Journal of Microbiological Methods 75 (2008) 109116

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j m i c m e t h

Colorimetric cell proliferation assay for microorganisms in microtiter plate using

water-soluble tetrazolium salts
Tadayuki Tsukatani a,, Hikaru Suenaga a, Tomoko Higuchi a, Tetsuyuki Akao a, Munetaka Ishiyama b,
Kimitoshi Ezoe b, Kiyoshi Matsumoto c
a
b
c

Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 839-0861, Japan
Dojindo Laboratories, Kumamoto 861-2202, Japan
Division of Food Biotechnology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School Kyushu University, Fukuoka 812-8581, Japan

A R T I C L E

I N F O

Article history:
Received 10 March 2008
Received in revised form 12 May 2008
Accepted 15 May 2008
Available online 24 May 2008
Keywords:
Cell proliferation assay
Electron mediator
Microorganism
Quinone
Tetrazolium salt

A B S T R A C T
A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using watersoluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble
tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efciency of
microorganisms and the inuence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8
as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to
medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to
formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be
applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Grampositive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and
viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change
reected the microbial cell proliferation.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Tetrazolium salts have become some of the most widely used tools
in cell biology for measuring the metabolic activity of cells ranging
from mammalian to microbial origin (Berridge et al., 2005). The
prototype tetrazolium salt, 2,3,5-triphenyl-2H-tetrazolium chloride
(TTC), rst developed more than a century ago. Then, various
tetrazolium salts such as 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl2H-tetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium
chloride (CTC) have been synthesized on the base of the structure of
TTC. MTT and CTC have usually been used as an indicator of cellular
viability in spectrophotometric and uorometric assays (Bhupathiraju
et al., 1999; Gruden et al., 2003a,b; Lveill et al., 1993; Mosmann,
1983; Stowe et al., 1995). These tetrazolium salts form insoluble
formazans. The need to solubilize MTT-formazan crystal prior to
spectrophotometric assay limits some applications. Recently, tetrazolium salts that form water-soluble formazans have been developed.
They are used in conjunction with intermediate electron acceptors that
facilitate the reduction reactions. They form soluble formazans and
consequently can be used in real-time assays. The vast majority of
cellular applications of tetrazolium salts involve microplate assay that
measures cell proliferation. Furthermore, they have been used in rapid
Corresponding author. Tel.: +81 942 6644; fax: +81 942 30 7244.
E-mail address: tukatani@tc.pref.fukuoka.jp (T. Tsukatani).
0167-7012/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2008.05.016

colorimetric assays for antimicrobial susceptibility testing of both

bacteria and fungi (Meletiadis et al., 2001; Tunney et al., 2004). The
most commonly used tetrazolium salt in colorimetric assays for
microorganism has been 2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), which,
after reduction, yields a water-soluble formazan derivative that can be
easily quantied colorimetrically (Paull et al., 1988; Scudiere et al.,
1988). More recently, a new generation of water-soluble tetrazolium
salts, which includes 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1; Ishiyama et al., 1993),
2,2-dibenzothiazolyl-5,5-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3(3,3-dimethoxy-4,4-biphenylene)ditetrazolium, disodium salt (WST-5;
Ishiyama et al., 1996a), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8;
Ishiyama et al., 1997) has been developed. A few of these tetrazolium
salts have been successfully used in assays for cell viability and
cytotoxicity (Ishiyama et al., 1996b; Tominaga et al., 1999). However,
this WST series had not been applied to microbial cell proliferation assays
until quite recently.
Comparisons of WST-1 or WST-8 in the assays for microbial cell
viability and antimicrobial susceptibility with other tetrazolium salts,
including XTT, were reported (McCluskey et al., 2005; Brady et al.,
2007). However, a detailed study on an intermediate electron
acceptor, which is called as electron mediator, has not been
performed. These tetrazolium salts require an electron mediator for

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T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

the cellular reduction because they are characterized by a net negative

charge and therefore are largely cell-impermeable (Berridge et al.,
2005). Thus, the selection of an electron mediator is very important

for the reduction of tetrazolium salts by microorganisms to formazan.

