This section contains a thorough description of Invitrogen™ viability and cytotoxicity kits. Fluorometric assays of cell viability and cytotoxicity are easy to perform with the use of a fluorescence microscope, fluorometer, fluorescence microplate reader or flow cytometer, and they offer many advantages over traditional colorimetric and radioactivity-based assays. Also discussed in this section are our unique single-step kits for assessing gram sign and for simultaneously determining gram sign and viability of bacteria, as well as the Invitrogen™ FilmTracer™ LIVE/DEAD™ Biofilm Viability Kit designed specifically for bacterial biofilms.
To facilitate use of our unique cell viability and cytotoxicity assay technology, we have developed several important products (Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2) that combine fluorescent reagents to yield, in most cases, two-color discrimination of the population of live cells from the dead-cell population by simply adding the reagents, incubating for a brief period and observing the results without any wash steps required. These facile assays are ideal for high-throughput screening applications and, in most cases, for imaging, fluorometry and flow cytometry.
LIVE/DEAD Viability/Cytotoxicity Kit for Animal Cells
Our Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit (L3224) for animal cells provides an exceptionally easy fluorescence-based method for determining viability of adherent or nonadherent cells and for assaying cytotoxicity. The kit comprises two probes: calcein AM and ethidium homodimer-1. Calcein AM is a fluorogenic esterase substrate that is hydrolyzed to a green-fluorescent product (calcein); thus, green fluorescence is an indicator of cells that have esterase activity as well as an intact membrane to retain the esterase products. Ethidium homodimer-1 is a high-affinity, red-fluorescent nucleic acid stain that is only able to pass through the compromised membranes of dead cells. The LIVE/DEAD viability/cytotoxicity assay offers several advantages:
- Simplicity. The reagents are simultaneously added to the cell suspension, which is then incubated for 30–45 minutes. No wash steps are required before analysis.
- Specificity and reliability. Green-fluorescent cells are live; red-fluorescent cells are dead (, ).
- Versatility. The LIVE/DEAD viability/cytotoxicity assay is compatible with adherent cells such as astrocytes, nonadherent cells and certain tissues. Results can be analyzed by fluorescence microscopy using standard fluorescein longpass filter sets, as well as by flow cytometry (Figure 15.3.1) or fluorometry. The fluorescence emissions of the two probes are easily resolved (Figure 15.3.2).
- Simple quantitation. Flow cytometric measurements yield only two populations; there are rarely any doubly stained cells (Figure 15.3.1). Quantitative assays of bulk cells can be made using a fluorescence microplate reader or fluorometer.
- Suitability for high-throughput screening. The ease, reliability and low cost of the LIVE/DEAD Viability/Cytotoxicity Kit make it an economical assay for high-throughput screening of cytotoxic agents.
Several laboratories have established the validity of the LIVE/DEAD viability/cytotoxicity assay for use with animal cells and tissues. Published applications have included measuring the toxic effects of tumor necrosis factor (TNF), β-amyloid protein, adenovirus E1A proteins, tetrodotoxin (TTX) binding to Na+ channels, methamphetamines, mitogenic sphingolipids and photodynamic therapy. This assay has also been adapted to quantitate lymphocyte-mediated cytotoxicity by flow cytometry, cell-mediated cytotoxicity by fluorescence microscopy and the viability of boar sperm by fluorescence microscopy.
The LIVE/DEAD Viability/Cytotoxicity Kit is intended for use with animal cells that can be analyzed within about an hour of adding the dyes to the cells. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2. This kit's two viability probes—calcein AM (C1430, C3099, C3100MP) and ethidium homodimer-1 (E1169)—are also available separately (see Viability and Cytotoxicity Assay Reagents—Section 15.2) and may be used in combination with other probes for discrimination of live and dead cells. When assays need to be conducted over longer periods or when hazardous samples are being analyzed, we recommend our Invitrogen™ LIVE/DEAD™ Reduced Biohazard Cell Viability Kit #1 (L7013, see below).
Figure 15.3.1 Flow cytometric viability assay using the LIVE/DEAD Viability/Cytotoxicity Kit (L3224). A 1:1 mixture of live and ethanol-fixed human B cells was stained with calcein AM and ethidium homodimer-1 according to the kit protocol. After 5 minutes, flow cytometric analysis was carried out with excitation at 488 nm. The resulting bivariate frequency distribution shows the clear separation of the green-fluorescent (530 nm) live-cell population from the red-fluorescent (585 nm) dead-cell population.
LIVE/DEAD Reduced Biohazard Cell Viability Kit
Rigorous precautions are necessary during analysis of biohazardous specimens. Therefore, fixation procedures that inactivate cells yet produce minimal distortion of their characteristics are highly advantageous. The LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 (L7013) provides a two-color fluorescence assay for animal cell viability that is designed to reduce the risk associated with handling potential biohazards such as viral, bacterial or protozoan pathogens.
Viability analysis with the LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 is provided by the cell-permeant, green-fluorescent SYTO 10 and cell-impermeant, red-fluorescent DEAD Red (ethidium homodimer-2) nucleic acid stains. The dye concentrations and their relative affinities are balanced so that a cell population exposed simultaneously to both dyes becomes differentially stained—live cells fluoresce green and dead cells fluoresce red (). This assay is simple, fast and can be carried out using a fluorescence microscope, flow cytometer or fluorescence microplate reader. Moreover, the staining pattern of a cell population is retained for several hours after fixation (Figure 15.3.3).
Our LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 has several unique features:
- Reduced handling risks. This kit allows viability staining to take place while the potentially pathogenic sample is well contained. Subsequent treatment with 4% glutaraldehyde (or less effectively with formaldehyde) permits safer handling during analysis, without disrupting the distinctive staining pattern. Glutaraldehyde is known to inactivate cells and viruses, while preserving their overall morphology. In addition, the high sensitivity and specificity of the assay mean that sample sizes can be very small, further reducing potential biohazards.
- Specificity and reliability. Live cells initially fluoresce green, and dead cells fluoresce red. With time, this discrimination is reduced but can still be detected, even after 24 hours (Figure 15.3.3).
- Independence from enzymatic activity. Because it relies on two nucleic acid stains that differ in their membrane permeability, this assay equates loss of cell viability with loss of membrane integrity. Consequently, the assay is totally independent of variations in enzymatic activity or electrical potential of the cell.
- Versatility. The analysis is readily quantitated with a fluorescence microscope or flow cytometer (Figure 15.3.3). This kit's protocol includes methods for analyzing the viability of nonadherent cells, as well as adherent cells on coverslips.
- Convenience. Cells can be stained and fixed at various times during the experiment, and the results can be analyzed several hours later, without loss of the discrimination pattern.
