colorimetric microbial viability assay

8/17/2019 Colorimetric Microbial Viability Assay

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Colorimetric microbial viability assay based on reduction of water-soluble

tetrazolium salts for antimicrobial susceptibility testing and screening

of antimicrobial substances

Tadayuki Tsukatani a,*, Tomoko Higuchi a, Hikaru Suenaga a, Tetsuyuki Akao a, Munetaka Ishiyama b,Takatoshi Ezoe b, Kiyoshi Matsumoto c

a Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 839-0861, Japanb Dojindo Laboratories, Kumamoto 861-2202, Japanc 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 8 May 2009

Available online 26 June 2009

Keywords:

Electron mediator

Microorganism

Naphthoquinone

Screening

Susceptibility testing

Tetrazolium salt

a b s t r a c t

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt

{2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H -tetrazolium, monosodium

salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for deter-

mining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was

investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-

1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and

pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the

minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and

those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric

assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics.

In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria

and its efficiency was demonstrated. 2009 Elsevier Inc. All rights reserved.

Recently, the need for rapid and accurate antimicrobial suscep-

tibility tests has been highlighted by the significant increase in the

number of antibiotic-resistant microorganisms causing clinical

infection. Standard methods, such as the broth microdilution

methods approved by the Clinical and Laboratory Standard Insti-

tute (CLSI)1 [1], take 18 to 22 h to determine the final antimicrobial

susceptibility of bacteria. Thus, methods for a rapid susceptibility

test using tetrazolium salts as indicator reagents have been devel-

oped [2–5].

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 [6]. The most

commonly used tetrazolium salt in colorimetric assays for micro-

organisms has been 2,3-bis (2-methyloxy-4-nitro-5-sulfophenyl)-

5-[(phenylamino)carbonyl]-2H -tetrazolium hydroxide (XTT); after

reduction, XTT yields a water-soluble formazan derivative that can

be easily quantified colorimetrically [7,8]. XTT have been used in

rapid colorimetric assays for antimicrobial susceptibility testing

of both bacteria and fungi [2,3]. However, a detailed study on an

electron mediator has not been performed. XTT requires an elec-

tron mediator for the cellular reduction because it is characterized

by a net negative charge and, therefore, is largely cell-impermeable

[6]. Thus, the selection of an electron mediator is very important

for the reduction of tetrazolium salts by microorganisms to forma-

zan. In addition, a detailed investigation on the influence with

medium components has not been carried out. XTT may be easily

reduced by components such as peptones and glycated proteins

in culture medium. We have developed the colorimetric method

based on the reduction of the tetrazolium salt {2-(2-methoxy-4-

nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazo-

lium, monosodium salt [WST-8]} for a microbial viability assay by

standardizing various factors that affect WST-8 conversion [9].

WST-8 and XTT are similar sulfonated tetrazolium salts, as shown

in Fig. 1. In this method, the 2-methyl-1,4-naphthoquinone (2-

0003-2697/$ - see front matter 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2009.06.026

* Corresponding author. Fax: +81-942-30-7244.

E-mail address: [email protected] (T. Tsukatani).1

Abbreviations used: CLSI, Clinical and Laboratory Standard Institute; XTT, 2,3-bis

(2-methyloxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H -tetrazolium

hydroxide; WST-8, {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulf-

ophenyl)-2 H -tetrazolium, monosodium salt; 2-methyl-1,4-NQ, 2-methyl-1,4-naph-

thoquinone; CFU, colony-forming units; MIC, minimum inhibitory concentration;

MTT, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H -tetrazolium bromide; Mops, 3-

morpholinopropanesulfonic acid; MRS, de Man–Rogosa–Sharpe; DMSO, dimethyl

sulfoxide; NBRC, Biological Resource Center in the National Institute of Technology

and Evaluation; JCM, Japan Collection of Microorganisms in RIKEN BioResource

Center; ATCC, American Type Culture Collection; AMP, ampicillin; CPFX, ciproflox-

acin; CFX, cefotaxime; CP, chloramphenicol; GM, gentamicin.