In addition, a detailed investigation on the inuence with medium
components has not been carried out. Certain tetrazolium salts may be

Fig. 1. Effects of electron mediators on the reduction of tetrazolium salts in the presence of microorganisms. Microbial cells were incubated in standard medium (pH 7.0) containing
0.1 mM WST-5 and 40 M of each electron mediator for 60 min at 30 or 37 C. Formazans produced by microorganisms were measured at 550 nm with a microplate reader. Electron
mediator: B-1, 1,4-BQ; B-2, 2-methyl-1,4-BQ; B-3, 2,5-dimethyl-1,4-BQ; B-4, 2,6-dimethyl-1,4-BQ; B-5, 2,3,5-trimethyl-1,4-BQ; B-6, 2,3,5,6-tetramethyl-1,4-BQ; B-7, 2,5-dimethoxy1,4-BQ; B-8, 2,6-dimethoxy-1,4-BQ; B-9, 2,3-dimethoxy-5-methyl-1,4-BQ; B-10, 2-tert-butyl-1,4-BQ; B-11, 2,6-tert-butyl-1,4-BQ; B-12, 2,5-tert-butyl-1,4-BQ; N-1, 1,4-NQ; N-2, 1,4NQ-2-sulfate; N-3, 2-methyl-1,4-NQ; N-4, 6-methyl-1,4-NQ; N-5, 5-hydroxy-2-methyl-1,4-NQ; N-6, 2-hydroxy-1,4-NQ; N-7, 5-hydroxy-1,4-NQ; N-8, 5,8-dihydroxy-1,4-NQ; N-9, 2amino-3-chloro-1,4-NQ; N-10, 1,2-NQ-4-sulfate; N-11, 4-amino-1,2-NQ; A-1, AQ-2-sulfate; A-2, AQ-2,6-disulfate; P-1, PMS; P-2, 1-methoxy-PMS. BQ, NQ, AQ and PMS mean
benzoquinone, naphthoquinone, anthraquinone and phenazine methosulfate, respectively.

T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

easily reduced by components such as peptones and glycated proteins

in culture mediums. Therefore, the modication of a tetrazolium
method with regard to these factors may lead to the development of a
valuable method to assay microbial cell proliferation.
In the present study, we evaluated combinations of six different
tetrazolium salts, namely WST-1, WST-3, WST-5, WST-8, WST-9, and
XTT, with electron mediators for cell proliferation assays of microorganisms in a 96-well microtiter plate, considering the metabolic
efciency of microorganisms and the inuence with medium
components.
2. Materials and methods
2.1. Chemicals and medium
2,3-Bis(2-methyloxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) was obtained from Sigma
Chemicals (St. Louis, MO, USA). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (WST-4), 2,2-dibenzothiazolyl-5,5-bis
[4-di(2-sulfoethyl)carbamoylphenyl]-3,3-(3,3-dimethoxy-4,4-biphenylene)ditetrazolium, disodium salt (WST-5), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt (WST-8), and 2-(4-nitrophenyl)-5-phenyl-3-[4-(4sulfophynylazo)-2-sulfophenyl]-2H-tetrazolium, monosodium salt
(WST-9) were special gifts from Dojindo (Kumamoto, Japan). The 27
electron mediators shown in Fig. 1 were bought from Sigma Chemicals
(St. Louis, MO, USA), Tokyo Kasei Co. (Tokyo, Japan), Toronto Research
Chemical (Toronto, ON, Canada), and Wako Pure Chemical Industries
(Osaka, Japan). A culture medium containing 0.25% yeast extract, 0.5%
peptone and 0.1% glucose was used as the standard medium for the cell
proliferation assay. Other commercial mediums shown in Fig. 5 were
purchased from Difco Laboratories (Detroit, MI, USA), Kyokuto Pharmaceutical Industrial Co. (Tokyo, Japan), Nissui Pharmaceutical Co. (Tokyo,
Japan), Oxoid Limited (Basingstoke, Hampshire, UK), respectively. All
other chemicals were of analytical reagent grade and were used without
further purication.

