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Molecular Probes™ Bacteria Counting Kit, for flow cytometry

Catalog No. 361055540
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Catalog No. 361055540 Supplier Molecular Probes™ Supplier No. B7277
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Description

Bacteria Counting Kit, for flow cytometry

The Bacteria Counting Kit accurately enumerates bacteria by flow cytometry. The kit includes the SYTO™ BC dye, which is also available separately (Cat No. S34855), a high-affinity nucleic acid stain that easily penetrates both gram-negative and gram-positive bacteria, producing an exceptionally bright green-fluorescent signal. The kit also includes a calibrated suspension of polystyrene microspheres. Signals from both the stained bacteria and the beads are easily detected in the green fluorescence channel and can be distinguished on a plot of forward scatter versus fluorescence. The density of the bacteria in the sample can be determined from the ratio of bacterial signals to microsphere signals in the cytogram.

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

Specifications

Product Type Bacteria Counting Kit
Detection Method Fluorescence
Quantity 1 kit
Sample Type Bacteria
Diameter (Metric) Bead 6 μm
Content And Storage Contains 1 vial of SYTO™ BC bacterial stain (100 μL, 1000x in DMSO), 1 vial microsphere standard (1 mL, 6 μm).

Store in refrigerator (2–8°C) and protect from light.
For Use With (Application) Flow Cytometry
For Use With (Equipment) Flow Cytometer
Includes BC Bacterial Stain, Microsphere Standard
Shipping Condition Room Temperature

Frequently Asked Questions (FAQs)

Is the SYTO BC bacteria stain from the Bacteria Counting Kit available as a standalone product?

Yes, the SYTO BC bacteria stain can be purchased separately (Cat. No. S34855).
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).
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).
I am using counting beads to count cells, but I cannot find the beads on my scatter plots. What do I do?

The first thing to do is check your threshold and see if it is set on forward scatter. If so, the beads are probably being excluded by the threshold. Reducing the threshold setting should reveal your beads.

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)
I want to count my cells using flow cytometry. How do I do this?

Cell counting using flow cytometry can be accomplished by adding an internal microsphere counting standard to the flow cytometric sample. The number of reference beads that are collected reflects a known volume. This allows you to calculate cell concentration.
What is the smallest size that I can detect with the Attune NxT Acoustic Focusing Cytometer?

The smallest size that you can detect with the Attune NxT Acoustic Focusing Cytometer is 0.5 µm.
What is the Attune NxT Autosampler?

The Attune NxT Autosampler, an optional accessory for the Attune NxT Acoustic Focusing Cytometer, enables rapid processing of up to 384 samples. It has broad compatibility with different plate formats, both 96- and 384-well plates. It has an intelligent probe designed to minimize clogging and carryover (<0.5%) and to prevent damage to the instrument. It mixes by aspiration rather than shaking to ensure homogeneity of the sample and maintain cell viability. Is performs automated cleaning as part of the shutdown process of the Attune NxT Cytometer. It provides consistent data regardless of sampling method (tube vs. plate) and collection rate.
What are the advantages of acoustic-assisted hydrodynamic focusing in flow cytometry?

-Modular design - Multiple configurations available - field upgradable.
-Save time - 10X faster speeds with no loss in data quality.
-Simplified sample prep - No wash, no lyse options, non-clogging fluidics.
-Enables unique applications - Complex protocols on a broad range of cell types and samples.
How is the Attune NxT Acoustic Focusing Cytometer different from traditional flow cytometers?

With the option to be configured with up to 4 lasers and 14 colors for multi-parameter analysis the Attune NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of four spatially separated lasers onto the sample stream enabling consistency in data over time, superior performance, and superior reliability. The instrument can be configured with up to 4 solid-state lasers (405 nm, 488 nm, 561 nm, and 637 nm) with flat top beam profiles.

The Attune NxT Flow Cytometer's acoustic focusing uses ultrasonic radiation pressure (> 2 MHz) to transport particles into the center of the sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional hydrodynamic pressure to the sample. The combination of these two forces- termed acoustic-assisted hydrodynamic focusing-results in a narrow core stream and uniform laser illumination, regardless of the sample input rate. In traditional cytometers that rely solely on hydrodynamic focusing, the sample core widens to accommodate the increases in flow rate, which results in less uniform laser light illumination.
What is flow cytometry?

Cytometry is the measurement of physical or chemical characteristics of cells or particles. Flow cytometry measures these characteristics of cells or particles as they individually pass lasers in a flow cytometer instrument. Flow cytometry is performed on single cells, providing discrete measurements for each cell in the sample. It also provides a statistical distribution of the measured characteristics of the sample.

A flow cytometer is made up of three subsystems: fluidics, optics, and electronics. Fluidics moves the cells and introduces them for interrogation. Optics generates and collects the light signals. Electronics converts the optical signals to proportional electronic signals for computer analysis.

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