Product DescriptionColony formation using an agar plate is a very common and reliable method for counting bacterial cells. However, it takes quite a long time to form a colony. Therefore, alternative detection methods have been developed. Bacteria-specific gene amplification methods such as PCR, LAMP, and nucleus staining are quite rapid, but these methods count dead bacteria as well. Therefore, detection of live cell functions is essential to determining the actual number of living bacteria in a sample. Tetrazolium salts can be used to detect respiratory activity of bacterial cells or mitochondria. CTC is a tetrazolium salt and is reduced by this respiratory activity to form fluorescent CTC formazan on the cell surface. Therefore, CTC is used for specific staining of aerobic live bacteria and can be applied to hard-to-culture bacteria (VNC: viable but non-culturable). CTC forms a fluorescent formazan by an electron transfer system. However, CTC alone is not sensitive enough to stain single cells. Therefore, the CTC-Rapid Staining Kit contains an enhancing reagent that improves the CTC staining efficiency. Compared with staining with CTC only, this staining kit enables rapid and sensitive staining of microorganisms. Maximum wavelengths of the CTC formazan dye are 430 nm or 480 nm for excitation and 630 nm for emission.

Fig. 1 Bacterial cell viability detection mechanism with CTC

General Staining ProtocolMicroscopy detection1. Centrifuge bacteria culture and remove the supernatant, and then resuspend the bacteria pellet with PBS(-).2. Add CTC + Enhancing reagent-B. Incubate at 37°C for 1 hour.3. Prepare a slide and detect fluorescence by B-excitation filter set.

Flow cytometry detection1. Centrifuge bacteria culture and remove the supernatant, and then resuspend the bacteria pellet with PBS(-).2. Add CTC + Enhancing reagent-A. Incubate at 37°C for 1 hour.3. Analyze the cells with a flow cytometry: 488 nm excitation, 630 nm emission.

1. A. Hiraishi, et al., An Improved Redox Dye-Staining Method Using 5-Cyano-2,3-Ditoryl Tetrazolium Chloride for Detection of Metabolically Active Bacteria in Activated Sludge. Microbes Environ. 2004;19:61-70.2. A. Kitaguchi, et al., Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization Following Formazan Reduction. Appl Environ Microbiol. 2005;71:2748-2752.

FACS DataE. coli

B. Subtilis

Candida albicans

E. faecalisFig. 2 CTC staining of various microorganisms with and without Enhancing reagent.

Experimental ExampleObservation by Microscopy

Fig. 3 E. coli staining (left) and L. casei staining (right) with CTC and DAPI. Bacterial cells were stained with CTC Rapid Staining Kit first, and then 1 μl of DAPI solution was added. The cells were incubated at room temperature for 5 min. Formaldehyde fixation with 1-4% formaldehyde can be performed before DAPI staining.