Product Description
Viable bacterial cell detections are very important for analyzing bacteria contamination in food or evaluating the cleanliness of facilities in order to protect us from food poisoning and infections. Bacterial cell detections are also used for the screening of sanitizing agents and drug resistance detections. Generally, counting the number of colonies on an agar plate is the standard method for determining the number of viable bacterial cells in samples. However, colony formations require one to several days. Dojindo’s Microbial Viability Assay Kit-WST can be used to determine the number of viable bacterial cells in a sample by a colorimetric method and can be applied to 96-well microplate assays. The electron mediator in the kit receives electrons from viable bacterial cells and transfers the electrones to WST, one of the water-soluble tetrazolium salts developed by Dojindo. Bacterial cell viability then can be determined by monitoring the color intensity of WST formazan dye. Since several types of media used for bacterial cell cultures and components do not interfere with the assay(Fig. 5), simply add the assay solution and incubate for one to several hours to determine the initial number of viable bacterial cells in the sample(Fig. 2). This assay kit was co-developed with the Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center.


Fig. 1 Bacterial cell viability detection mechanism.


Assay Data

Fig. 2 Correlation between initial number of E. coli and time-dependent O.D. increase. The initial number of viable E.coli were determined by a colony counting method.



Fig. 3 Correlation between the initial number of SA and time-dependent O.D. increase. The initial number of viable SA were determined by the colony counting method.


Comparison Chart

Fig. 5 Influence of culture media or substances used for bacterial cell culture.
The data indicated that WST is less sensitive to various culture media or substances which are used for bacterial cell culture. WST is a better tetrazolium salt than XTT for bacterial cell viability assays.

General Procedure 2
Determination of the susceptibility of Staphylococcus aureus to oxacillin
Oxacillin: antimicrobial agent: 0-64 μg/ml
Microorganism: Staphylococcus aureus (SA)
Methicillin-resistant Staphylococcus aureus (MRSA)
1. Culture SA or MRSA with Mueller-Hintonmedium containing various concentrations of Oxacillin for 6 hours at 35ºC.
2. Add Microbial Viability Assay solution equal to 1/20 the volume of the culture medium.
3. Incubate for 2 hours at 35^ºC.
4. Measure the O.D. at 450 nm to determine the MIC (Minimum inhibitory concentration).

The list of samples measured by Microbial Viability Kit

Mould	Yeast
Alternaria alternata NBR+B40:B56C31805 Aspergillus flavus NBRC4295 Aspergillus fumigatus NBRC33022 Aspergillus niger NBRC105649 Aspergillus terreus NBRC6346 Aspergillus ustus NBRC4128 Aureobasidium pullulans NBRC6353 Chaetomium globosum NBRC6347z Cladosporium cladosporioides NBRC6348 Exophiala dermatitidis NBRC6421 Paecilomyces variotii NBRC33284 Penicillium citrinum NBRC6352 Penicillium pinzophilum NBRC33285 Phoma citricarpa NBRC5287 Pseudallescheria boydii NBRC32229 Rhizopus oryzae NBRC31005 Sporothrix schenckii NBRC32961 Trichoderma virens NBRC6355 Trichophyton rubrum NBRC 5807	Candida albicans ATCC90028 Candida albicans JCM 1542 Candida albicans SC5314 Candida krusei NBRC1395 Candida parapsilosis NBRC1396 Candida utilis Cryptococcus albidus NBRC0378 Cryptococcus neoformans ATCC66031 Saccharomyces cerevisiae NBRC2347 Trichosporon asahii NBRC103889 Trichosporon asahii M9459 Trichosporon asahii M9925 Zygosaccharomyces rouxi
Gram-positive	Gram-negative
Bacillus cereus Bacillus subtillis Corynebacterium glutamicum Enterococcus faecalis Enterococcus faecalis FSCC 146002 Lactobacillus casei Listeria monocytogenes Micrococcus luteus Staphylococcus aureus Staphylococcus aureus NBRC13276 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis Staphylococcus epidermidis ATCC 14990 Staphylococcus epidermidis FSCC 223011 Streptococcus mutans NCTC 10449 Streptococcus mutans UA 159	Acetobacter sp. Acinetobacter baumannii ATCC 19606 Campylobacter concisus ATCC 33237 Enterobacter cloacae FSCC145003 Escherichia coli 1.1369 Escherichia coli ATCC 25922 Escherichia coli BW25113 Escherichia coli K-12 Fusobacterium nucleatum subsp. polymorphum JCM 12990 Klebsiella pneumoniae FSCC167002 Proteus mirablils Pseudomonas aeruginosa Pseudomonas aeruginosa NBRC13275 Pseudomonas aeruginosa ATCC 27853 Salmonella enteritidis Salmonella typhimurium Serratia marcescens Stenotrophomonas maltophilia ATCC 51331 Vivrio parahaemolyticus Yersinia enterocollica