The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
LIVE/DEAD Fixable Dead Cell Stain Kits
The Invitrogen™ LIVE/DEAD™ Fixable Dead Cell Stain Kits (three of which were formerly named LIVE/DEAD Reduced Biohazard Cell Viability Kits #2, #3 and #4) employ an amine-reactive fluorescent dye to evaluate mammalian cell viability by flow cytometry (Figure 15.3.4). The LIVE/DEAD Fixable Dead Cell Stain Kits are identical except for the fluorescent color of the amine-reactive dye:
- LIVE/DEAD Fixable Blue Dead Cell Stain Kit *for UV excitation* (L23105, excitation/emissionm ~350/450 nm)
- LIVE/DEAD Fixable Aqua Dead Cell Stain Kit *for 405 nm excitation* (L34957, excitation/emission maxima ~367/526 nm)
- LIVE/DEAD Fixable Yellow Dead Cell Stain Kit *for 405 nm excitation* (L34959, excitation/emission maxima ~400/575 nm)
- LIVE/DEAD Fixable Violet Dead Cell Stain Kit *for 405 nm excitation* (L34955, excitation/emission maxima ~416/451 nm)
- LIVE/DEAD Fixable Green Dead Cell Stain Kit *for 488 nm excitation* (L23101, excitation/emission maxima ~495/520 nm)
- LIVE/DEAD Fixable Red Dead Cell Stain Kit *for 488 nm excitation* (L23102, excitation/emission maxima ~595/615 nm)
- LIVE/DEAD Fixable Far Red Dead Cell Stain Kit *for 633 or 635 nm excitation* (L10120, excitation/emission maxima ~650/665 nm)
- LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit *for 633 or 635 nm excitation* (L10119, excitation/emission maxima ~750/775 nm)
- LIVE/DEAD Fixable Dead Cell Stain Sampler Kit *for flow cytometry* (L34960), which contains one vial of each of the eight different fluorescent reactive dyes
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity between the live and dead cell populations is typically greater than 50 fold (Figure 15.3.5), and this fluorescence intensity discrimination is completely preserved following formaldehyde fixation, using conditions that inactivate pathogens. These single-color assays use only one channel of a flow cytometer, making the reactive dyes in the LIVE/DEAD Fixable Dead Cell Stain Kits compatible with multiparameter staining experiments; appropriate flow cytometer channels may vary depending on the instrument. The assays can also be used to detect dead cells by fluorescence microscopy; however, the difference in fluorescence intensity of the live and dead cells can be appreciable, making it relatively difficult to simultaneously photograph the two populations. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
Figure 15.3.4 Principle of our LIVE/DEAD Fixable Green Dead Cell Stain Kit (L23101). Live cells (left) react with the kit's green-fluorescent, amine-reactive dye only on their surface to yield weakly fluorescent cells. Cells with compromised membranes (right) react with the dye throughout their volume, yielding brightly stained cells. In both cases, the excess reactive dye is subsequently washed away. Other LIVE/DEAD Fixable Dead Cell Stain Kits operating on the same principle are available, providing aqua (L10119), yellow (L10120), blue (L23105), violet (L34955), red (L23102), far-red (L34957), and near-IR (L34959) fluorescence.
Figure 15.3.5 Live and dead cells distinguished by flow cytometry using the LIVE/DEAD Fixable Green Dead Cell Stain Kit (L23101). The LIVE/DEAD Fixable Green Dead Cell Stain Kit was used to differentially stain live and dead Jurkat cells taken from a healthy culture (top panel), an aged culture (middle panel) and a heat-killed culture (bottom panel). Following the staining reaction, the cells were fixed in 3.7% formaldehyde and analyzed by flow cytometry. Nearly identical results were obtained using unfixed cells (data not shown).
Figure 15.3.5 Live and dead cells distinguished by flow cytometry using the LIVE/DEAD Fixable Green Dead Cell Stain Kit (L23101). The LIVE/DEAD Fixable Green Dead Cell Stain Kit was used to differentially stain live and dead Jurkat cells taken from a healthy culture (top panel), an aged culture (middle panel) and a heat-killed culture (bottom panel). Following the staining reaction, the cells were fixed in 3.7% formaldehyde and analyzed by flow cytometry. Nearly identical results were obtained using unfixed cells (data not shown).
ArC Amine-Reactive Compensation Bead Kit
Optimized for use with the LIVE/DEAD Fixable Dead Cell Stain Kits, the Invitrogen™ ArC™ Amine Reactive Compensation Bead Kit (A10346) is a tool designed to remove spectral overlap of the fixable dead-cell stains with other standard fluorophores. This kit provides two polystyrene microsphere samples: ArC Reactive Beads, which are reactive to all dyes in the Live/Dead Fixable Dead Cell Stain Kits, and Negative Control Beads, which have no reactivity. The two components provide negative and positive populations that can be used to accurately set compensation when using the Live/Dead Fixable Dead Cell Stains.
LIVE/DEAD Cell-Mediated Cytotoxicity Kit
Cytotoxicity triggered by a natural defense mechanism may be much slower than cell lysis triggered by a cytotoxic reagent. Our Invitrogen™ LIVE/DEAD™ Cell-Mediated Cytotoxicity Kit (L7010) is intended for cytotoxicity assessments extending over time periods that are too long for effective use of cytoplasmic markers, such as calcein, which may leak out or become sequestered. This kit is based directly on procedures developed by Kroesen and colleagues for measuring natural killer (NK) cell–mediated, lymphokine-activated killer (LAK) cell–mediated and T cell–mediated cytotoxicity by fluorescence microscopy. The assay has also been adapted for rapid flow cytometric analysis of NK cell activity.
Analysis of cell-mediated cytotoxicity using this kit is easy. In order to distinguish target cells, cultures are labeled overnight with DiOC18(3), a green-fluorescent membrane stain (Tracers for Membrane Labeling—Section 14.4). Target cells are then washed free of excess DiOC18(3) and combined in various proportions with effector cells. After a suitable incubation period, propidium iodide, a red-fluorescent, membrane-impermeant nucleic acid stain, is added. Propidium iodide labels dead effector cells, as well as dead target cells once their plasma membranes are compromised. Because the target cells retain the green-fluorescent membrane stain, both live and dead effector cells and live and dead target cells can readily be discriminated with a fluorescence microscope. Dead target cells exhibit both green-fluorescent membrane staining and red-fluorescent nuclear staining, whereas dead effector cells show only red-fluorescent nuclear staining. Live target cells have only green-fluorescent membrane staining, and live effector cells are unstained. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
LIVE/DEAD Sperm Viability Kit
The Invitrogen™ LIVE/DEAD™ Sperm Viability Kit (L7011), developed in collaboration with Duane L. Garner, provides a novel fluorescence-based method for analyzing the viability of sperm in different species. The LIVE/DEAD Sperm Viability Kit contains the membrane-permeant SYBR 14 nucleic acid stain, along with the conventional dead-cell stain, propidium iodide. Using this combination of dyes, researchers can rapidly distinguish live and dead cells with visible-light excitation (), thus avoiding the harmful effects of UV exposure and allowing flow cytometric analysis of sperm viability to be performed using an argon-ion laser excitation source. When semen is incubated briefly with these two stains, live sperm with intact membranes fluoresce bright green, whereas sperm cells with damaged membranes fluoresce red. Garner and colleagues assessed bovine sperm viability with flow cytometry and with fluorescence microscopy; both techniques allowed live and dead cells to be visualized simultaneously. Furthermore, it was reported that neither the ability to fertilize oocytes nor the development of the embryos was affected by SYBR 14 staining of porcine sperm. The effect of two-photon illumination on the viability of human sperm stained with these reagents has also been analyzed. This assay is particularly useful for evaluating the viability of cryopreserved sperm.
