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MICROBIOLOGY STAINS & KITS

General bacterial stains, fluorescent gram stains, and viability dyes
General yeast stains, yeast viability dyes and kits, and organelle stains
PMA & PMAxx™ for fast, quantitative microbial viability by PCR

At Biotium, our specialty is creating fluorescent dyes that simplify and improve your scientific experiments. For Microbiology, we offer a wide range of fluorescent dyes and assays kits to stain bacteria, yeast and fungi, including dyes to assess cell viability and bacterial gram stain type. We have also developed a complete toolkit of reagents for viability PCR, a revolutionary method for determining microbial viability using qPCR.

Also visit our Cellular Stains Table for a comprehensive list of cellular stains with their ability to stain various cell types.


BACTERIAL STAINS

For bacteria, we offer fluorescent dyes to stain live cells, dead cells, and gram+ cells. Combining these dyes in multi-color microscopy or flow cytometry experiments allows several parameters to be assessed at one time.

Bacterial Live Cell Stains

Often it can be useful to visualize or detect all bacteria present in a sample, regardless of species or metabolic state. For this purpose, we offer a selection of dyes that generally stain all bacteria, both gram-positive and gram-negative. The dyes are available in various colors and localizations:

  • BactoView™ Live, RedDot™1, DMAO, Thiazole Orange, and Hoechst are cell membrane permeant nucleic acid binding dyes.
  • SynaptoGreen™, SynaptoRed™, and CellBrite™ Fix are all cell membrane/cell periphery stains. CellBrite™ Fix dyes are a set of fixable membrane dyes available in multiple colors.

Live Cell Bacterial Stains

Features

BactoView™ Live Fluorescent Bacterial Stains• Nucleic acid binding dyes
• Green and red options
• Useful for fluorescence microscopy or flow cytometry
RedDot™1 Far-Red DNA Dye• Nucleic acid binding dye
• Far-red fluorescence
• Useful for fluorescence microscopy
DMAO, 2 mM in DMSO• Green nucleic acid binding dye
• Useful for fluorescence microscopy or flow cytometry
Hoechst 33342, 10 mg/mL in H2O• Blue nucleic acid binding dye
• Useful for fluorescence microscopy or flow cytometry
Thiazole Orange• Nucleic acid binding dye
• Green fluorescence
• Useful for fluorescence microscopy or flow cytometry
CellBrite™ Fix Membrane Stains• Fixable membrane dyes
• Green, red and far-red options
• Useful for fluorescence microscopy
SynaptoGreen™ C4• Red fluorescent membrane dye
• Useful for fluorescence microscopy
SynaptoRed™ C2• Green fluorescent membrane dye
• Useful for fluorescence microscopy
E. coli stained with BactoView™ Live Green (stains DNA) and SynaptoRed™ (stains the membrane)
Bacillus subtilis stained with CellBrite™ Fix 555, fixed in paraformaldehyde, and imaged the next day.

Fluorescent Gram Stains

A traditional colorimetric gram staining protocol is laborious, requiring up to 5 different solutions to stain cells, including toxic alcohol and acetone to decolorize the sample, and precise timing. In contrast, Wheat Germ Agglutinin (WGA) has been shown to bind specifically to N-acetylglucosamine in the peptidoglycan layer of the cell wall of gram-positive bacteria, and can be labeled with a number of different fluorescent dyes for one-step fluorescent cell labeling. We offer fluorescent WGA conjugates in 13 color options, as well as combination gram stain/viability kits and DNA dyes.

Product NameFeatures
Wheat Germ Agglutinin (WGA) CF® Dye conjugates•   Fast and sensitive gram stains
•   Available in 13 colors
Live Bacterial Gram Stain Kit•   CF®594 WGA stains gram-positive cells red
•   DAPI stains all cells blue
Bacterial Viability and Gram Stain Kit•   CF®488A WGA stains gram-positive cells green
•   EthD-III stains dead cells red
•   DAPI stains all cells blue

 