Analytical Biochemistry 393 (2009) 117–125

Contents lists available at ScienceDirect

Analytical Biochemistry

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 / y a b i o

http://dx.doi.org/10.1016/j.ab.2009.06.026mailto:[email protected]://www.sciencedirect.com/science/journal/00032697http://www.elsevier.com/locate/yabiohttp://www.elsevier.com/locate/yabiohttp://www.sciencedirect.com/science/journal/00032697mailto:[email protected]://dx.doi.org/10.1016/j.ab.2009.06.026


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methyl-1,4-NQ) used as an electron mediator was reduced by

microorganisms, and WST-8 was then reduced by the naphthohy-

droquinone produced to its formazan, which exhibits a maximum

absorbance at 460 nm. Our results indicated that WST-8 is superior

to XTT with regard to the reactive efficiency with electron media-

tors (reduced form) produced by microorganisms and the effects of medium components. However, further investigation suggested

that high concentrations of 2-methyl-1,4-NQ suppressed some

microbial proliferation. In particular, microbial proliferation was

inhibited at relatively low cell density (


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The current method is based on the measurement of the metabolic

activity of microorganisms. Therefore, it is thought that the meta-

bolic activity of the total microbial cell mass is proportional to the

absorbance obtained by the current method. Fig. 2 shows the rela-

tionship between microbial cell density and the absorbance in

E. coli and B. cereus in the logarithmic growth phase. Linear rela-

tionships were obtained between the cell density and the absor-

bance with good correlation coefficients. In all microorganisms

shown in Table 1, linear relationships were obtained (data not

shown). Therefore, it seems reasonable to suppose that the meta-bolic activity measured by the WST-8 colorimetric method reflects

the microbial cell proliferation in the logarithmic growth phase.

The cultivated bacteria and yeast were diluted with cation-ad-

justed Mueller–Hinton broth and Mops-buffered RPMI-1640 med-

ium, respectively, to adjust the microbial cell density. The

microbial suspension (190ll) was added to each well of a 96-well

microtiter plate. Then the detection reagent (10 ll), 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 mea-

sured temporally as absorbance at 460 nm with a microplate read-

er (VersaMax, Molecular Devices, Sunnyvale, CA, USA).

Susceptibility testing

Reference MICs were determined by the broth microdilution

method currently recommended by the CLSI [1]. Serial 2-fold dilu-

tions of each antibiotic were prepared in cation-adjusted Mueller–

Hinton broth with dilutions of 0.007 to 256 lg/ml. Ampicillin

(AMP), ciprofloxacin (CPFX), cefotaxime (CFX), chloramphenicol

(CP), and gentamicin (GM) were used as the antibiotics. Bacteria

were adjusted with phosphate-buffered saline to a turbidity equal

to that of the 0.5 McFarland standard and then were diluted 10-fold. The prepared bacteria suspension was further diluted with

antibiotic solution to provide a final inoculum density of approxi-

mately 105 CFU/ml in each well. Each well of a plate was inocu-

lated with 100 ll of inoculum, and the plate was incubated for

22 h at 35 C. After incubation, the MIC was read as the lowest con-

centration of antibiotic at which there was no visible growth.

In the susceptibility tests using the proposed method, the inoc-

ulum (190ll) prepared as described above was incubated for 6 h at

35 C, and then 10 ll of the detection reagent was added to each

well. After incubation for 2 h at 35 C, the formazan produced

was measured at 460 nm with a microplate reader. The MIC was

Table 1

Effects of the final concentration of 2-methyl-1,4-NQ on the detection time (1 CFU/ml).

Microorganism 2-Methyl-1,4-NQ concentration (lM)

1 5 10 40 80

Bacteria

Gram-negative

Escherichia coli NBRC3972 – 8.8 8.5 8.0 8.2

Klebsiella pneumoniae NBRC3512 – 10.1 9.8 9.9 10.5

Proteus mirabilis NBRC13300 – 13.6 12.9 13.6 13.8

Pseudomonas aeruginosa NBRC13275 – 19.5 18.2 15.8 15.4

Salmonella enterica subsp. enterica NBRC3313 – 11.0 10.2 10.2 10.1

Serratia marcescens NBRC102204 – 10.9 10.6 10.5 10.5

Vibrio parahaemolyticus NBRC12711 17.1 19.3 26.5 n.d. –

Gram-positive

Bacillus cereus NBRC13494 6.2 7.1 9.1 n.d. –

Bacillus subtilis JCM1465 9.1 9.9 n.d. n.d. –

Enterococcus faecalis JCM5803 11.0 9.4 9.3 8.4 –

Listeria monocytogenes ATCC15313 22.7 23.1 22.7 30.3 –

Micrococcus luteus NBRC13867 31.1 29.1 27.3 30.2 –

Staphylococcus aureus subsp. aureus NBRC12732 13.9 16.1 22.6 n.d. –

Staphylococcus epidermidis NBRC12993 25.2 29.2 n.d. n.d. –

Yeast

Candida albicans JCM2085 26.2 26.5 25.6 34.2 –

Candida krusei NBRC1395 29.2 28.4 32.9 n.d. –Candida parapsilosis NBRC1396 27.8 28.5 n.d. n.d. –

Saccharomyces cerevisiae NBRC2347 27.5 31.2 n.d. n.d. –

Note. Values are in hours (h). n.d., not detected because the proliferation was inhibited.

