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MICROBIOLOGY STAINS & KITS

Fluorescent gram stain and viability kits
Yeast viability and organelle-specific stains
PMA & PMAxx™ dyes and kits for v-PCR

BACTERIAL STAINS

At Biotium, our specialty is creating fluorescent dyes that simplify and improve your scientific experiments. 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.

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
•   EthDIII stains dead cells red
•   DAPI stains alll cells blue
Fluorescent nucleic acid dyes•   Wide selection of nucleic acid dyes that stain bacteria
•   Green fluorescent DMAO, Acridine Orange, Hoechst, and others

 

Microbiology Stain - E. coli and Salmonella co-culture stained with DAPI and CF633-WGA
A co-culture of E. coli and Salmonella stained with DAPI (blue) and CF®633 WGA (red). Spherically-shaped gram-positive Salmonella  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 stains gram-positive Pseudomonas 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
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 absorb and 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
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 stains can be used in live yeast. We also offer stains for the fungal cell wall and bud scars of budding yeast.

Product NameFeatures
Live-or-Dye™ Fixable Viability Staining Kits•   Fixable dead cell stains
•   Choice of 8 fluorescent colors
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. cerevisiae stained with CF®488A WGA (green) and CF®594 ConA (red).
Vacuoles of S. cerevisiae stained with LysoView™ 633.
 
Mitochondrial staining of S. cerevisiae with MitoView™ 633.
 

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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.

Product NameSKUBacteria StrainGene NamePurchase
PMA Real-Time PCR Bacterial Viability Kit – E. coli (uidA)31050E. coliuidAPurchase 31050
PMAxx™ Real-Time PCR Bacterial Viability Kit – E. coli (uidA)31050-XPurchase 31050-X
PMA Real-Time PCR Bacterial Viability Kit – E. coli O157:H7 (Z3276)31037E. coli O157:H7Z3276Purchase 31037
PMAxx™ Real-Time PCR Bacterial Viability Kit – E. coli O157:H7 (Z3276)31037-XPurchase 31037-X
PMA Real-Time PCR Bacterial Viability Kit – Listeria monocytogenes (hly)31051L. monocytogeneshlyPurchase 31051
PMAxx™ Real-Time PCR Bacterial Viability Kit – Listeria monocytogenes (hly)31051-XPurchase 31051-X
PMA Real-Time PCR Bacterial Viability Kit – Salmonella enterica (invA)31033S. entericainvAPurchase 31033
PMAxx™ Real-Time PCR Bacterial Viability Kit – Salmonella enterica (invA)31033-XPurchase 31033-X
PMA Real-Time PCR Bacterial Viability Kit – Legionella pneumophila (mip)31053L. pneumophilamipPurchase 31053
PMA Real-Time PCR Bacterial Viability Kit – Mycobacterium tuberculosis (groEL2)31034M. tuberculosisgroEL2Purchase 31034
PMA Real-Time PCR Bacterial Viability Kit – Staphylococcus aureus (mecA)31036S. aureusmecAPurchase 31036
PMA Real-Time PCR Bacterial Viability Kit – Staphylococcus aureus (nuc)31035S. aureusnucPurchase 31035

 

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Bacterial Viability and Gram Stain Kit

From: $210 Sizes: 200 assaysCatalog #: 32001

Concanavalin A (Con A) CF Dye Conjugates

From: $137 Sizes: 5 x 1 mgCatalog #: 29015, 29075, 29074, 29058, 29020, 29019, 29018, 29017, 29016, - 29080View allHide

PMAxx, 20 mM in H2O

From: $121 Sizes: 100 uLCatalog #: 40069

Viability/Cytotoxicity Assay kit for Bacteria Live and Dead Cells

From: $276 Sizes: 100-1000 assaysCatalog #: 30027

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

From: $350 Sizes: 1,000 assaysCatalog #: 31064

Yeast Vitality Staining Kit

From: $210 Sizes: 1,000 assaysCatalog #: 31062

Wheat Germ Agglutinin (WGA) Conjugates

From: $63 Sizes: 1 mg, 5 x 1 mgCatalog #: 29021, 29024-1, 29025-1, 29026-1, 29027-1, 29028-1, 29029-1, 29059-1, 29064-1, 29077-1, 29077, 29076-1, 29023-1, 29022-1, 29021-1, 29022, 29023, 29024, 29025, 29026, 29027, 29028, 29029, 29059, 29064, 29073, - 29076View allHide

Live Bacterial Gram Stain Kit

From: $126 Sizes: 200 assays, 800 assaysCatalog #: 32000, - 32000-1View allHide