111

Table 1
Sensitivities for various microorganisms
Cell density (CFU/ml)a

Microorganism

Yeast
Candida utilis
Saccharomyces cerevisiae
Zygosaccharomyces rouxii
Gram-positive bacteria
Bacillus cereus
Bacillus subtilis
Corynebacterium glutamicum
Enterococcus faecalis
Lactobacillus casei
Listeria monocytogenes
Micrococcus luteus
Staphylococcus aureus
Staphylococcus epidermidis
Gram-negative bacteria
Acetobacter sp.
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella enteritidis
Salmonella typhimurium
Serratia marcescens
Vibrio parahaemolyticus
Yersinia enterocolitica

1h

4h

NBRC0626
NBRC2347
NBRC0505

5.53 107
8.70 105
1.65 105

6.18 106
2.65 105
2.47 104

NBRC13494
JCM1465
NBRC12168
JCM5803
NBRC15883
ATCC15313
NBRC13867
NBRC12732
NBRC12993

6.70 105
2.45 106
1.69 106
5.18 107
8.40 107
5.07 106
8.29 105
2.78 106
5.53 106

6.77 104
6.71 105
2.47 105
1.76 106
2.34 106
6.46 105
1.29 105
2.71 105
1.12 106

NBRC3283
NBRC3972
NBRC3512
NBRC13300
NBRC13275
NBRC3313
NBRC12529
NBRC102204
NBRC12711
JCM7577

2.53 107
1.31 107
1.76 107
7.42 106
1.76 108
2.55 107
1.73 107
7.15 107
2.90 107
1.92 107

7.39 106
2.86 105
5.59 105
1.35 106
1.78 107
1.06 106
2.60 106
5.08 106
1.03 107
5.46 106

a
Viable cell density that gives the absorbance change of 0.5 during the reaction time
of 1 and 4 h.

coccus faecalis (JCM5803), and Yersinia enterocolitica (JCM7577) were

bought from the Japan Collection of Microorganisms in RIKEN
BioResource Center (JCM, Tsukuba, Japan). Listeria monocytogenes
(ATCC15313) was purchased from the American Type Culture collection (ATCC, Rockville MD, USA). Batches of medium (5 ml in a glass
tube) were inoculated from fresh culture plates and incubated for 18 h
at the optimum temperature (2537 C), respectively. As culture
mediums, YM broth, tryptic soy broth plus yeast extract, and Man
RogasaSharpe (MRS) medium were used for yeast, bacteria except
lactic acid bacteria, and lactic acid bacteria, respectively.

2.2. Preparation of detection reagents

2.4. Colorimetric microbial assay using tetrazolium salts
Tetrazolium salts were dissolved in distilled water at a concentration of 11.1 mM and lter sterilized with a membrane lter (0.2 m).
Electron mediators were dissolved in DMSO at a concentration of
8.0 mM except for 2,5-dimethoxy-1,4-benzoquinone (BQ), 2,6dimethoxy-1,4-BQ and 2,3-dimethoxy-5-methyl-1,4-BQ. Because
these BQs were not soluble in DMSO, distilled water was used as the
solvent. Then, the tetrazolium salt solution was mixed with electron
mediators at a ratio of 9:1. The prepared detection reagent contained
10 mM tetrazolium salts and 0.8 mM electron mediators.
2.3. Microbial strains and growth conditions
The twenty-two microbial strains used in this study are shown in
Table 1. Candida utilis (NBRC0626), Saccharomyces cerevisiae
(NBRC2347), Zygosaccharomyces rouxii (NBRC0505), Bacillus cereus
(NBRC13494), Corynebacterium glutamicum (NBRC12168), Lactobacillus casei (NBRC15883), Micrococcus luteus (NBRC13867), Staphylococcus aureus (NBRC12732), Staphylococcus epidermidis (NBRC12993),
Acetobacter sp. (NBRC3283), Escherichia coli (NBRC3972), Klebsiella
pneumoniae (NBRC3512), Proteus mirabilis (NBRC13300), Pseudomonas
aeruginosa (NBRC13275), Salmonella enteritidis (NBRC3313), Salmonella typhimurium (NBRC12529), Serratia marcescens (NBRC102204),
and Vibrio parahaemolyticus (NBRC12711) were obtained from the
Biological Resource Center in the National Institute of Technology and
Evaluation (NBRC, Chiba, Japan). Bacillus subtilis (JCM1465), Entero-