0.0

0.5

1.0

1.5

2.0

2.5

Microbial cell density (×106

CFU/ml)

A b s o r b a n c e ( 4 6 0 n m )

0.0

0.5

1.0

1.5

2.0

2.5

0.0 0.5 1.0 1.5 2.0 2.5 3.00.0 1.0 2.0 3.0 4.0 5.0 6.0

Microbial cell density (×107

CFU/ml)

A b s o r b a n c e ( 4 6 0 n m )

A B

Fig. 2. Measurement of microbial cell proliferation by using WST-8 colorimetric microbial viability assay: (A) E. coli; (B) B. cereus. Microbial cells were incubated in cation-

adjusted Mueller–Hinton broth containing 0.5 mM WST-8 and 5 lM 2-methyl-1,4-NQ for 1 h at 37C. Formazan produced by microorganisms was measured at 460 nm witha microplate reader.

Colorimetric microbial viability assay/ T. Tsukatani et al. / Anal. Biochem. 393 (2009) 117–125 119


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read as the lowest concentration of antimicrobial agent at which

absorbance change was less than 0.05 versus the blank value that

was obtained without bacteria.

Screening of bacteriocin-producing lactic acid bacteria

For the screening of bacteriocin-producing lactic acid bacteria,

Lactobacillus lactis NBRC12007, NBRC100933, and other lactic acid

bacteria isolated in our laboratory were grown in MRS medium

at 30 C for 1 day. The incubated medium was adjusted to pH 6.5

to 6.8 with 8 M NaOH and then was filtered with a membrane filter

(0.2 lm).

In the proposed method, 90 ll of cation-adjusted Mueller–Hin-

ton broth was added to each well, and then 90 ll of the medium

prepared above was mixed in the well. After that, 10 ll of the test

organism was added to each well and then was incubated for 6 h at

37 C. After incubation, 10ll of the detection reagent was added to

each well. After incubation for 2 h at 37 C, the formazan produced

was measured at 460 nm with a microplate reader.

On the other hand, reference antimicrobial activity of the med-

ium incubated with lactic acid bacteria was measured using the

spot-on-lawn method as the conventional method [12,13].

Results

Effect of concentration of 2-methyl-1,4-NQ on microbial cell

proliferation

The WST-8 contains sulfonate groups giving them a net nega-

tive charge that reduces their ability to move across cell mem-

branes [6]. Thus, it is necessary to employ an electron mediator

to facilitate the cellular reduction of tetrazolium salts. We have

found that 2-methyl-1,4-NQ is metabolized most effectively by

various microorganisms [9]. However, further investigation sug-

gested that high concentrations of 2-methyl-1,4-NQ suppressed

some microbial proliferations. Fig. 3 shows the effects of 2-

methyl-1,4-NQ at the final concentration of 40 lM on the prolifer-

ation of E. coli, K. pneumoniae, B. cereus, and S. aureus. When E. coli

and K. pneumoniae were employed, the absorbance increased with

increasing cultivation times in the density range of 101 to 108 CFU/

ml. On the other hand, in the case of B. cereus

and S. aureus

, the

absorbance increase was inhibited less than the density of 103 to

104 CFU/ml. It is preferable to protect microorganisms from the

toxicity of quinones during the measurement so as to enhance

the sensitivity of the cell proliferation assay. Thus, the effect of

the final concentrations of 2-methyl-1,4-NQ on the cell prolifera-

tion assay of B. cereus was studied. As shown in Fig. 4, growth

was observed at the 2-methyl-1,4-NQ concentrations of 1, 5, and

10lM but not at 40 lM. This result suggested that the decrease

of the concentration of 2-methyl-1,4-NQ repressed its toxicity dur-

ing the measurement. At the final concentration of 5 lM, when the

detection time is defined as the time required to give an absor-

bance change of 0.5, the detection time ( y/h) could be expressed by

y ¼ 0:435log½ x þ 7:12;

