneumoniae Pseudomonas syringae
Staphylococcus aureus
Bacillus subtilisMicrococcus luteusSalmonella oranienburg Streptococcus pyogenes
Clostridium perfringens
Mycobacterium phlei
Serratia marcescens
 
Escherichia coliPseudomonas aeruginosaShigella sonnei 

 All tested species demonstrate a good correlation between the results obtained with the LIVE/DEAD® BacLight™ Bacterial Viability Kit and standard plate counts (Figure 2).

Flow cytometry analysis of bacterial cultures
Figure 2. Flow cytometry analysis of relative culture viability of gram-positive and gram-negative bacterial cultures. Bacterial cells were grown to log phase and stained with the LIVE/DEAD® BacLight™ Viability Kit, then analyzed using the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser for SYTO® 9 and propidium iodide (PI) excitation. Samples were run at a collection rate of 25 µL/min using a fluorescent threshold, and fluorescence emission was detected using a 530/30 bandpass filter for SYTO® 9 fluorescence and 640 longpass filter for PI fluorescence. Bacterial populations were identified using a dual-parameter scatter plot (side versus forward scatter). (A) Viability analysis of E. coli (gram-negative) cells. Both live (L) and dead (D) cells fluoresce green (SYTO® 9) but only dead cells fluoresce red (PI). Viability was calculated using the following equation: % viability = [number of live events/(number of live events + number of dead events)] x 100%. In this example, E. coli culture viability was determined to be 95.7 ± 0.5%. The bar graph shows a comparison of bacterial cell concentration determined using the LIVE/DEAD® BacLight™ Viability Kit vs. traditional plate counts. The concentration of live cells was determined to be 6.37 ± 0.29 x 104 events/mL using the Attune® software; the plating method indicated a viable cell concentration of 6.28 ± 0.67 x 104 CFU/mL, with no significant difference in viable cell concentration observed  between the two methods. (B) S. aureus (gram-positive) cells stained with the LIVE/DEAD® BacLight™ Viability Kit. In this example, culture viability was determined to be 87.5 ± 3.3%. The bar graph shows a comparison of bacterial cell concentration determined using the LIVE/DEAD® BacLight™ Viability Kit vs. traditional plate counts. Bacterial cell concentration was determined to be 6.83 ± 0.25 x 104 events/mL using the Attune® software; the plating method indicated a viable cell concentration of 7.60 ± 0.72 x 104 CFU/mL, with no significant difference in viable cell concentration observed between the two methods.

Diverse Applications in Microbiology

The LIVE/DEAD® BacLight™ Bacterial Viability Kit has been used in numerous flow cytometry applications, including  characterization of bacteria from different aquatic environments [4], detection of bacterial contamination [5], analysis of antimicrobial susceptibility testing [6], quantification of viable but unculturable organisms [7], and the study of vital bacterial cellular functions during solar disinfection [8]. Life Technologies offers a wide range of products specific for the study of microbiology using flow cytometry (Table 1). 

Table 1. Flow Cytometry Products for Microbiology.

Application  Product Attune® Cytometer Channel(s)
(Optical Filters)
Quantity Cat. No.
Bacterial viabilityLIVE/DEAD® BacLight™ Bacterial Viability KitBL1 and BL3 (530/30 and 640LP)1 kitL7007, L7012, L13152
Bacterial viability and cell countingLIVE/DEAD® BacLight™ Bacterial Viability and Counting KitBL1 and BL3 (530/30 and 640LP)1 kitL34856
Bacterial cell countingBacteria Counting Kit, for flow cytometryBL1 (530/30)1 kitB7277
Bacterial cell stainingBacLight™ Green bacterial stain, special packagingBL1 (530/30)20 x 50 µgB35000
BacLight™ Red bacterial stain, special packagingNA20 x 50 µgB35001
SYBR® Green I nucleic acid stainBL1 (530/30)500 µLS7563
SYTO® BC green fluorescent nucleic acid stain, 5 mM solution in DMSOBL1 (530/30)100 µLS34855
SYTO® 9 green fluorescent nucleic acid stain,
5 mM solution in DMSO
BL1 (530/30)100 µLS34854
Determination of bacterial
gram character
LIVE BacLight™ Bacterial Gram Stain Kit, for microscopy and quantitative assaysBL1 and BL3 (530/30 and 640LP)1000 assaysL7005
Microbial membrane potential/
microbial vitality
BacLight™ Bacterial Membrane Potential Kit,
for flow cytometry
BL1 and BL3 (530/30 and 640LP)100 assaysB34950
Microbial metabolismBacLight™ RedoxSensor™ Green Vitality Kit,
for flow cytometry
BL1 and BL3 (530/30 and 640LP)1 kitB34954
BacLight™ RedoxSensor™ CTC Vitality Kit,
for flow cytometry and microscopy
BL1 and BL3, or VL1 (530/30 and 640LP,  or 450/40)1 kitB34956

The Attune® Acoustic Focusing Cytometer

Attune® Acoustic Focusing CytometerPrecision and Sensitivity At All Speeds
The Attune® Acoustic Focusing Cytometer is the first cytometer that uses ultrasonic waves—rather than hydrodynamic forces—to position cells into a single focused line along the central axis of a capillary. Acoustic focusing is largely independent of the sample input rate, enabling cells to be tightly focused at the point of laser interrogation regardless of the sample-to-sheath ratio. This, in turn, allows cell velocity to be slowed to collect more photons for high-precision analysis at unprecedented volumetric sample throughput.  

The Attune® cytometer achieves sample throughput at rates over 10 times faster than other cytometers —up to 1000 µL per minute—enabling rapid detection of rare events with a reliable measure of accuracy. The Attune® cytometer is designed to collect up to 20 million events per run, and has adjustable collection rates of 25–1000 µL/min. With this technology you can control the flow rate, number of photons detected, length of your experiment, number of samples run, and more. As a result, the Attune® cytometer enables higher sensitivity when analyzing precious samples or detecting rare events.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.