The present method is thought to be based mainly on the following

reaction mechanism. First, quinone as an electron mediator is reduced
to hydroquinone by microbial cells. Then, the hydroquinone reduces
tetrazolium salts extracellularly to formazans, which exhibit absorbance maxima ranging from 438 to 550 nm, respectively, and can be
quantied spectrophotometrically.
The cultivated microorganisms were diluted with the standard
medium (pH 7.0) to adjust the microbial cell density. The microbial
suspension (190 l) was added to each well (96-well microtiter plate).
Then, the detection reagent (10 l), which consists of an electron
mediator and tetrazolium salt, was added to the well and incubated at
30 or 37 C. The formation of formazan was measured as absorbance at
438, 550, 550, 460, 495, and 470 nm for WST-1, WST-4, WST-5, WST-8,
WST-9, and XTT, respectively, with a microplate reader (VersaMax,
Molecular Devices Co., Sunnyvale, CA, USA).
2.5. Viability determined by colony counts
After serial dilution, microorganism cultures were plated with a
standard method agar (Nisui Pharmaceutical Co., Tokyo, Japan)
containing 0.25% yeast extract, 0.5% peptone, 0.1% glucose, and 1.5%
agar onto plastic plates (90 mm i.d.), and then incubated at the
optimum temperature (2537 C). Colonies were counted after 1 or
2 days.

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T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

2.6. Susceptibility test

Reference MICs were determined by the microdilution method
currently recommended by the National Committee for Clinical
Laboratory Standards. Serial two-fold dilutions of each antimicrobial
agent were prepared in MuellerHinton broth (Kyokuto Pharmaceutical
Industrial Co., Tokyo, Japan) within dilution schemes of 0.0625 to 64 g/ml.
As the antimicrobial agents, ciprooxacin (CFX), cefotaxime (CTX),
chloramphenicol (CP) and gentamicin (GM) were used, B. cereus was
adjusted with phosphate-buffered saline to a turbidity equal to that of 0.5
McFarland standard, and then diluted by 10-fold. The suspension was
further diluted with antimicrobial agent solution to provide a nal
inoculum density of approximately 105 CFU/ml. Each well of a plate was
inoculated with 100 l inoculum, and the plate was incubated for 24 h at
35 C. After incubation, the MIC was read as the lowest concentration of
antimicrobial agent at which there was no visible growth.
In the susceptibility tests using the proposed method, the
inoculum to be tested was prepared as described above, and 5 l of
the detection reagent was added to each well after 6 h of incubation at
35 C. After incubation for 2 h at 35 C, the produced formazans were
measured at 460 nm with a microplate reader. The MIC was read as the
lowest concentration of antimicrobial agent at which no absorbance
change occurred.

microorganisms. Hence, we decided to use 2-methyl-1,4-NQ as the

electron mediator in subsequent experiments. The selection is thought to
be reasonable because menaquinone molecules in microorganisms are
derivatives of 2-methyl-1,4-NQ.
In order to enhance the sensitivity of a cell proliferation assay, it is
preferable to protect microorganisms from the toxicity of quinones during
the measurement. The effect of the various concentrations (0120 M)
of 2-methyl-1,4-NQ on the amount of formazan produced by S. cerevisiae,
S. enteritidis, and B. cereus was studied as shown in Fig. 2. When
S. enteritidis and B. cereus were employed, the absorbance change
increased with increasing concentration of 2-methyl-1,4-NQ. However,
in the case of S. cerevisiae, the absorbance increase was inhibited above
the nal concentration of 80 M. Therefore, we decided to use 2-methyl1,4-NQ at the nal concentration of 40 M.
3.2. Comparison of tetrazolium salts
We studied combinations of 6 kinds of tetrazolium salts with 2methyl-1,4-NQ as the electron mediator in consideration of the