The dyes provided in the LIVE/DEAD Sperm Viability Kit stain cells more rapidly than conventional stains (within 5–10 minutes), and both label DNA, thereby avoiding the ambiguity that may arise from targeting separate cellular components. The membrane-permeant SYBR 14 stain provided in the LIVE/DEAD Sperm Viability Kit should also serve as a valuable tool for labeling and tracking live sperm, thus facilitating analysis of their motility and abundance in semen samples. Reliable viability measurements with bovine, porcine, ovine, murine, goat, turkey and human sperm have been published. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
Conventional sperm viability assays have employed mixtures of two or three dyes, including fluorescein diacetate derivatives, rhodamine 123 and reduced nucleic acid stains such as dihydroethidium (hydroethidine, D1168, D11347, D23107; Viability and Cytotoxicity Assay Reagents—Section 15.2). Acridine orange (A1301, A3568), which fluoresces at different wavelengths when bound to DNA and RNA, and the UV light–excitable nucleic acid stains Hoechst 33258 (H1398, H3569, H21491) and Hoechst 33342 (H1399, H3570, H21492) are frequently used to determine sperm viability and DNA content and to trace sperm–oocyte fusion. These nucleic acid stains are described in Nucleic Acid Stains—Section 8.1.
LIVE/DEAD Violet Viability/Vitality Kit
The Invitrogen™ LIVE/DEAD™ Violet Viability/Vitality Kit (L34958) provides a two-color cell viability and vitality assay that enables the simultaneous identification of live and dead cells. The assay employs two fluorescent probes―calcein violet AM and aqua-fluorescent reactive dye―to indicate recognized parameters of cell health (intracellular esterase activity and plasma membrane integrity, respectively). Both dyes utilize 405 nm violet diode laser excitation, allowing other laser lines to be used for conventional fluorophores. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
LIVE/DEAD Cell Vitality Assay Kit
The Invitrogen™ LIVE/DEAD™ Cell Vitality Assay Kit (L34951) provides a simple two-color fluorescence assay that distinguishes metabolically active cells from injured cells and dead cells. The assay is based on the reduction of C12-resazurin to red-fluorescent C12-resorufin in metabolically active cells and the uptake of the cell-impermeant Invitrogen™ SYTOX™ Green nucleic acid stain in cells with compromised plasma membranes (usually late apoptotic and necrotic cells). Dead cells emit mostly green fluorescence, whereas the healthy, metabolically active cells emit mostly red fluorescence (Figure 15.3.6). The injured cells have lower metabolic activity and, consequently, reduced red-fluorescent emission. Because they possess intact membranes, injured cells accumulate little SYTOX Green dye and, therefore, also emit very little green fluorescence.
Nonfluorescent resazurin, which can be reduced by viable cells to red-fluorescent resorufin, has been extensively used to detect the metabolic activity of many different cell types, from bacteria to higher eukaryotes. Resazurin is nontoxic and stable in culture media, allowing researchers to continuously monitor proliferating cells and to investigate cytotoxicity in both conventional and high-throughput applications. The LIVE/DEAD Cell Vitality Assay Kit includes a lipophilic version of resazurin, C12-resazurin, which is more permeable to live cells and, after reduction to C12-resorufin, is far better retained than the nonlipophilic resorufin. These characteristics result in brighter signals and better detection limits. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
Figure 15.3.6 Flow cytometric analysis of Jurkat cells using the LIVE/DEAD Cell Vitality Assay Kit (L34951). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for 4 hours at 37°C, 5% CO2. The cells were then treated with the reagents in the LIVE/DEAD Cell Vitality Assay Kit as specified in the kit protocol (Product Information Sheet) and analyzed by flow cytometry. This dot plot of SYTOX Green fluorescence versus resorufin fluorescence shows resolution of live-, injured- and dead-cell populations.
Vybrant Cell Metabolic Assay Kit
One potential drawback in the use of resazurin as a substrate that can detect metabolic activity in live cells (R12204, Viability and Cytotoxicity Assay Reagents—Section 15.2) is the relatively poor uptake of the substrate and poor cellular retention of the reduced by-product, resorufin. We have found that the lipophilic dodecylresazurin (C12-resazurin), included as a component in the Invitrogen™ Vybrant™ Cell Metabolic Assay Kit (V23110), surpasses resazurin alone in cell permeability and its reduction product (C12-resorufin) is very well retained, permitting single-cell analysis of the substrate's turnover by flow cytometry (Figure 15.3.7). This enhanced uptake, turnover and retention of C12-resazurin by metabolically active cells translates into much brighter signals and far better detection limits when compared with assays using resazurin alone (Figure 15.3.8).
C12-resazurin can be used in any viability/cytotoxicity assay that employs resazurin for both conventional and high-throughput applications. C12-resorufin, which is the reduction product of C12-resazurin, has the same absorption/emission maxima as unmodified resorufin (~571/ 585 nm); therefore, no changes in instrumentation are required in order to use the kit in place of a resazurin-based assay. The Vybrant Cell Metabolic Assay Kit (V23110) contains:
- Dodecylresazurin (C12-resazurin)
- Detailed protocols for the assay (Vybrant Cell Metabolic Assay Kit)
Each kit contains sufficient reagents for 500–1000 assays in a fluorescence microplate or about 10,000 flow cytometry assays.
Figure 15.3.7 Flow cytometric analysis of Jurkat cells stained with C12-resazurin. Cells were loaded with 0.1 µM C12-resazurin, a component of the Vybrant Cell Metabolic Assay Kit (V23110), and 1 mM SYTOX Green (S7020). After a 15-minute incubation, the cells were analyzed. Healthy (live) cells reduce C12-resazurin into red-fluorescent C12-resorufin and exclude the cell impermeant green-fluorescent SYTOX Green. Dead cells show little reduction of the C12-resazurin, but strong staining by SYTOX Green. Cells indicated in the figure as dying are of indeterminate viability, showing both reduction of C12-resazurin and compromised membrane integrity.
Figure 15.3.8 Detection limit of C12-resazurin and linear response with increasing cell number using our Vybrant Cell Metabolic Assay Kit (V23110). Jurkat cells were loaded with 5 µM C12-resazurin for 15 minutes. The resulting signal was measured in a fluorescence microplate reader with excitation/emission at 530/590 nm. For comparison, the detection limit for resazurin in a similar experiment was ~8000 cells/well (data not shown).
Vybrant Cytotoxicity Assay Kit
The LIVE/DEAD kits generally assay cell death via probes that gain entry to the interior of the cell as a result of plasma membrane damage. In contrast, the Invitrogen™ Vybrant™ Cytotoxicity Assay Kit (V23111) monitors the release of the cytosolic enzyme glucose 6-phosphate dehydrogenase (G6PD) from damaged cells into the surrounding medium. G6PD is a ubiquitous enzyme that is part of the pentose phosphate pathway, and is crucial for cellular antioxidant defenses via its production of NADPH. Detection of G6PD is by a two-step enzymatic process that leads to the reduction of resazurin into the red-fluorescent resorufin (Figure 15.3.9). The resulting signal is proportional to the amount of G6PD released into the cell medium, which correlates with the number of dead cells in the sample (Figure 15.3.10).
