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Molecular Probes™ LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry

Catalog No. 361054994
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361054994 100 reactions kit
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Catalog No. 361054994 Supplier Molecular Probes™ Supplier No. L34856
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Description

The LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit 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.

The LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit 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 uses a mixture of two nucleic acid stains - green-fluorescent SYTO™ 9 dye and red-fluorescent propidium iodide - for viability determinations, and a calibrated suspension of microspheres for accurate sample volume measurements.

Any physiological buffer between pH 7.0–8.0, including PBS, can be used to dilute the SYTO dyes for the staining solution.

Specifications

Content And Storage Contains 1 vial SYTO™ 9 nucleic acid stain (200 μL, 3.34 mM in DMSO), 1 vial of propidium iodide (200 μL, 20 mM in DMSO), and 1 vial of a microsphere standard (1.0 mL of 6.0 μm diameter microspheres).

Store in refrigerator (2–8°C) and protect from light.
Detection Method Fluorescence
Format Tube
Type Bacterial Viability and Counting Kit
Label or Dye SYTO™ 9, propidium iodide
Product Line BacLight, LIVE/DEAD
Quantity 100 reactions kit
Shipping Condition Room Temperature
Wavelength SYTO™ 9: 485/498, PI: 535/617

Frequently Asked Questions (FAQs)

When using the LIVE/DEAD BacLight kit for bacterial viability, with SYTO 9 and propidium iodide (PI), why are some cells detected with both dyes? Shouldn't live cells be green and dead cells be red?

SYTO 9 dye will enter all cells, live or dead. PI only enters cells with compromised membranes (dead cells or damaged cells). First, perform single color staining and examine under both filter sets. Longpass filters or the use of too much dye may result in excessive bleedthrough of the green dye emission into the red channel and the emission of PI in the green channel. Use narrower bandpass filters if possible, and use lower concentrations of the dye. Some live cells may take up PI by engulfment; avoid extended incubation times with the dye. Apply PI to a live cell culture and optimize incubation times to limit engulfment.

I want to use propidium iodide and SYTO 9 to do LIVE/DEAD testing of a bacterial sample. Will anaerobic conditions adversely affect the assay?

Oxygen content should not affect the binding of propidium iodide and SYTO 9 to nucleic acids. SYTO 9 will label all cells, and propidium iodide will label only dead cells or cells with a compromised membrane.
I have a LIVE/DEAD BacLight Bacterial Viability kit that has SYTO 9 and propidium iodide in it. Will I be able to stain eukaryotic cells that have engulfed bacteria and determine if the bacteria are alive or dead using this kit?

Unfortunately, no. SYTO 9 will label the nuclei of live or dead cells, including the eukaryotic cells. Propidium iodide is cell impermeant, and will only enter dead cells. If the eukaryotic cells are dead, they will label with propidium iodide as well. If the eukaryotic cells are alive, propidium iodide will not be able to enter and thus will not label the bacteria inside, whether the bacteria are alive or dead. We are not aware of any way to do a viability assay of bacteria once they have been engulfed by cells.
When using the Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry, some cells seem to have both red and green signal. Are these cells dead or alive or dying?

The green dye in the kit will label all the cells as it is a cell-permeant nucleic acid stain. The red dye is not cell permeant, and will only stain the cells with compromised membranes (dead cells). Therefore, any cells with red signal will be considered dead. It is possible that you will have some cells that are only red, some that are red and green, and some that are only green. Sometimes the red dye will displace the green dye. In any case, any red cells are dead.

Also, the green dye emission may bleed through into the red channel. Do a single-color staining and examine under both green and red filter sets to determine the level of bleedthrough. To avoid this bleedthrough, use a lower concentration of dye, and, if possible, use narrow bandpass filters.
How can I count my bacteria by flow cytometry?

There are several options. We have two fluorescence based kits that are useful for bacterial counting: Live/Dead BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856) and Bacteria Counting kit, for flow cytometry (Cat. No. B7277). Another option is the Flow Cytometry Sub-micron Particle Size Reference Kit (Cat. No. F13839).
I am having a hard time separating my bacteria from instrument noise. How can I optimize my signal?

Electronic noise is due to stray light collected by the system optics and/or low-level electronic signals. Noise may be eliminated or reduced through correct setup of instrument threshold and photomultiplier tube (PMT) voltage. The easiest way to differentiate a bacterial population from other small particles and electronic noise is to label the bacteria with a fluorescent stain and use the fluorescence emission to identify the population of interest. SYTO 9 is a great stain to label all cells and is detected in the FITC channel.
What bacterial parameters can I look at by flow cytometry?

You can stain bacteria with a general stain such as BacLight Green Bacterial Stain (Cat. No. B35000) or BacLight Red Bacterial Stain (Cat. No. B35001). You can look at gram character (Cat. No. L7005), cell viability (Cat. Nos. L7007, L7012, and L13152), cell count (Cat. Nos. L34856 and B7277), and cell vitality. Cell vitality can be measured by membrane potential (Cat. No. B34950) or by metabolism (Cat. Nos. B34954 and B34956).
Can I use a bacterial stain such as the LIVE/DEAD BacLight Bacterial Viability and Counting Kit (Cat. No. L34856) to detect bacteria in my human cell sample?

This will not work well because the dyes in this kit will also stain mammalian cells. You can probably distinguish cells based on their scatter properties, but the stain will not confirm your selection.
Can I look at bacteria and yeast on a flow cytometer?

Yes, you can. We offer:

-Counting assays: Bacteria Counting Kit, for flow cytometry (Cat. No. B7277) or LIVE/DEAD BacLight Bacterial Viability and Counting Kit, for flow cytometry (Cat. No. L34856).
-Viability/vitality assays: LIVE/DEAD BacLight Bacterial Viability Kit (Cat. No. L13152).
-Membrane potential: BacLight Bacterial Membrane Potential Kit (Cat. No. B34950).
-Yeast viability/vitality assays: LIVE/DEAD FungaLight Yeast Viability Kit, for flow cytometry (Cat. No. L34952), FungaLight Yeast CFDA, AM/Propidium Iodide Vitality Kit (Cat. No. F34953)

For Research Use Only. Not for use in diagnostic procedures.