Bacteria Counting and Enumeration Assays for Flow Cytometry

Our bacteria counting and enumeration assays for flow cytometry contain a mixture of fluorescent nucleic acid stains for bacterial identification, as well as a calibrated suspension of beads for accurate sample volume measurements. Accurate detection and enumeration of the live and dead bacteria in a sample is an important aspect of many experimental procedures in biotechnology.

Bacteria Cell Counting Assay

The Bacteria Counting Kit for flow cytometry accurately enumerates bacteria . The kit includes the SYTO® BC dye, 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.

Figure 1. Enumeration of Bacillus cereus using the Bacteria Counting Kit for flow cytometry. Bacteria stained with the SYTO® BC bacterial cell stain and mixed with known concentrations of weakly fluorescent 6 µm polystyrene microsphere standards produce a bivariate frequency distribution for forward light scatter versus green fluorescence intensity that allows the bacterial population number to be determined by reference to the clearly separated microsphere standard population, indicated by red data points.

Conjugate Laser Ex/Em* Cat. No.  
Bacteria Counting Kit488 nm480/500 nmB7277product details

*Indicates excitation and emission.

Publications Where this Product is Referenced

McIver, AM, et al. (2008) Microbial Oxidation of Naphthalene to cis-1,2-Naphthalene Dihydrodiol Using Naphthalene Dioxygenase in Biphasic Media. Biotechnology Progress 24:593-598.

İleri, N., et al. (2007) Phosphate enrichment and fed-batch operation for prolonged β-lactamase production by Bacillus licheniformis. J. of Applied Micro. (102) 1418–1426.

Berney, M., et al. (2007) Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry.  App. Env. Micro., 73:3283-3290.

El-Nezami, H. et al. (2004) Chemical Moieties and Interactions Involved in the Binding of Zearalenone to the Surface of Lactobacillus rhamnosus Strains GG.  J. Agric. Food Chem., 52: 4577–4581.

Bacteria Cell Counting and Viability Assay

The LIVE/DEAD® BacLight™ Bacterial Viability and Bacteria Counting Kit allows researchers to reliably distinguish and quantitate live and dead bacteria with the aid by flow cytometry, 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.

Figure 2. Analysis of bacterial cultures using the LIVE/DEAD® BacLight™ Bacterial Viability and Bacteria Counting Kit. Suspensions of live (untreated) and dead (alcohol-treated) Staphylococcus aureus (panels A and C) and Escherichia coli (panels B and D) were stained with the SYTO® 9 nucleic acid stain and propidium iodide and then analyzed by flow cytometry according to the kit protocol. The green or red fluorescence versus side scatter cytogram (panel A or B) was used to gate the bacterial population and the bead population (left and right boxes, respectively). Events in the bacteria region of each cytogram are also displayed in red fluorescence versus green fluorescence cytograms (panels C and D). Live and dead bacteria/mL can be calculated from either the fluorescence versus side scatter cytogram or the green fluorescence versus red fluorescence cytogram, depending on which one shows the best separation of the live and dead populations. The position of the live and dead populations in these cytograms may be dependent on cell type and gram character. Some samples may exhibit events that fall outside the defined regions and should be evaluated appropriately (e.g., see panel D).

Product Laser Ex/Em* Apoptotic Cell Stain
Dead Cell Stain
Cat. No.
LIVE/DEAD® BacLight™ Bacterial Viability and Bacteria Counting Kit488nm488 nm    480/500 nm (L),
490/635 nm (D)
SYTO®9Propidium Iodide
*Indicates excitation and emission. L = Live, D = Dead.

Publications Where this Product is Referenced

F. Hegler and A. Kappler (2010) Cryopreservation of anoxygenic phototrophic Fe(II)-oxidizing bacteria. Cryopreservation 61:158-160.

Orth, R. et al, (2010) An efficient method for enumerating oral spirochetes using flow cytometry. J. Micro. Methods. 80:123-128.

Kramer, M., et al. (2009) Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. App. Micro. Biotech. 84:1137-1147.

Pascaud, A., et al. (2009) A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil. J. Micro. Methods 76:81-87.