The Vybrant Cytotoxicity Assay Kit contains all enzymes and substrates needed to detect the release of G6PD from damaged and dying cells. The assay can be completed in less than an hour and is effective with as few as 500 cells per sample. Resorufin, the end product of the G6PD cytotoxicity assay, has absorption and emission maxima at ~571 nm and 585 nm, respectively, placing the fluorescent signal beyond the autofluorescence of most biological samples. In addition, the levels of G6PD in serum commonly used for cell culture are lower than those of lactate dehydrogenase (LDH), an enzyme often used in similar assays, thus resulting in lower background signals (Figure 15.3.11). The Vybrant Cytotoxicity Assay Kit (V23111) contains:
- Resazurin (5 vials)
- Reaction mixture (diaphorase, glucose 6-phosphate and NADP+)
- Reaction buffer
- Cell-lysis buffer
- Detailed protocols for the assay (Vybrant Cytotoxicity Assay Kit)
Sufficient reagents are provided for about 1000 assays in a fluorescence microplate reader.
Figure 15.3.10 Detection of dead and dying cells using the Vybrant Cytotoxicity Assay Kit (V23111). Jurkat cells were treated with 10 µM camptothecin for six hours, then assayed for glucose 6-phosphate dehydrogenase release. An untreated control sample is shown for comparison. The fluorescence was measured in a microplate reader (excitation/emission ~530/590 nm). A background of 55 fluorescence units was subtracted from each value.
Figure 15.3.11 10% bovine serum was assayed for the presence of lactate dehydrogenase (LDH, blue) and glucose 6-phosphate dehydrogenase (G6PD, red). G6PD was assayed using the Vybrant Cytotoxicity Assay Kit (V23111); LDH was detected using a similar method, in which LDH reduces lactate to generate NADH. The result clearly shows that, over the time course of the experiment, the serum generates a much lower signal in the G6PD assay than in the LDH assay.
LIVE/DEAD Yeast Viability Kit
Our Invitrogen™ LIVE/DEAD™ Yeast Viability Kit (L7009) provides an extremely simple and sensitive assay for discriminating viable yeast and fungi in complex mixtures or in pure cultures. This kit contains our unique two-color fluorescent viability probe, the Invitrogen™ FUN™ 1 dye, which has low intrinsic fluorescence, moderate affinity for nucleic acids and exceptional membrane permeability. Also included is the UV light–excitable counterstain Calcofluor White M2R, which labels the cell walls of yeast and fungi fluorescent blue, regardless of the cell's metabolic state.
The FUN 1 viability probe displays some extraordinary spectral properties when used to stain metabolically active yeast and fungal cells, exploiting normal endogenous biochemical processing mechanisms that appear to be well conserved among different fungal species. The FUN 1 stain passively diffuses into a variety of cell types and initially stains the cytoplasm with a diffusely distributed green fluorescence. However, in several common species of yeast and fungi, subsequent processing of the dye by live cells results in the formation of distinct vacuolar structures with compact form that exhibit a striking red fluorescence, accompanied by a reduction in the green cytoplasmic fluorescence (Figure 15.3.12). Formation of the red-fluorescent intravacuolar structures requires both plasma membrane integrity and metabolic capability. Dead cells fluoresce bright yellow-green, with no discernable red structures.
FUN 1 stain can be used alone or together with Calcofluor White M2R to determine the metabolic activity of single fungal cells by manual or automated fluorescence microscopy (, ). Both live and dead cells may be viewed simultaneously by fluorescence microscopy using a standard fluorescein longpass optical filter set. FUN 1 dye staining can also be used to assay the viability of suspensions of fungal cells using a fluorescence microplate reader or a fluorometer. The FUN 1 reagent has been extensively used for flow cytometric analysis of yeast, including of their susceptibility to antifungal agents.
The LIVE/DEAD Yeast Viability Kit has been tested on several fungal species, including Candida albicans, Candida pseudotropicalis and several strains of Saccharomyces cerevisiae, under a variety of experimental conditions. Formation of the red-fluorescent structures was observed not only in logarithmically growing cells but also in nonculturable cells with residual metabolic activity. The LIVE/DEAD Yeast Viability Kit should be particularly useful for detecting very low numbers of live or dead fungal cells, even in complex mixtures such as blood. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2. The FUN 1 cell stain is also available separately (F7030, Viability and Cytotoxicity Assay Reagents—Section 15.2).
Figure 15.3.12 Fluorescence emission spectra of a Saccharomyces cerevisiae suspension that has been stained with the FUN 1 cell stain, which is available separately (F7030) or in our LIVE/DEAD Yeast Viability Kit (L7009). After the FUN 1 reagent was added to the medium, the fluorescence emission spectrum (excited at 480 nm) was recorded in a spectrofluorometer at the indicated times during a 30-minute incubation period. The shift from green (G) to red (R) fluorescence reflects the processing of FUN 1 by metabolically active yeast cells.
LIVE/DEAD FungaLight Yeast Viability Kit
The Invitrogen™ LIVE/DEAD™ FungaLight™ Yeast Viability Kit (L34952) allows researchers to easily, reliably and quantitatively distinguish live and dead yeast in minutes. The kit contains solutions of SYT0 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 yeast cells. When used alone, SYTO 9 stain generally labels all yeast in a population—those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates yeast with damaged membranes, displacing SYTO 9 stain. Thus, with an appropriate mixture of the SYTO 9 and propidium iodide stains, yeast with intact cell membranes stain fluorescent green, whereas yeast with damaged membranes stain fluorescent red. The excitation/emission maxima for these dyes are 480/500 nm for SYTO 9 stain and 530/635 nm for propidium iodide. The background remains virtually nonfluorescent. Furthermore, this kit also accommodates fine-tuning of the dye combinations so that optimal staining of yeast can be achieved under a variety of experimental conditions. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
FungaLight CFDA AM/Propidium Iodide Yeast Vitality Kit
The Invitrogen™ FungaLight™ CFDA AM/Propidium Iodide Yeast Vitality Kit (F34953) combines a cell-permeant esterase substrate with a membrane integrity indicator to evaluate the vitality of yeast cells by flow cytometry or microscopy. The acetoxymethyl ester (AM) of the esterase substrate 5-carboxy-fluorescein diacetate (CFDA) allows this reagent to permeate cell membranes. Once inside the cell, the lipophilic blocking and diacetate groups are cleaved by nonspecific esterases, resulting in a fluorescent, charged form that leaks out of cells very slowly. In contrast, the membrane integrity indicator, propidium iodide, penetrates yeast with damaged membranes. With an appropriate mixture of the CFDA AM and propidium iodide stains, esterase-active yeast with intact cell membranes stain fluorescent green, whereas yeast with damaged membranes stain fluorescent red. The excitation/emission maxima for these dyes are 492/517 nm for CFDA AM and 530/635 nm for propidium iodide. The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
LIVE/DEAD BacLight Bacterial Viability Kits
The original LIVE/DEAD Viability/Cytotoxicity Kit (L3224, see above) is a proven tool for assessing viability of animal cells but is generally not suitable for use with bacterial and yeast cells. Consequently, we have developed the Invitrogen™ LIVE/DEAD™ BacLight™ Bacterial Viability Kits (L7007, L7012, L13152), which provide two different nucleic acid stains—the SYTO 9 dye and propidium iodide—to rapidly distinguish live bacteria with intact plasma membranes from dead bacteria with compromised membranes (, ). This assay has several significant features:
- Ease of use. The reagents are simultaneously added to the bacterial suspension, which is then incubated for 5–10 minutes. No wash steps are required before analysis.