3. Results and discussion

3.1. Comparison of electron mediators
The WST series and XTT contain sulfonate groups giving them a net
negative charge that reduces their ability to move across cell membranes
(Berridge et al., 2005). Thus, it is necessary to employ an electron mediator
to facilitate the cellular reduction of tetrazolium salts. Quinones are lipid
molecules present in all species of respiratory and photosynthetic
microorganisms. Quinones have been used not only as chemotaxonomic
markers in microbial systematics, but also as good measures of microbial
populations in the environment in terms of quantity, quality, and activity
(Hiraishi, 1999). Microbial quinones are categorized into two major
structural classes, the NQs and the BQs, which are represented by
menaquinone and ubiquinone. Therefore, the use of quinones as an
electron mediator for the microbial cell proliferation assay is thought to be
effective. Various derivatives of BQ, NQ, anthraquinone (AQ), or phenazine
were applied to the microbial assay. Five kinds of fermentation
microorganisms and 6 kinds of food-poisoning bacteria were used to
compare the electron mediators. Fig. 1 shows the effects of electron
mediators on the amount of formazan produced in the presence of WST-5
as the tetrazolium salt. WST-5 was chosen as the tetrazolium salt for the
sensitive detection of reductive substances produced by microorganisms
from electron mediators since WST-5 is more readily reduced by reductive
chemicals such as 2,3,5-trimethyl-1,4-hydroquinone and 2,3,5,6-tetramethyl-1,4-hydroquinone than other tetrazolium salts used in this study
(data not shown). As shown in Fig. 1, it would seem that the outcomes are
quite strain dependent. In BQs, 2,3,5-trimethl-1,4-BQ (B-5), 2,3,5,6tetramethyl-1,4-BQ (B-6), 2,5-dimethoxy-1,4-BQ (B-7), and 2,6dimethoxy-1,4-BQ (B-8) were effectively metabolized by food-poisoning
bacteria. However, the reduction rate of some BQs by S. enteritidis was
relatively low. On the other hand, only 2,3,5,6-tetramethyl-1,4-BQ was
effectively metabolized by fermentation microorganisms. In NQs, 1,4-NQ
(N-1), 2-methyl-1,4-NQ (N-3), 6-methyl-1,4-NQ (N-4), and 4-amino-1,2NQ (N-11) were effectively metabolized by food-poisoning bacteria.
However, only 2-methyl-1,4-NQ (N-3) was effectively metabolized by
fermentation microorganisms. AQs were hardly reduced by all microorganisms used here. Phenazines could be reduced by food-poisoning
bacteria except for L. monocytogenes but not fermentation microorganisms. These results indicated that 2-methyl-1,4-NQ was most effectively
metabolized by all microorganisms tested here. The purpose of the present
study was to develop an applicable detection method for various species of

Fig. 2. Effect of concentration of 2-methyl-1,4-NQ on the reduction of WST-8 by

microorganisms. Microbial cells were incubated in standard medium (pH 7.0) containing 0.5 mM WST-8 and 0120 M 2-methyl-1,4-NQ for 60 min at 30 or 37 C. Formazans
produced by microorganisms were measured at 460 nm with a microplate reader.
Concentration (M) of 2-methyl-1,4-NQ: 0, ; 20, ; 40, ; 80, ; 120, . Microbial cell
density: Saccharomyces cerevisiae, 3.48 10 6 CFU/ml; Salmonella enteritidis,
5.11 107 CFU/ml; Bacillus cereus, 3.20 105 CFU/ml.