Microbiology Stain - E. coli and Salmonella co-culture stained with DAPI and CF633-WGA
A co-culture of E. coli and Staphylococcus stained with DAPI (blue) and CF®633 WGA (red). Spherically-shaped gram-positive Staphylococcus  stain with WGA, while rod-shaped gram-negative E. coli do not stain. DAPI stains all cells blue.
E. coli and Staphylococcus stained with CF594-WGA and DAPI
Co-culture of E. coli and Staphylococcus stained with CF®594 WGA  and DAPI. Gram-positive Staphylococcus show blue DAPI staining and red cell surface staining with WGA, while gram-negative E. coli show only DAPI staining.
Heat-killed Pseudomonas and Micrococcus stained with the Bacterial Viability and Gram Stain Kit
Heat-killed Pseudomonas and Micrococcus stained with the Bacterial Viability and Gram Stain Kit. CF®488A WGA stains gram-positive Micrococcus green, EthDIII stains dead cells red, and DAPI stains all cells blue.

Bacterial Viability Stains

Biotium offers a selection of different fluorescent viability dyes and kits, to allow for the differentiation of live and dead cells by microscopy or flow cytometry (see the Viability PCR section below for detection of viable bacteria by qPCR).

Product NameFeatures
Live-or-Dye™ Fixable Viability Staining Kits•   Fixable dead cell stains
•   Available in 8 fluorescent colors
•   Useful for flow cytometry or microscopy
Live-or-Dye NucFix™ Red Staining Kit •   Fixable, DNA-binding dead cell stain
•   Works in gram-negative bacteria only
•   Stains dead cells red
RedDot™2 Far-Red DNA Dye•   Nuclear-specific dead cell stain
•   Stains dead cells with far-red fluorescence
Ethidium Homodimer III (EthD-III)•   Nuclear-specific dead cell stain
•   Stains dead cells red
Viability/Cytotoxicity Assay Kit •   Green fluorescent DMAO stains DNA in all bacteria
•   Ethidium Homodimer III (EthD-III) stains dead cells red
Bacterial Viability and Gram Stain Kit•   CF®488A WGA stains gram-positive cells green
•   EthDIII stains dead cells red
•   DAPI stains alll cells blue
5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) •   Substrate for measuring bacterial respiratory activity
•   Healthy cells reduce CTC into an insoluble red fluorescent formazan product

 

A mixture of live and dead E. coli stained with Live-or-Dye™ 568/583 and DAPI. Live-or-Dye™ 568/583 stains dead cells red, and DAPI stains all cells blue
A mixture of live and dead E. coli stained with the Viability/Cytotoxicity Kit. DMAO stains all cells green and EthD-III stains dead cells red.

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YEAST STAINS AND KITS

Fluorescent dyes and kits to stain yeast cells, including viability dyes, combination staining kits, and organelle stains. Combining these dyes in multi-color microscopy or flow cytometry experiments allows maximum experimental versatility.

Yeast Viability Stains

Biotium offers a selection of different fluorescent viability dyes and kits, to allow for the differentiation of live and dead yeast cells by microscopy or flow cytometry (see the Viability PCR section below for detection of viable yeast by qPCR).

Product NameFeatures
Live-or-Dye™ Fixable Viability Staining Kits•   Fixable dead cell stains
•   Choice of 8 fluorescent colors
Propidium Iodide•   Red dead cell stain
•   Not fixable
Ethidium Homodimer III (EthD-III)•   Far-red dead cell stain
•   Not fixable
RedDot™2 Far-Red Nuclear Stain•   Far-red, nuclear dead cell stain
•   Not fixable
NucSpot® 470•   Green, nuclear dead cell stain
•   Not fixable
ViaVac™ Red/Green•   Yeast-specific vitality dye
•   stains the vacuole red in metabolically active cells
Yeast Live-or-Dye™ Fixable Live/Dead Staining Kit•   Thiazole Orange stains all cells green
•   Live-or-Dye™ 568/583 stains dead cells red
•   Fixable staining
Yeast Viability Staining Kit •   CF® Dye-labeled Concanavalin A (ConA) to label the cell walls of all cells
•   Live-or-Dye™ viability dye to label dead cells
•   Choose blue/green, green/red, or red/far-red color combinations
Yeast Vitality Staining Kit •   Calcofluor White labels all cell walls blue
•   ViaVac™ Red/Green stains the vacuole red in metabolically active cells

