-Bacstain- CTC Rapid Staining Kit 　 (for Flow cytometry)BS01 Product Manual

-Bacstain - series offer several kinds of bacterial fluorescence staining dye and can be applied for microbial cell viability
assay in different principles.
CTC has been used by many researchers to evaluate the microbial respiratory activity.
-Bacstain - CTC Rapid Staining Kits allows quick and high-sensitivity CTC-staining.

 

CTC (5-cyano-2,3-ditolyl tetrazolium chloride)	10 mg x 3
Enhancing reagent A	100 μl (DMSO solution) x 1

Store at 0-5ºC

• Incubator
• Micropipette (20 μl, 1,000 μl)
• Flow cytometer (488 nm laser, red emission filter)

1. 1. Add 750 μl of distilled water to one CTC tube and vortex gently to dissolve (final concentration 50 mmol/l) ^a) .
2.  Resuspend the organism with PBS(-) or saline and adjust the number of cells to 10⁶ cells/ml (flow cytometry) ^b) .
3. Add each reagent into the 1 ml of microbial cell suspension and vortex gently to mix.
   Refer to the conditions in the following tables.

   	CTC solution	Enhancing reagent A
   Enhancing reagent A	20 μl	1 μl

4. Incubate the microbial cells at 37 ℃ for 30 min^c).
5.  Analyze the stained cells by flow cytometer or under a microscope.

a)	This solution is stable at -20°C for 2 weeks.
b)	Since remaining culture medium in the sample undergoes unspecific colored-reaction, it should be duly removed.
c)	When CTC-staining is insufficient, add extra CTC solution or increase the incubation time. In this case, CTC solution should be limited less or equal to 100 μl/sample.

 

Enhancer (+)

 

Enhancer (-)

-Bacstain- DAPI solution is utilized for the nucleotide staining of bacteria either with membrane impairment or with intact membrane. Since CTC only stains viable aerobic cells, double staining using CTC and DAPI enable to identify viable aerobic cells among the entire cell population. Formaldehyde fixation (1–4% final concentration) is not required for the DAPI staining. However, if fixation is necessary for the downstream experiment or if the formaldehyde fixation is a standard protocol to prepare samples, it can be done in between CTC staining and DAPI staining. In this case, please add 1 μl of DAPI solution to CTC-stained cell suspension (approx. 1 ml) and then incubate the sample at room temperature for 5 min.

This kit can perform at least 100 tests for the flow cytometric assay and the microscopic assay by following the protocol described herein.

1. 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. , 2004, 19 (1), 61.
2. A. Kitaguchi, N. Yamaguchi and M. Nasu, "Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by
   Fluorescence In Situ Hybridization Following Formazan Reduction", Appl. Environ. Microbiol ., 2005, 71 (5), 2748.

-Bacstain - CFDA solution

CFDA is widely used as an indicator for the measurement of esterase activity.
-Bacstain- CFDA solution is provided as Ready-to-Use DMSO solution.
Fluorescent carboxyfluorescein is produced from non fluorescent CFDA by the esterase in the microbial cell.

 

-Bacstain - DAPI solution, AO solution

DAPI and AO are nucleic acid staining dyes and can be applied for bacteria either with membrane impairment or with
intact membrane.

 

-Bacstain - PI solution

PI is a nucleic acid staining dye. Membrane-injured cells are stained with red emission by PI.

 

Products	Code	Maximum Ex/Em(nm)	Number of assays
CTC Rapid Staining Kit (for Flow cytometry)	BS01	430, 480/630	100
CTC Rapid Staining Kit (for Microscopy)	BS02	430, 480/630	100
CFDA solution	BS03	493/515	100
DAPI solution	BS04	360/460	100
AO solution	BS05	420-460/630-650(ssDNA) 500/520(dsDNA)	100
PI solution	BS07	530/620	100

These products were developed by joint-research with Fukuoka Industrial Technical Center in Japan.