T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

113

reactive efciency between tetrazolium salts and the hydroquinone

produced by microorganisms from 2-methyl-1,4-NQ, and the inuence with medium components.
Fig. 3 shows the effects of tetrazolium salts on the amount of
formazan produced in the presence of 2-methyl-1,4-NQ. S. cerevisiae,
S. enteritidis, and B. cereus were employed as representative yeast,
Gram-negative bacteria, and Gram-positive bacteria, respectively. At
the nal concentration of 0.1 mM, WST-1, WST-5, WST-8, and XTT gave
relatively high absorbances, as shown in Fig. 3(A). When tetrazolium
salts were used at the nal concentration of 0.5 mM, the amount of
formazan produced increased with increasing concentration of the
tetrazolium salt except with WST-5, as shown in Fig. 3(B). Especially,
in the case of B. cereus, formazan was hardly produced from WST-5. It
is likely that the inhibition is due to the cell toxicity of WST-5, whereas
the maximum absorbance was obtained in all microorganisms used
here when WST-8 was employed as the tetrazolium salt at a nal
concentration of 0.5 mM.
Culture mediums used for microbial cultivation contain various
components that may affect non-cellular reduction of tetrazolium
salts. In particular, the concentrations of yeast extract, peptones,
sugars, and lipids in culture mediums are relatively high. The noncellular reduction of tetrazolium salts leads to an overestimation of
microbial cell density. Therefore, the inuences of each co-existing
component on the reduction of tetrazolium salts WST-5, WST-8, and
XTT were compared (Fig. 4). The concentration of each component
was adjusted with phosphate-buffered saline (PBS, pH 7.0) and then

Fig. 4. Inuences of medium components on the non-cellular reduction of tetrazolium

salts. Tetrazolium salts (0.5 mM) were incubated in PBS (pH 7.0) containing each
medium component with 2-methyl-1,4-NQ (40 M) for 24 h at 37 C. The produced
formazans were measured at 550, 460, or 470 nm for WST-5, WST-8, or XTT,
respectively, with a microplate reader.

Fig. 3. Effects of tetrazolium salts on the amount of formazan produced by

microorganisms in the presence of 2-methyl-1,4-NQ. Microbial cells were incubated
in standard medium (pH 7.0) containing 0.1 or 0.5 mM tetrazolium salts and 40 M 2methyl-1,4-NQ for 60 min at 30 or 37 C. Formazans produced by microorganisms were
measured at 438, 550, 550, 460, 495, or 470 nm for WST-1, WST-4, WST-5, WST-8, WST9, or XTT, respectively, with a microplate reader. Microbial cell density: Saccharomyces
cerevisiae, 2.95 106 CFU/ml; Salmonella enteritidis, 6.05 107 CFU/ml; Bacillus cereus,
1.58 105 CFU/ml.

autoclaved for 15 min at 121 C. When WST-5 and XTT were employed,
the non-cellular reductions by yeast extract, malt extract, or soy
peptone in the absence of microorganisms were marked. In particular,
the absorbances increased remarkably when soy peptone and glucose
were autoclaved together. In this case, the Maillard reaction, a nonenzymatic reaction between protein amino groups and reducing
sugars, was promoted. It has been reported that certain tetrazolium
salts are reduced easily by the ketoamine moiety of glycated proteins
or by oxygen free radicals generated from glycated proteins (Azevedo
et al., 1988; Mashiba et al., 1992; Ukeda et al., 2002), whereas, in the
case of WST-8, the non-cellular reduction due to co-existing
components in culture mediums was hardly observed.
In this investigation, we also studied the inuences of commonly
used commercial culture mediums on the non-cellular reduction of
tetrazolium salts in the absence of microorganisms. As shown in Fig. 5,
WST-5 and XTT were reduced remarkably when brain heart infusion
broth, EC broth, or tryptic soy broth was used. These culture mediums
also slightly affected the reduction of WST-8. These culture mediums
contain large amounts of nutrient components such as peptones,
amino acids, various extracts, and sugars. However, no reduction of
WST-8 was observed after these culture mediums were stored for a

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T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

Fig. 6. Cell proliferation assays for Saccharomyces cerevisiae (), Salmonella enteritidis (),
and Bacillus cereus (). Microbial cells were incubated in standard medium (pH 7.0)
containing 0.5 mM tetrazolium salts and 40 M 2-methyl-1,4-NQ for 4 h at 30 or 37 C.
Formazans produced by microorganisms were measured at 460 nm with a microplate reader.