- Specificity. Live bacteria fluoresce green and dead bacteria fluoresce red. Live and dead bacteria can be distinguished and quantitated in minutes, even in a mixed population of bacterial species ().
- Reliability. The LIVE/DEAD BacLight Bacterial Viability Kits yield consistent results in tests on a variety of eubacterial genera (Some organisms that have been successfully stained with our LIVE/DEAD BacLight Bacterial Viability Kits—Table 15.3). It can also be used to assess the viability of Eurioplasma eurilytica and Mycoplasma hominus mycoplasma as well as cysts of the protozoan parasite Giardia muris ().
- Validity. Viability measurements in fresh cultures of bacteria typically correlate well with enumeration techniques involving growth in liquid or solid media. However, variable results have been found using the LIVE/DEAD BacLight reagents to assess viability in some marine bacteria from environmental samples.
- Versatility. Bacteria can be stained in suspension or immobilized on microscope slides or filter membranes. Protocols are provided for bacterial viability analysis using a fluorescence microscope, fluorometer (Figure 15.3.13) or fluorescence microplate reader.
The intensities of the fluorescence signals produced by the SYTO 9 and propidium iodide nucleic acid stains can be adjusted by mixing different proportions of the dye solutions provided in the LIVE/DEAD BacLight Kits. We have balanced the dye concentrations so that, for most bacteria, equal volumes of the two solutions provided give balanced staining of most species. The background remains virtually nonfluorescent, allowing live and dead cells to be easily differentiated in any fluorescence microscope equipped with a longpass fluorescein or comparable optical filter set. Under certain conditions, bacteria with compromised membranes may recover and reproduce, even though such bacteria may be scored as dead in this assay. Conversely, some bacteria with intact membranes may be unable to reproduce in nutrient medium, yet be scored as live. Combining several different measures of viability, such as membrane permeability, enzyme activity and redox potential, offers a more thorough assessment of bacterial viability and eliminates the inherent limitations of any single viability assay.
The LIVE/DEAD BacLight viability assay has been used to estimate total and viable bacteria in drinking water, to quantitate total and viable concentrations of aerosolized Pseudomonas fluorescens by fluorescence microscopy and to measure the reliability of disinfection agents on reducing the viability of Cryptosporidium parvum and Giardia muris cysts. The number of live natural planktonic bacteria, as determined with the LIVE/DEAD BacLight Bacterial Viability Kit, reportedly correlated well with the number of bacteria with high DNA content (HDNA), as determined with SYTO 13 green-fluorescent nucleic acid stain (S7575), leading to the recommendation that %HDNA be used as an index of the percentage of actively growing bacterial cells in marine plankton samples. The reagents in the LIVE/DEAD BacLight Bacterial Viability Kit have been utilized in a high-throughput fluorescence-based screen for bacterial mechanosensitive ion-channel (MscL) activity that replaces otherwise tedious and difficult assays.
Our original packaging of the LIVE/DEAD BacLight Bacterial Viability Kit (L7007), in which the dyes were mixed at different proportions in two solutions, is still available for customers who have already developed protocols using that formulation. However, we recommend use of the LIVE/DEAD BacLight Bacterial Viability Kit (L7012), which is more flexible because it provides separate solutions of the SYTO 9 and propidium iodide nucleic acid stains, thus facilitating calibration of bacterial fluorescence at each of the two emission wavelengths in quantitative assays. Kit L7007 was designed primarily for use in fluorescence microscopy; Kit L7012 is equally well suited for use in fluorescence microscopy and is better suited than Kit L7007 for use in quantitative analysis with a fluorometer (Figure 15.3.13), fluorescence microplate reader, flow cytometer or other instrumentation.
Figure 15.3.13 Viability analysis of bacterial suspensions comprising various proportions of live and isopropyl alcohol–killed Escherichia coli using the reagents in the LIVE/DEAD BacLight Bacterial Viability Kit (L7007, L7012, L13152). Live and dead bacteria are stained fluorescent green (G) by SYTO 9 and fluorescent red (R) by propidium iodide, respectively. Bacterial suspensions that have been incubated in the two stains simultaneously and then excited at 470 nm exhibit a fluorescence spectral shift from red to green as the percentage of live bacteria in the sample is increased.
For added convenience, our LIVE/DEAD BacLight Bacterial Viability Kit (L13152) provides the separate stains dry and premeasured into pairs of polyethylene transfer pipettes (Figure 15.3.14). Kit L13152 has several advantages:
- The stains are dry, without DMSO or other potentially harmful solvents, allowing viability determination of solvent-sensitive microorganisms—just dissolve the dyes in virtually any aqueous medium and then add them to the cells.
- The stains are premeasured and supplied in sealed polyethylene transfer pipettes, eliminating the need for pipetting microliter volumes—perfect for educational settings, where there is a need to simplify handling and minimize equipment expenditures.
- This stain formulation does not require refrigeration and is chemically stable, even in poor conditions—storage at 37°C for more than six months produces no detectable changes, making the assay well suited to field testing and other situations where storage or use conditions are less than optimal.
Each of our LIVE/DEAD BacLight Bacterial Viability Kits includes a procedure for mounting bacteria stained with the reagents in the LIVE/DEAD BacLight Bacterial Viability Kit on filter membranes and a proprietary mounting oil that we have found to be useful for the direct epifluorescence filter technique (DEFT). The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2. The SYTO 9 nucleic acid stain is also available separately (S34854, Viability and Cytotoxicity Assay Reagents—Section 15.2).
Figure 15.3.14 The convenient and versatile procedure for using the specially packaged LIVE/DEAD BacLight Bacterial Viability Kit (L13152). Simply dissolve the premeasured dyes in buffer, mix with the bacterial sample and observe the fluorescence.
LIVE/DEAD BacLight Bacterial Viability and Counting Kit
Accurate detection and enumeration of the live and dead bacteria in a sample is an important aspect of many experimental procedures in biotechnology. Because of the marked differences in morphology, cytology and physiology among the many bacterial genera, a universally applicable direct-count viability assay has been very difficult to attain. 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, and methods for assessing bacterial membrane integrity commonly have high levels of background fluorescence. Both types of determinations suffer from being very sensitive to growth and staining conditions. The Invitrogen™ LIVE/DEAD™ BacLight™ Bacterial Counting and Viability Kit (L34856) allows researchers to reliably distinguish and quantitate live and dead bacteria with the aid of a flow cytometer, even in a mixed population containing a range of bacterial types.