 

S. cereviciae stained with the Yeast Vitality Staining Kit: Calcofluor White stains all cell walls blue and ViaVac™ stains the vacuoles of metabolically active cells red and the cytoplasm of all cells green (not shown).
Yeast Live-or-Dye Fixable Live/Dead Staining Kit
S. cerevisiae stained with the Yeast Live-or-Dye™ Fixable Live/Dead Staining Kit: Thiazole Orange to stain all cells (green) and Live-or-Dye™ 568/583 to stain dead cells (red, appears yellow in overlay).
Yeast Viability Staining Kit
S. cerevisiae stained with the Yeast Viability Staining Kit: CF®640R ConA stains cell walls of all cells with far-red fluorescence (pseudocolored blue in image), while Live-or-Dye™ 568/583 stains dead cells red.

Organelle Stains for Yeast

A number of our organelle and cellular stains can be used to stain yeast cells. We also offer stains for the fungal cell wall and bud scars of budding yeast.

Product NameFeatures
MitoView™ Dyes •   Mitochondrial dyes in five colors:
•   MitoView™ 633 is mitochondrial membrane potential-dependent
LysoView™ 540 & 633•   Label vacuoles in live yeast cells
•   2 LysoView dyes stain the yeast vacuole LysoView™ 540 & LysoView™ 633
LipidSpot™ 488•   Label vacuoles in live yeast cells
•   Stains intracellular membrane structures
Calcofluor White•   Blue fluorescent chitin stain stains cell walls
•   Strongly stains bud scars
CellBrite™ Fix•   Stains the cell membrane
•   Available in 3 dye colors
•   Staining is fixable
MemBrite™ Fix•   Stains the cell periphery
•   Available in 8 dye colors
•   Staining is fixable
CF® Dye Concanavalin A (Con A) Conjugates•   Con A lectin binds to a-mannopyranosyl and a-glucopyranosy in the cell wall of yeast and fungi
•   Available in 10 colors
CF® Dye Wheat Germ Agglutinin (WGA) Conjugates•   WGA lectin binds sialic acid and N-acetylglucosamine
•   Strongly labels bud scars
•   Available in 13 colors
Thiazole Orange•   Green nucleic acid binding dye
•   Can be used as an all-cell yeast stain
•   Staining in yeast is nuclear in unfixed cells and cytoplasmic after fixation
NucSpot® 470 Nuclear Stain•   Green nucleic acid binding dye
•   Stains the nucleus of dead or fixed and permeabilized yeast
ViaFluor® CFSE •   Can be used as an all-cell cytoplasmic stain in yeast
Phalloidin conjugates •   Phalloidin stains F-actin cables and patches in fixed and permeabilized yeast
•   Available in 20 colors and biotin

 

S. cerevisiae stained with CF®488A WGA (green) to stain bud scars and CF®594 ConA (red) to stain the cell wall.
S. cerevisiae stained with LysoView™ 633 to visualize the vacuoles. Imaged on a Zeiss confocal microscope.
S. cereviciae stained separately with MemBrite™ Fix 488/515, 568/580, and 640/660. Combined and imaged.

Mitochondrial staining of S. cerevisiae with MitoView™ 633. Imaged on a Zeiss confocal microscope.
Fixed and permeabilized S. cerevisiae stained with CF®568-conjugated phalloidin
Heat-killed S. cerevisiae stained with NucSpot®470 Nuclear Stain.

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VIABILITY PCR WITH PMA & PMAXX™


Viability PCR (v-PCR)

Viability PCR is a powerful technology for the sensitive and rapid detection of viable microorganisms. Unlike time-consuming culturing methods, qPCR is a fast and sensitive method of detection. However, normal PCR does not distinguish between live and dead cells. With v-PCR using PMAxx™ or PMA, you get the speed, sensitivity and specificity of PCR, plus quantifiable viability. And because no culturing is required, you can even detect viable but not culturable (VBNC) bacteria.The v-PCR technology can be applied not only to bacteria but to other organisms like yeast and viruses.

How does v-PCR work?