Fig. 5. Inuences of commercial culture mediums on the non-cellular reduction of

tetrazolium salts. Tetrazolium salts (0.5 mM) were incubated in commercial culture medium
with 2-methyl-1,4-NQ (40 M) for 24 h at 37 C. The formazans produced were measured at
550, 460, or 470 nm for WST-5, WST-8, or XTT, respectively, with a microplate reader.

month at 4 C (data not shown). This result showed that the reductive
reactivity of the culture mediums disappeared during storage. Thus,
these culture mediums should be stored for certain periods before use
to prevent the non-cellular reduction of tetrazolium salts. Signicant
inhibition of growth of microorganisms used here on mediums stored
for a month was hardly observed.
From the results described above, it is thought that the combination of WST-8 and 2-methyl-1,4-NQ is most appropriate for the
microbial cell proliferation assay in consideration of the reactive
efciency between tetrazolium salts and the hydroquinones produced
by microorganisms and the inuences with medium components.

measured by colony counting were obtained with good correlation

coefcients (r N 0.998). In addition, linear relationships with good
correlation coefcients (r N 0.994) were obtained in other microorganisms as well as the three kinds of microorganisms described above
(data not shown), suggesting that absorbance changes reected the
cell proliferation of various microorganisms.
Table 1 shows the viable cell density that gives an absorbance
change of 0.5 during the reaction times of 1 and 4 h. The cell density
was estimated from the calibration curve obtained by the present
method. In general terms, some microorganisms gave absorbance
changes of 0.5 at lower cell density (about 105106 CFU/ml), the others
gave the values at higher cell density of (about 106107 CFU/ml)
within 1 h. By the latter, it took 4 h at least to give the value at lower
cell density (about 105106 CFU/ml). Except for lactic acid bacteria,
Gram-positive bacteria showed about 10-fold greater sensitivity than
Gram-negative bacteria. It is well-known that Gram-negative bacteria
have thicker plasma membrane than Gram-positive bacteria. It is
thought that the difference of sensitivity between Gram-positive and
Gram-negative bacteria is due to the thickness of the plasma
membrane since an electron mediator must move across the plasma
membrane and then be functioned by reductases. The thick plasma
membrane would suppress the metabolic efciency of an electron
mediator. Signicant difference between strains having the same
Gram staining character was hardly observed. The sensitivity for lactic
acid bacteria such as E. faecalis and L. casei was almost equal to that for
Gram-negative bacteria. One explanation for the lower sensitivity
comparing other Gram-positive bacteria may be that the pH of a culture
medium decreased by the produced lactic acid since the reduction of

3.3. Cell proliferation assay and sensitivity

To test the applicability of the present method, the cell proliferation assay was investigated with various microorganisms including 3
kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of
Gram-negative bacteria. Fig. 6 shows the cell proliferation assays for S.
cerevisiae, S. enteritidis, and B. cereus. Linear relationships between the
absorbance obtained by the present method and viable cell density

Fig. 7. Time course of the reduction of tetrazolium salt during the cultivation of Salmonella
enteritidis at different cell densities. Salmonella enteritidis prepared at different cell
densities were incubated in standard medium (pH 7.0) containing 0.5 mM WST-8 and
40 M 2-methyl-1,4-NQ for12 h at 30 C. Formazans produced by Salmonella enteritidis
were measured at 460 nm with a microplate reader.

