06 -Bacstain- Series

-Bacstain- CTC Rapid Staining Kit (for Microscopy)

Bacterial Staining

  • Product code
    BS02  -Bacstain- CTC Rapid Staining Kit (for Microscopy)
Unit size Price Item Code
100 assays $261.00 BS02-10

ORDER
Component
100 assays ・CTC
・Enhancing Reagent B
10 mg × 3
500 μl× 1

Product Description

Colony 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

Manual

Technical info

Microscopy detection
1. 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.

Experimental Example

Observation by Microscopy

Fig. 1 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.

References

Open References

1) A. W. Coleman, "Enhanced Detection of Bacteria in Natural Environments by Fluorochrome Staining of DNA", Limnol. Oceanogr., 198025, 948.
2) E. Severin, J. Stellmach and H. -M. Nachtigal, "Fluorimetric Assay of Redox Activity in Cells", Anal. Chim. Acta1985170, 341.
3) G. G. Rodriguez, D. Phipps, K. Ishiguro and H. F. Ridgway, "Use of a Fluorescent Redox Probe for Direct Visualization of Actively Respiring Bacteria", Appl. Environ. Microbiol.199258(6), 1801.
4) G. Schaule, H. C. Flemming and H. F. Ridgway, "Use of 5-Cyano-2,3-ditolyl Tetrazolium Chloride for Quantifying Planktonic and Sessile Respiring Bacteria in Drinking Water", Appl. Environ. Microbiol., 1993, 59(11), 3850.
5) R. A. Bovill, J. A. Shalloross and B. M. Markey, "Comparison of the Fluorescent Redox Dye 5-Cyano-2,3-ditolyltetrazolium Chloride with p-Iodonitrotetrazolium Violet to Detect Metabolic Activity in Heat-stressed Listeria monocytogenes Cells", J. Appl. Bacteriol., 199477(4), 353.
6) M. T. E. Suller and D. Lloyd, "Flow Cytometric Assesment of the Postantibiotic Effect of Methicillin on Staphylococcus aureus", Antimicrob. Agents Chemother., 199842(5), 1195.
7) M. Kawai, N. Yamaguchi and M. Nasu "Rapid Enumeration of Physiologically Active Bacteria in Purified Water Used in the Pharmaceutical Manufacuturing Process", J. Appl. Microbiol., 199986, 496. 
8) N. Yamaguchi, M. Sasada, M. Yamanaka and M. Nasu, "Rapid Detection of Respiring Escherichia coli O157:H7 in Apple Juice, Milk and Ground Beef by Flow Cytometry", Cytometry200354A, 27.
9) A. Kitaguchi, N. Yamagauchi and M. Nasu, "Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization Following Formazan Reduction", Appl. Environ. Microbiol., 200571(5), 2748.
10)A. Hiraishi and N. Yoshida, "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.200419(1), 61.

Handling and storage condition

Handling and storage condition
0-5°C, Protect from light
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Product Classification

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