No.	Sample	Reference
1)	<Yeast> ・Candida albicans JCM 1542	A. Azuma, N. Akiba and S. Minakuchi, "Hydrophilic surface modification of acrylic denture base material by silica coating and its influence on Candida albicans adherence", J. Med. Dent. Sci.., 2012, 59, (1), 1-7.
2)	<Gram-negative> ・Escherichia coli BW25113	H. Y. Oh, J. O. Lee and O. B. Kim, "Increase of organic solvent tolerance of Escherichia coli by the deletion of two regulator genes, fadR and marR", Appl. Microbiol. Biotechnol.., 2012, 96, (6), 1619-27.
3)	<Gram-negative> ・Escherichia coli K-12	T. Nakayashiki, N. Saito, R. Takeuchi, H. Kadokura, K. Nakahigashi, B. L. Wanner and H. Mori, "The tRNA thiolation pathway modulates the intracellular redox state in Escherichia coli", J. Bacteriol.., 2013, 195, (9), 2039-49.
4)	<Gram-positive> ・Streptococcus mutans UA 159 <Gram-negative> ・Escherichia coli 1.1369	S. He, P. Zhou, L. Wang, X. Xiong, Y. Zhang, Y. Deng and S. Wei, "Antibiotic-decorated titanium with enhanced antibacterial activity through adhesive polydopamine for dental/bone implant", J. R. Soc. Interface., 2014, 11, (95).
5)	<Mould> ・Trichophyton rubrum NBRC 5807	S. Arai, T. Yoshino, T. Fujimura, S. Maruyama, T. Nakano, A. Mukuno, N. Sato and K. Katsuoka, "Mycostatic effect of recombinant dermcidin against Trichophyton rubrum and reduced dermcidin expression in the sweat of tinea pedis patients", J. Dermatol.., 2015, 42, (1), 70-6.
6)	<Mould> ・Trichophyton rubrum	S. Arai, T. Yoshino, T. Fujimura, S. Maruyama, T. Nakano, A. Mukuno, N. Sato and K. Katsuoka, "Mycostatic effect of recombinant dermcidin against Trichophyton rubrum and reduced dermcidin expression in the sweat of tinea pedis patients", J. Dermatol.., 2015, 42, (1), 70-6.
7)	<Gram-positive> ・Staphylococcus aureus NBRC13276 <Gram-negative> ・Pseudomonas aeruginosa NBRC13275	T. Tsukatani, T. Kawaguchi, H. Suenaga, M. Shiga and T. Ikegami, "RAPID AND SIMPLE DETERMINATION OF MINIMUM BIOFILM ERADICATION CONCENTRATION BY A COLORIMETRIC MICROBIAL VIABILITY ASSAY BASED ON REDUCTION OF A WATER-TETRAZOLIUM SALT AND COMBINATED EFFECT OF ANTIBIOTICS AGAINST MICROBIAL BIOFILM", J Microbiol Biotechnol Food Sci.., 2016, 6, (1), 677.
8)	<Gram-positive> ・Streptococcus mutans NCTC 10449	M. Hashimoto, H. Yanagiuchi, H. Kitagawa and Y. Honda, "Inhibitory Effect of Platinum Nanoparticles on Biofilm Formation of Oral Bacteria", Nano Biomed., 2017, 9, (2), 77-82.
9)	<Gram-positive> ・Enterococcus faecalis FSCC 146002 ・Staphylococcus aureus ATCC 25923 ・Staphylococcus epidermidis FSCC 223011 <Gram-negative> ・Acinetobacter baumannii ATCC 19606 ・Enterobacter cloacae FSCC 145003 ・Escherichia coli ATCC 25922 ・Klebsiella pneumoniae FSCC 167002 ・Pseudomonas aeruginosa ATCC 27853 ・Stenotrophomonas maltophilia ATCC 51331	Z. Xu, X. Zhao, X. Chen, Z. Chen and Z. Xia, "Antimicrobial effect of gallium nitrate against bacteria encountered in burn wound infections", RSC Adv.., 2017, 7, 52266-52273.
10)	<Yeast> ・Candida albicans SC5314	J. Nagao, T. Cho, M. Mitarai, K. Iohara, K. Hayama, S. Abe and Y. Tanaka, "Antifungal activity in vitro and in vivo of a salmon protamine peptide and its derived cyclic peptide against Candida albicans", FEMS Yeast Res.., 2017, 17, (1).
11)	<Mould> ・Trichosporon asahii M9459 ・Trichosporon asahii M9925	T. Ichikawa, C. Hirata, M. Takei, N. Tagami, H. Murasawa and R. Ikeda, "Cell surface hydrophobicity and colony morphology of Trichosporon asahii clinical isolates", Yeast., 2017, 34, (3), 129-137.
12)	Soil Microorganisms	M. Kamitakahara, S. Takahashi, T. Yokoi, C. Inoue and K. Ioku, "Preparation of spherical porous hydroxyapatite granules as support materials for microorganisms", JCS Japan., 2018, 126, (9), 732-735.
13)	<Gram-positive> ・Staphylococcus epidermidis ATCC 14990	F. Boschetto, T. Adachi, S. Horiguchi, D. Fainozzi, F. Parmigiani, E. Marin, W. Zhu, B. McEntire, T. Yamamoto, N. Kanamura, O. Mazda, E. Ohgitani and G. Pezzotti, "Monitoring metabolic reactions in Staphylococcus epidermidis exposed to silicon nitride using in situ time-lapse Raman spectroscopy", J Biomed Opt.., 2018,(5), 1-10.
14)	<Gram-negative> ・Campylobacter concisus ATCC 33237 ・Fusobacterium nucleatum subsp. polymorphum JCM 12990	T. Nambu, D. Wang, C. Mashimo, H. Maruyama, K. Kashiwagi, K. Yoshikawa, K. Yamamoto and T. Okinaga, "Nitric Oxide Donor Modulates a Multispecies Oral Bacterial Community-An In Vitro Study", Microorganisms., 2019, 7, (9), 353.