T. Tsukatani et al. / Journal of Microbiological Methods 75 (2008) 109116

tetrazolium salts is usually suppressed under acid conditions. Actually,

the pH of a culture medium decreased less than 6.0 after the assay. This
study showed that there was a more than double-digit difference for the
sensitivity to detect strains used in this study.
These results suggested that the present method can be applied as
a cell proliferation assay in various species of microorganisms,
although its sensitivity varied with the species of microorganism.
3.4. Long-term cultivation
Fig. 7 shows the time course for the absorbance obtained by the
proposed method during the cultivation of S. enteritidis at different
cell densities. At a high cell density (1.93 108 CFU/ml), the absorbance
increased rapidly and became constant within 2 h. The time required
to reduce all tetrazolium salts increased proportionally with decreasing initial cell density. At a cell density of 1.93 101 CFU/ml, it took 12 h
to nish the reduction of tetrazolium salts. When the detection time
(h) is dened as the time required to give an absorbance change of 0.5,
the detection time (y) could be expressed by
y 0:629logx 11:929
where [x] is the initial cell density (CFU/ml). A linear relationship
between the detection time (y) and the initial cell density (x) with a
good correlation coefcient (r = 0.9991) was obtained. This equation
shows that it takes about 12 h to produce an absorbance change of 0.5
by a single cell of S. enteritidis. This means that the test is Salmonellanegative when no change of absorbance is observed within 12 h. This
result suggested that the initial cell density can be estimated from the
time required to give a certain absorbance change.
3.5. Susceptibility curves and MIC determination
To evaluate the applicability of the proposed method for a
susceptibility test, susceptibility test of B. cereus against four
antimicrobial agents was carried out using the proposed method and
the broth microdilution method. The plots of the absorbances at
various concentrations of antimicrobial agents produced smooth
susceptibility curves of B. cereus (Fig. 8). The MICs estimated by the
present method were 0.12, 2.0, 4.0, and 0.24 g/ml for CFX, CP, CTX, and
GM, respectively. On the other hand, the MICs obtained by the broth
microdilution method were 0.12, 2.04.0, 4.0, and 0.120.24 g/ml for
CFX, CP, CTX, and GM, respectively. The MICs obtained by the proposed
method coincided well with those obtained by the conventional
method within one dilution. This result suggests that the present
method can provide a useful means for the rapid susceptibility test.

Fig. 8. Susceptibility test of Bacillus cereus to a range of antimicrobial agent doses.

Antimicrobial agent: CFX, ; CP, ; CTX, ; GM, . Bacillus cereus was incubated in
MuellerHinton broth containing 0.062564 g/ml antimicrobial agents for 6 h at 35 C
and then the detection reagent was added. After 2 h of incubation at 35 C, the produced
formazans were measured at 460 nm with a microplate reader.

115

4. Conclusion
A method to assay microbial cell proliferation in 96-well microtiter
plate using water-soluble tetrazolium salts and electron mediators
was developed, and its efciency was demonstrated. From the results
of this study, we found that 2-methyl-1,4-NQ was most effectively
metabolized by various microorganisms. The use of 2-methyl-1,4-NQ
as an electron mediator could give a method available for various
kinds of strains. Furthermore, it became clear that WST-8 was superior
to conventional tetrazolium salts such as XTT with regard to the
reactive efciency with the hydroquinones produced by microorganisms and the inuences with medium components. The use of WST-8
could lead to a more sensitive and reliable method than conventional
tetrazolium methods. From the point of view of the improvement for
applicability, sensitivity and reliability, the novel tetrazolium method
based on the combination of 2-methyl-1,4-NQ and WST-8 could serve
as the colorimetric technique for the cell proliferation assay of various
microorganisms.
By using microtiter plates, the advantages of simplicity and highthroughput are gained because samples must be handled individually in
the other quantication methods. Furthermore, the use of microtiter
plates allows large amounts of data to be generated quickly and with a
single assay procedure. Thus, the tetrazolium method using a microtiter
plate is a valuable method to assay microbial cell proliferation. The
proposed method would have various applications such as antimicrobial
susceptibility testing, and screening of antimicrobial substances.
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