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Cat. No.: BVAK-200 (for 200T)

Cat. No.: BVAK-1k (for 1000T)

Description

Bacterial Viability Assay Kit or Bacterial Cytotoxicity Assay Kit is an efficient, convenient, and sensitive dual-fluorescence staining method for detecting bacterial viability based on the green fluorescent DNA dye DMAO and the red fluorescent dye propidium iodide (PI). When using this assay kit, live bacteria exhibit green fluorescence, while dead bacteria exhibit both green and red fluorescence. The kit can be used with fluorescence microscopy, laser confocal microscopy, fluorescence plate readers, flow cytometers, and other fluorescence detection systems.

DMAO, or N,N-dimethylaniline N-oxide, is a green fluorescent nucleic acid dye suitable for both Gram-positive and Gram-negative bacteria. It stains both live and dead bacteria and has membrane permeability, allowing it to penetrate cell membranes and preferentially bind to double-stranded DNA. DMAO is highly stable and resistant to degradation during routine handling and storage at room temperature. PI is a non-permeable fluorescent dye that cannot penetrate biologically active cytoplasmic membranes, so it cannot stain bacteria with intact cell membranes. However, in necrotic bacteria, the integrity of the cell membrane is lost, allowing PI to enter the cell nucleus, bind to double-stranded DNA, and embed into the bacterial DNA double helix to form PI-DNA complexes, resulting in red fluorescence. This binding occurs with little or no sequence preference, with approximately one molecule of PI dye inserted every 4-5 base pairs. By using DMAO and PI together, the assay detects bacterial viability, with live bacteria with intact cell membranes exhibiting green fluorescence, while dead bacteria with damaged membranes exhibit both green and red fluorescence.

The maximum excitation wavelength of the DMAO-DNA complex is 503nm, with a maximum emission wavelength of 530nm, while the maximum excitation wavelength of the PI-DNA complex is 535nm, with a maximum emission wavelength of 617nm.

The DMAO and PI provided in this assay kit are both stored as 1000X stock solutions. The solutions have been optimized for most bacteria, but for optimal results with different types of bacteria, some experimentation with concentrations may be necessary. The final concentrations of DMAO and PI typically range from 0.5-2X, with a recommended concentration of 1X for best results. Additionally, the kit provides a detection buffer that can be used to prepare the staining working solution for bacterial viability assays.

Composition

• DMAO (1000X)
• PI (1000X)
• Detection Buffer

Storage

Store at -20°C for up to one year. Both DMAO (1000X) and PI (1000X) should be protected from light.

Notes

• Before bacterial staining, remove the culture medium, as components in the medium can bind to DMAO and PI dyes, reducing staining efficiency.
• Fluorescent dyes are susceptible to quenching; therefore, avoid exposure to light as much as possible to minimize fluorescence quenching.
• For initial use, it is recommended to aliquot DMAO (1000X) and PI (1000X) and store them at -20°C to avoid repeated freeze-thaw cycles.
• The detection buffer has been filtered to remove bacteria. Be careful to avoid microbial contamination during use, as it can significantly affect staining results. If the detection buffer becomes cloudy or shows obvious microbial contamination, it should not be used.
• When using a fluorescence plate reader, use a suitable black or white plate for fluorescence detection. 
• This product is for scientific research by professionals only and is not intended for clinical diagnosis or treatment, food or drug applications, or storage in residential areas.
• For your safety and health, wear laboratory attire and disposable gloves during handling.

Only for research and not intended for treatment of humans or animals

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