This kit utilizes a mixture of two nucleic acid stains—the green-fluorescent SYTO 9 dye and red-fluorescent propidium iodide—for viability determinations, as well as a calibrated suspension of beads for accurate sample volume measurements. The SYTO 9 and propidium iodide stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain generally labels all bacteria in a population—those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain fluorescence when both dyes are present. With an appropriate mixture of the SYTO 9 and propidium iodide stains, bacteria with intact cell membranes fluoresce bright green, whereas bacteria with damaged membranes exhibit significantly less green fluorescence and they often also fluoresce red. The cell type and the gram character influence the amount of red-fluorescent staining exhibited by dead bacteria. Both the SYTO 9 and propidium iodide stains are efficiently excited by the 488 nm spectral line of the argon-ion laser found in most flow cytometers, and their nucleic acid complexes can be detected in the green and red channels, respectively; the background remains virtually nonfluorescent.
The calibrated suspension of microspheres serves as a reference standard for sample volume. The size and fluorescence of the beads in this microsphere standard have been carefully chosen to ensure that they will be clearly distinguishable from any stained bacteria population in a fluorescence versus side scatter cytogram. A bacterial culture is simply stained with the optimal mixture of SYTO 9 dye and propidium iodide, and then a fixed number of microspheres are added before analyzing the sample on a flow cytometer. Live and dead bacteria and the microspheres are all easily distinguished in a plot of fluorescence versus side scatter (Figure 15.3.15). The concentration of both the live bacteria and the dead bacteria can then be determined from the ratio of bacteria events to microsphere events in the cytogram (Figure 15.3.16).
The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2. The SYTO 9 nucleic acid stain is also available separately (S34854, Viability and Cytotoxicity Assay Reagents—Section 15.2).
Figure 15.3.15 Analysis of bacterial cultures using the LIVE/DEAD BacLight Bacterial Viability and Counting Kit (L34856). Suspensions of live (untreated) and dead (alcohol-treated) Staphylococcus aureus (panels A and C) and Escherichia coli (panels B and D) were stained with the SYTO 9 nucleic acid stain and propidium iodide and then analyzed by flow cytometry according to the kit protocol. The green or red fluorescence versus side scatter cytogram (panel A or B) was used to gate the bacterial population and the bead population (left and right boxes, respectively). Events in the bacteria region of each cytogram are also displayed in red fluorescence versus green fluorescence cytograms (panels C and D). Live and dead bacteria/mL can be calculated from either the fluorescence versus side scatter cytogram or the green fluorescence versus red fluorescence cytogram, depending on which one shows the best separation of the live and dead populations. The position of the live and dead populations in these cytograms may be dependent on cell type and gram character. Some samples may exhibit events that fall outside the defined regions and should be evaluated appropriately (e.g., see panel D).
Figure 15.3.16 Best-fit linear regression analysis generated using the LIVE/DEAD BacLight Bacterial Viability and Counting Kit (L34856). Suspensions of live (untreated, solid line (r2=0.9982)) and dead (alcohol-treated, dashed line (r2=0.9974)) Staphylococcus aureus were mixed at various live:dead ratios. Mixtures were stained according to the kit protocol and analyzed in triplicate by flow cytometry. Values of bacteria/mL were calculated according to the equation provided in the kit protocol; the mean values are shown above. This experiment may be performed to determine optimal dye concentrations, to practice the cell-staining procedure or to generate a standard curve for unknown samples.
BacLight RedoxSensor Green Vitality Kit
Bacterial oxidation–reduction activity is an informative parameter for measuring cell vitality. Bacterial oxidases and reductases engage in important functions involving the electron transport chain, catabolic and anabolic pathways and xenobiotic compound metabolism. The Invitrogen™ RedoxSensor™ Green reagent, available in the Invitrogen™ BacLight™ RedoxSensor™ Green Vitality Kit (B34954), is an indicator of bacterial reductase activity; this reductase activity is, in turn, a reliable marker for changes in electron transport chain function and for changes in vitality that occur following antibiotic treatment. The RedoxSensor Green reagent penetrates both gram-positive and gram-negative bacteria, although differences in signal intensity may be observed based upon cell wall characteristics. Following reduction, the RedoxSensor Green reagent will produce a stable green-fluorescent signal (excitation/emission maxima ~490/520 nm) in 10 minutes that is compatible with formaldehyde fixation techniques. The BacLight RedoxSensor Green Vitality Kit is useful for measuring the effects of antimicrobial agents and for monitoring cultures in fermenters.
The fluorescence intensity of cells stained with the RedoxSensor Green reagent is altered when cells are treated with reagents that disrupt electron transport, such as sodium azide or carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which are both included in this kit. Species differences in responses may be observed. For example, sodium azide disrupts the fluorescence response in Escherichia coli, and CCCP disrupts fluorescence response in Staphylococcus aureus. The BacLight RedoxSensor Green Vitality Kit has been tested on logarithmically growing cultures of the following bacterial species: Micrococcus luteus, Staphylococcus aureus, Bacillus cereus, B. subtilis, Klebsiella pneumoniae, Escherichia coli and Salmonella cholerasuis. Most gram-positive bacteria stain more efficiently than many gram-negative bacteria; and the response of each bacterial system should be investigated and optimized.
Each BacLight RedoxSensor Green Vitality Kit contains:
- RedoxSensor Green reagent
- Propidium iodide, an indicator of membrane integrity
- Sodium azide
- Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
- Detailed protocols (BacLight RedoxSensor Green Vitality Kit)
At the recommended reagent dilutions and volumes, this kit contains sufficient material to perform ~200 tests by flow cytometry. Although the BacLight RedoxSensor Green Vitality Kit has been developed for flow cytometric analysis, it may be appropriate for other analysis platforms.
BacLight RedoxSensor CTC Vitality Kit
The Invitrogen™ BacLight™ RedoxSensor™ CTC Vitality Kit (B34956) provides effective reagents for evaluating bacterial health and vitality, and these staining reagents can withstand fixation procedures. This kit contains 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which has been used by researchers to evaluate the respiratory activity of many bacterial populations derived from environmental sources including food, soil, stone and marine and fresh water, as well as populations undergoing drug efficacy evaluations. Briefly, healthy cells respiring via the electron transport chain will absorb and reduce CTC into an insoluble, red-fluorescent formazan product. Cells not respiring or respiring at slower rates will reduce less CTC, and consequently produce less fluorescent product, giving a semiquantitative estimate of healthy versus unhealthy bacteria. SYTO 24 green-fluorescent nucleic acid stain and DAPI are provided as counterstains to assist the researcher in differentiating cells from debris and in calculating total cell numbers. Bacteria labeled with CTC and either counterstain may be evaluated immediately or after storage, with or without fixation, using a flow cytometer equipped with appropriate excitation sources (CTC in combination with SYTO 24 green-fluorescent nucleic acid stain: single 488 nm argon-ion laser; CTC in combination with DAPI: dual UV and 488 nm lasers). Bacteria stained using the BacLight RedoxSensor CTC Vitality Kit may also be viewed using most standard epifluorescence microscopes equipped with the appropriate filters; for visualizing CTC, SYTO 24 and DAPI staining, we recommend optical filters optimized for the Invitrogen™ Texas Red™ dye, FITC and DAPI, respectively.