PMAxx™ and PMA are photoreactive dyes with high affinity for DNA. The dyes intercalate into dsDNA and form a covalent linkage upon exposure to intense visible light. PMAxx™ and PMA inhibit PCR amplification of modified DNA templates by a combination of removal of modified DNA during purification and inhibition of template amplification by DNA polymerases. Because PMAxx™ and PMA are cell membrane-impermeable, when a sample containing both live and dead bacteria is treated with dye, only dead bacteria with compromised cell membranes are susceptible to DNA modification. In a real-time PCR reaction, dead cell DNA will show delayed amplification and higher Ct than live cells. In a mixed population, v-PCR permits quantitation of cell viability.

Principal of viability PCR with PMA or PMAxx™ dyes.The membrane impermeable dyes selectively enter dead cells and modify their DNA. Modified DNA is not amplified, allowing quantitation of viable cell DNA.
Example of viability qPCR of untreated or heat-killed <em>E. coli</em>with PMA dye. PMA treatment eliminates dead cell DNA, resulting in a large delay in Ct compared to live or untreated cells. NTC: no template control.

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PMA & PMAxx™ Dyes for Viability PCR

PMA is the original dye developed at Biotium for v-PCR. It has been validated in a wide variety of bacterial strains, as well as yeast and viruses (see PMA References). However, while PMA is generally effective at differentiating between live and dead bacteria by qPCR, it does not completely eliminate PCR products from dead cell DNA. This could potentially give false positive results.

PMAxx™ was designed by Biotium scientists to be a superior alternative to PMA. PMAxx™ is much more effective at eliminating PCR amplification of dead cell DNA, and therefore provides the best discrimination between live and dead bacteria.

Download the PMA™ and PMAxx™ flyer to learn more.

Live or dead bacteria treated with either PMA or PMAxx™ prior to qPCR. Dead bacteria treated with either PMA or PMAxx™ show delays in amplification compared to either live cells, or untreated dead cells. Bacteria treated with PMAxx™ show a larger delay than those treated with PMA. Therefore PMAxx™ is better able to differentiate live vs dead bacteria by viability PCR.

PMA & PMAxx™ for Viability PCR
Product NameCatalog No.Features
PMAxx™, 20 mM in H2O40069•    Next-generation vPCR dye
•    Gives the best live/dead discrimination
PMA Dye40013•    Original vPCR dye developed at Biotium
•    Validated in hundreds of publications and a wide variety of organisms
PMA Dye, 20 mM in H2O40019

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PMA Enhancer for Gram-Negative Bacteria

PMA Enhancer for Gram Negative Bacteria was designed to improve discrimination between live and dead gram-negative bacteria during viability PCR. PMA Enhancer is provided as a 5X solution, and is added to a sample before the addition of viability dye. When a sequence from a gram-negative bacteria is amplified by PCR, samples pre-treated with Enhancer show a decrease in the signal from dead cells, with no change in the signal from live cells. PMA Enhancer is compatible with PMAxx™ as well as PMA. PMAxx™ plus Enhancer is the optimal way to perform viability PCR on gram-negative bacteria. PMA Enhancer is available as a stand-alone product, and is also a component of our PMA Bacterial Viability Kits (gram-negative strains only) (see below).

E. coli were killed with mild heat treatment (56°C for 3 hrs) and treated with PMAxx™ or PMAxx™ + Enhancer, followed by light exposure using PMA-Lite™, DNA purification, and qPCR with Fast EvaGreen® qPCR Master Mix. dCt values were calculated by subtracting the Ct without dye from the Ct with dye. Only dead cells treated with PMAxx™ + Enhancer showed a large dCt, indicating that the dye successfully inhibited PCR of dead cell DNA.

PMA Enhancer for Gram-Negative Bacteria

Product NameCatalog No.Features
PMA Enhancer for Gram Negative Bacteria, 5X Solution31038•    Improves live/dead discrimination in gram-negative strains

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Photoactivation Devices

PMA-Lite™

The PMA-Lite™ LED photolysis device allows for time- and temperature-controlled light treatment of PMAxx- or PMA-treated samples in microcentrifuge tubes.

