With the CountBright Plus Absolute Counting Beads/CountBright Absolute Counting Beads, instead of a tight cluster of beads on the scatter plot, I am seeing the beads cover a broad area. What is causing this?
Prior to taking an aliquot, make certain to thoroughly mix the beads. This may also be caused if the beads were ever frozen or the stock solution has any microbial contamination.
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Which buffers can I use with the CountBright Plus Absolute Counting Beads and CountBright Absolute Counting Beads?
You may use any physiological buffer or media in a pH range from pH 4 to 10. Avoid extremes of pH and high salt concentrations.
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How are CountBright Plus Absolute Counting Beads different from CountBright Absolute Counting Beads?
Here are the differences:
CountBright Plus Absolute Counting Beads:
Bead Size: 4 µm
Excitation Range: 385 - 810 nm
Emission Range: 385 - 860 nm
CountBright Absolute Counting Beads:
Bead Size: 7 µm
Excitation Range: 350 - 635 nm
Emission Range: 350 - 810 nm
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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.
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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).
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