Each BacLight RedoxSensor CTC Vitality Kit contains:
- 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)
- SYTO 24 green-fluorescent nucleic acid stain
- 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)
- Detailed protocols (BacLight RedoxSensor CTC Vitality Kit)
At the recommended reagent dilutions and volumes, this kit contains sufficient material to perform at least 50 tests by flow cytometry or microscopy.
BacLight Bacterial Membrane Potential Kit
The Invitrogen™ BacLight™ Bacterial Membrane Potential Kit (B34950) provides a fluorescent membrane-potential indicator dye, DiOC2(3), along with a proton ionophore (CCCP) and premixed buffer. At low concentrations, DiOC2(3) exhibits green fluorescence in all bacterial cells, but it becomes more concentrated in healthy cells that are maintaining a membrane potential, causing the dye to self-associate and the fluorescence emission to shift to red. The red- and green-fluorescent bacterial populations are easily distinguished using a flow cytometer. CCCP is included in the kit for use as a control because it eradicates the proton gradient, eliminating bacterial membrane potential.
Using the BacLight Bacterial Membrane Potential Kit, we have detected membrane potentials in all bacteria tested (including logarithmically growing cultures of Micrococcus luteus, Staphylococcus aureus, Bacillus cereus, Staphylococcus warnerii, Escherichia coli and Salmonella choleraesuis), although the magnitude varies with species (Figure 15.3.17). For many gram-positive species, such as M. luteus and S. aureus, the DiOC2(3) red:green ratio has been shown to vary with the intensity of the proton gradient (Figure 15.3.18). In gram-negative bacteria, such as E. coli and S. choleraesuis, a DiOC2(3) response is observed in the presence of a membrane potential but the response does not appear to be proportional to proton gradient intensity.
Each BacLight Bacterial Membrane Potential Kit contains:
- DiOC2(3) in dimethylsulfoxide (DMSO)
- CCCP in DMSO
- Phosphate-buffered saline (PBS)
- Detailed protocols (BacLight Bacterial Membrane Potential Kit)
Using the recommended reagent dilutions and volumes, this kit provides sufficient DiOC2(3) to perform approximately 100 individual assays by flow cytometry; sufficient CCCP is provided for 30 depolarized control samples. The BacLight Bacterial Membrane Potential Kit is designed to assay bacterial concentrations between 105 and 107 organisms per mL. Note that DiOC2(3) and CCCP are inhibitors of respiration, rendering the cells nonculturable beyond the brief time window required for staining and analysis.
Figure 15.3.17 Detection of membrane potential in various bacteria with the BacLight Bacterial Membrane Potential Kit (B34950). Red/green fluorescence ratios were calculated using population mean fluorescence intensities for gram-positive (Micrococcus luteus, Staphylococcus aureus, Bacillus cereus and Staphylococcus warnerii) and gram-negative (Escherichia coli and Salmonella choleraesuis) bacteria incubated with 30 µM DiOC2(3) for 30 minutes in either the presence or absence of 5 µM CCCP, according to the kit protocol.
Figure 15.3.18 Response of Staphylococcus aureus to valinomycin and external potassium ions, as measured by flow cytometry using the BacLight Bacterial Membrane Potential Kit (B34950). Samples containing S. aureus were treated with 5 µM valinomycin in different concentrations of potassium buffer, and then stained using 30 µM DiOC2(3) for 30 minutes, according to the kit protocol. Data are expressed either using a ratiometric parameter based on the formula provided in the kit protocol (triangles, right axis) or as the ratio of population mean red-fluorescence intensity/mean green-fluorescence intensity (circles, left axis).
LIVE BacLight Bacterial Gram Stain Kit
The Invitrogen™ LIVE BacLight™ Bacterial Gram Stain Kit (L7005) is based on differential nucleic acid staining of live gram-negative and gram-positive bacteria. The gram stain is one of the most important and widely used differential stains for the taxonomic classification of bacteria in both clinical and research settings. The original method involves several steps, including heat fixation of the bacteria, a two-step staining protocol, alcohol extraction and counterstaining. Over the years, several improved gram-staining techniques have been developed, but most still involve cell-fixation or cell-permeabilization steps that kill the bacteria being tested. Our single-step LIVE BacLight Bacterial Gram Stain Kits can overcome many of the problems inherent in these labor-intensive, fixation-dependent procedures.
Unlike conventional gram stain procedures, the LIVE BacLight Bacterial Gram Stain Kit allows researchers to rapidly classify bacteria as either gram-negative or gram-positive in minutes using a single staining solution, no fixatives and no wash steps. The LIVE BacLight Bacterial Gram Stain Kit contains our green-fluorescent SYTO 9 and red-fluorescent hexidium iodide nucleic acid stains. These two dyes differ in both their spectral characteristics and in their ability to label live gram-negative and gram-positive bacteria. When a mixed population of live gram-negative and gram-positive bacteria is simultaneously stained with the membrane-permeant SYTO 9 dye in combination with hexidium iodide, gram-negative bacteria fluoresce green, and the gram-positive bacteria fluoresce red-orange (, ). Dead bacteria do not exhibit predictable staining patterns. Gram-negative and gram-positive organisms can be easily differentiated in any fluorescence microscope equipped with a standard fluorescein longpass optical filter set or by flow cytometry. The assay provides a sensitive indicator system for analyzing low numbers of bacteria in the presence of background material because the unbound reagents exhibit low fluorescence when not bound to nucleic acids. The LIVE BacLight Bacterial Gram Stain Kit should be a useful tool for measuring dynamic changes in the composition of bacterial populations.
Our LIVE BacLight Bacterial Gram Stain Kit provides separate solutions of the SYTO 9 and hexidium iodide nucleic acid stains, thus facilitating calibration of bacterial fluorescence at each of the two emission wavelengths in quantitative assays. The kit is well suited for use in fluorescence microscopy, as well as for use in quantitative analysis with a fluorometer (Figure 15.3.19), fluorescence microplate reader, flow cytometer or other instrumentation. The kit includes a procedure for mounting bacteria stained with the LIVE BacLight Bacterial Gram Stain Kit on filter membranes, as well as the proprietary BacLight mounting oil that we have found to be useful for the direct epifluorescence filter technique (DEFT). The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2. Hexidium iodide (H7593), the gram-positive bacteria–selective nucleic acid stain, and the SYTO 9 nucleic acid stain (S34854) are both available separately (Viability and Cytotoxicity Assay Reagents—Section 15.2). The validity of using hexidium iodide in combination with the SYTO 13 green-fluorescent nucleic acid stain to correctly predict the gram sign of 45 clinically relevant organisms, including several known to be gram variable, has been demonstrated.