PMA-Lite™ Features:

  • Provides even illumination to up to 18 microcentrifuge tubes
  • Internal fan keeps temperature < 37°C
  • Four timer settings for 10, 15, 20 or 30 minutes of light exposure
  • Durable LED lights with 465-475 nm emission
  • For efficient activation of PMAxx™, PMA, EMA, or similar dyes

Glo-Plate™ Blue

The Glo-Plate™ Blue LED Illuminator is designed for even light treatment of PMAxx™- or PMA-treated samples in plates or larger tubes.

Glo-Plate™ Blue Features:

  • Provides even illumination across a flat surface, ideal for multi-well plates
  • Four timer settings for 10, 15, 20 or 30 minutes of light exposure
  • Durable LED lights with blue emission
  • Light exposure surface stays cool
  • For use with of PMAxx™, PMA, or Biotium’s DNAzure® Visible Gel Stain

PMA-Lite LED Photolysis Device
PMA-Lite™ LED photolysis device
Glo-Plate™ Blue LED Illuminator

Photoactivation Devices

Product NameCatalog No.Features
PMA-Lite™ LED Photolysis DeviceE90002•    Photoactivation device, holds 16 microcentrifuge tubes
Glo-Plate™ Blue LED IlluminatorE90004•    Photoactivation device for plates or larger tubes

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Strain-Specific PCR Bacterial Viability Kits

Strain-specific v-PCR kits are designed for selective detection of viable bacteria from a specific strain using PMAxx or PMA dye and real-time PCR. The kits contain PMA dye OR PMAxx™ dye, Forget-Me-Not™ EvaGreen® qPCR Master Mix, and validated PCR primers for detection of selected strains of bacteria that are of widespread interest to food safety, public health, and/or antibacterial research.

Kits include:
• PMA dye OR PMAxx™ dye
• Forget-Me-Not™ EvaGreen® qPCR Master Mix
• ROX reference dye
• PMA Enhancer (gram-negative strains only)
• Validated PCR primers for specific bacterial strain

One kit can be used to treat 80 bacterial cultures with PMA or PMAxx™ and perform 200 PCR reactions.

Strain-Specific Bacterial Viability PCR Kits

Bacteria StrainGene nameKit with PMA (catalog #)Kit with PMAxx™ (catalog #)
Salmonella entericainvA3103331033-X
Mycobacterium tuberculosisgroEL231034N/A
Staphylococcus aureusnuc31035N/A
MRSAmecA31036N/A
E. coli O157:H7Z32763103731037-X
E. coliuidA3105031050-X
Listeria monocytogeneshly3105131051-X
Legionella pneumophilamip31053N/A

 

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Bacterial Viability and Gram Stain Kit

From: $216 Sizes: 200 assaysCatalog #: 32001

Concanavalin A (Con A) CF® Dye Conjugates

From: $148 Sizes: 5 x 1 mgCatalog #: 29015, 29016, 29017, 29018, 29019, 29020, 29058, 29074, 29075, - 29080View allHide

PMAxx™ dye

From: $131 Sizes: 100 uLCatalog #: 40069

Viability/Cytotoxicity Assay kit for Bacteria Live and Dead Cells

From: $284 Sizes: 100-1000 assaysCatalog #: 30027

Yeast Live-or-Dye™ Fixable Live/Dead Staining Kit

From: $361 Sizes: 1,000 assaysCatalog #: 31064

Yeast Vitality Staining Kit

From: $216 Sizes: 1,000 assaysCatalog #: 31062

Wheat Germ Agglutinin (WGA) Conjugates

From: $68 Sizes: 1 mg, 5 x 1 mgCatalog #: 29021, 29022, 29023, 29024, 29025, 29026, 29027, 29028, 29029, 29059, 29064, 29073, 29021-1, 29022-1, 29023-1, 29024-1, 29025-1, 29026-1, 29027-1, 29028-1, 29029-1, 29059-1, 29064-1, 29077-1, 29077, 29076-1, - 29076View allHide

Live Bacterial Gram Stain Kit

From: $130 Sizes: 200 assays, 800 assaysCatalog #: 32000, - 32000-1View allHide