Figure 15.3.19 Analysis of the percentage of gram-negative Escherichia coli in mixed suspensions containing gram-positive Staphylococcus aureus using the reagents in the LIVE BacLight Bacterial Gram Stain Kit (L7005). Live gram-negative and gram-positive bacteria are stained fluorescent green (G) by SYTO 9 and fluorescent red (R) by hexidium iodide, respectively. Bacterial suspensions that have been incubated in the two stains simultaneously and then excited at 470 nm exhibit a fluorescence spectral shift from red to green as the percentage of gram-negative bacteria in the sample is increased.
ViaGram Red+ Bacterial Gram Stain and Viability Kit
The Invitrogen™ ViaGram™ Red+ Bacterial Gram Stain and Viability Kit (V7023) provides an easy, three-color fluorescent staining protocol that differentially stains many gram-negative and gram-positive bacterial species and, at the same time, discriminates live from dead cells based on plasma membrane integrity. This kit contains three reagents: two nucleic acid stains for viability determination—the blue-fluorescent cell-permeant DAPI and the green-fluorescent cell-impermeant SYTOX Green nucleic acid stain—as well as the red-fluorescent Texas Red-X wheat germ agglutinin (WGA) for gram sign determination. Bacteria with intact cell membranes stain fluorescent blue with DAPI, whereas bacteria with damaged membranes stain fluorescent green with the SYTOX Green nucleic acid stain. The background remains virtually nonfluorescent. The Texas Red-X WGA component selectively binds to the surface of gram-positive bacteria, providing a red-fluorescent cell-surface stain that effectively distinguishes them from gram-negative bacteria, even in the presence of the viability stains. Thus, with three fluorescent colors, the four possible combinations of live or dead, gram-negative and gram-positive cells are discriminated with a fluorescence microscope (, ). This kit also includes a procedure for mounting bacteria on filter membranes and the BacLight mounting oil, which we have found to be useful for the direct epifluorescence filter technique (DEFT). The kit components, number of assays and assay principles are summarized in Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2.
FilmTracer LIVE/DEAD Biofilm Viability Kit
The FilmTracer LIVE/DEAD Biofilm Viability Kit (L10316) provides a two-color fluorescence assay of bacterial viability based on membrane integrity, and has proven useful for a diverse array of bacterial genera, including those growing in biofilm communities. The kit utilizes a mixture of SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which differ both in their spectral characteristics and in their ability to penetrate healthy bacteria. SYTO 9 stain generally labels all bacteria in a population―those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes; SYTO 9 stain fluorescence is thereby reduced in cells where both dyes are present. When treated with an appropriate mixture of these stains, bacteria with intact cell membranes display green fluorescence while those with damaged membranes display red fluorescence.
We offer several individual fluorescent FilmTracer biofilm stains, which are described in Viability and Cytotoxicity Assay Reagents—Section 15.2):
- FilmTracer calcein red-orange biofilm stain (F10319)
- FilmTracer calcein violet biofilm stain (F10320)
- FilmTracer calcein green biofilm stain (F10322)
- FilmTracer FM 1-43 green biofilm cell stain (F10317)
- FilmTracer SYPRO Ruby biofilm matrix stain (F10318)
Wheat Germ Agglutinin Sampler Kit
Fluorescent lectins have proven useful in microbiology applications. Fluorescent wheat germ agglutinin (WGA) conjugates selectively stain chitin in fungal cell walls, as well as the surface of gram-positive but not of gram-negative bacteria. Fluorescent WGA has also been shown to bind to sheathed microfilariae and has been used to detect filarial infection in blood smears. Our Wheat Germ Agglutinin Sampler Kit (W7024) provides 1 mg samples of four of our brightest fluorescent WGA conjugates, spanning the spectrum from blue to red. Included in this kit are conjugates of the blue-fluorescent Invitrogen™ Alexa Fluor™ 350, green-fluorescent Oregon Green™ 488, orange-fluorescent tetramethylrhodamine and red-fluorescent Texas Red-X dyes (Molecular Probes lectin conjugates—Table 7.10). See Lectins and Other Carbohydrate-Binding Proteins—Section 7.7 for more information on lectins, including additional WGA and concanavalin A (Con A) conjugates.
The Invitrogen™ EnzChek™ Reverse Transcriptase Assay Kit (E22064) is designed to provide a convenient and efficient method for measuring reverse transcriptase activity. This kit has been used to determine HIV-1 reverse transciptase heterodimer activity, to measure SARS-CoV viral titers in 293T host cultures and to quantitate reverse transciptase activity in Friend virus (FV) culture supernatants.
The key to this assay is our PicoGreen dsDNA quantitation reagent, which preferentially detects dsDNA or RNA–DNA heteroduplexes over single-stranded nucleic acids or free nucleotides. In the assay, the sample to be measured is added to a mixture of a long poly(A) template, an oligo(dT) primer and dTTP. Reverse transcriptase activity in the sample results in the formation of long RNA–DNA heteroduplexes, which are detected by the Invitrogen™ PicoGreen™ reagent at the end of the assay. In less than an hour, samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluorescein (FITC). The assay is sensitive, detecting as little as 0.02 units of HIV reverse transcriptase and has about a 50-fold linear range.
The EnzChek Reverse Transcriptase Assay Kit contains sufficient reagents for approximately 1000 assays using a fluorescence microplate reader:
- PicoGreen dsDNA quantitation reagent
- Bacteriophage lambda DNA standard
- Poly(A) ribonucleotide template
- Oligo dT16 primer
- TE buffer, polymerization buffer and an EDTA solution
- Detailed protocols (EnzChek Reverse Transcriptase Assay Kit)
The luciferin–luciferase bioluminescence assay is extremely sensitive; most luminometers can detect as little as 1 picomole of pre-existing ATP or ATP as it is generated in kinetic systems. This sensitivity has led to its widespread use for detecting ATP in various enzymatic reactions, as well as for measuring viable cell number, for monitoring ATP release from cells and for detecting low-level bacterial contamination in samples such as blood, milk, urine, soil and sludge. The luciferin–luciferase bioluminescence assay has also been used to determine cell proliferation and cytotoxicity in both bacterial and mammalian cells, and to distinguish cytostatic versus cytocidal potential of anticancer drugs on malignant cell growth.
We offer a convenient ATP Determination Kit (A22066) for the sensitive bioluminescence-based detection of ATP with recombinant firefly luciferase and its substrate luciferin. This assay is based on luciferase's absolute requirement for ATP to produce light. In the presence of Mg2+, luciferase catalyzes the reaction of luciferin, ATP and O2 to form oxyluciferin, AMP, CO2, pyrophosphate and ~560 nm light (Figure 15.3.20).
The ATP Determination Kit (A22066) contains:
- Luciferin (5 × 3 mg)
- Dithiothreitol (DTT)
- Concentrated reaction buffer
- Detailed protocols for ATP quantitation (ATP Determination Kit)
Unlike most other commercially available ATP detection kits, our ATP Determination Kit provides the luciferase and luciferin packaged separately, which enables researchers to optimize the reaction conditions for their particular instruments and samples. The ATP Determination Kit provides sufficient reagents to perform 200 ATP assays using 0.5 mL sample volumes or 500 ATP assays using 0.2 mL sample volumes.
For Research Use Only. Not for use in diagnostic procedures.