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 coefficient (r = 0.9997) was obtained. This equa-

tion shows that it takes 7.12 h to produce an absorbance change of

0.5 by a single cell of B. cereus. Likewise, the effects of the final con-

centrations (1–80 lM) of 2-methyl-1,4-NQ on the cell proliferation

assays of various microorganisms were studied. Table 1 shows the

detection times required to obtain an absorbance change of 0.5 by

a single cell of various microorganisms. As described above, the

detection time was estimated from the calibration curve obtained

by the current method. Except for V. parahaemolyticus, the prolifer-

0.0

1.0

2.0

3.0

4.0

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

0 2 4 6 8 10 12 0 2 4 6 8 10 12

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

0 2 4 6 8 10 0 2 4 6 8 10

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

A B

DC

Fig. 3. Effects of 2-methyl-1,4-NQ (40lM)on theproliferation assaysof various microorganisms:(A) E. coli; (B) K. pneumoniae; (C) B. cereus; (D) S. aureus. Microbial cells were

incubated in cation-adjusted Mueller–Hinton broth containing 0.5 mM WST-8 and 40lM 2-methyl-1,4-NQ at 37 C. Formazan produced by microorganisms was measured

temporally at 460 nm with a microplate reader. Cell density (CFU/ml): E. coli, 4.2 10n; K. pneumoniae, 4.1 10n; B. cereus, 4.2 10n; S. aureus, 1.9 10n. 10n = 108 (j), 107(h), 106 (), 105 (e), 104 (N), 103 (D), 102 (d), or 101 (s).

120 Colorimetric microbial viability assay / T. Tsukatani et al. / Anal. Biochem. 393 (2009) 117–125


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ations of gram-negative bacteria were hardly affected by the con-

centration of 2-methyl-1,4-NQ. This result shows that gram-nega-

tive bacteria have resistance to the toxicity of 2-methyl-1,4-NQ.

Therefore, we decided to use 2-methyl-1,4-NQ at the final concen-

tration of 40lM for gram-negative bacteria except for V. parahae-molyticus. In gram-positive bacteria and yeast, the proliferations

of some microorganisms were inhibited above the final concentra-

tion of 10lM. At less than 5lM, good proliferations were observed

in all microorganisms, although the sensitivity varied with the spe-

cies of microorganism. Decreasing the concentration of 2-methyl-

1,4-NQ tended to diminish the detection time. However, a further

decrease of the concentration of 2-methyl-1,4-NQ increased thedetection time in the case of relatively high cell density (106 CFU/

ml). Table 2 shows the detection times that give an absorbance

0.0

1.0

2.0

3.0

4.0

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

0 2 4 6 8 10 12 0 2 4 6 8 10 12

Time (h)

A b s o r b

a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

Time (h)

A b s o r

b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

0 2 4 6 8 10 12 0 2 4 6 8 10 12

Time (h)

A b s o r b a n c e ( 4 6 0 n m )

A B

DC

Fig. 4. Effects of the final concentration of 2-methyl-1,4-NQ on the proliferation assay of B. cereus: (A) 40 lM; (B) 10 lM; (C) 5 lM; (D) 1 lM. Microbial cells were incubated

in cation-adjusted Mueller–Hinton broth containing 0.5 mM WST-8 and 1 to 40lM 2-methyl-1,4-NQ at 37 C. Formazan produced by microorganisms was measured

temporally at 460 nm with a microplate reader. Cell density (CFU/ml): B. cereus, 4.2 10n. 10n = 108 (j), 107 (h), 106 (), 105 (e), 104 (N), 103 (D), 102 (d), or 101 (s).

Table 2

Effects of the final concentration of 2-methyl-1,4-NQ on the detection time (106 CFU/ml).

Microorganism 2-Methyl-1,4-NQ concentration (lM)

1 5 10 40 80

Bacteria

Gram-negative

Escherichia coli NBRC3972 – 2.6 2.4 2.1 1.9

Klebsiella pneumoniae NBRC3512 – 2.8 2.6 2.2 2.2

Proteus mirabilis NBRC13300 – 2.5 2.4 2.0 2.1

Pseudomonas aeruginosa NBRC13275 – 6.6 5.9 4.8 4.4

Salmonella enterica subsp. enterica NBRC33l3 – 3.5 3.3 2.8 2.6Serratia marcescens NBRC102204 – 3.4 3.1 2.8 2.8

Vibrio parahaemolyticus NBRC12711 4.7 4.4 5.9 9.4 –

Gram-positive

Bacillus cereus NBRC13494 1.7 1.1 1.1 1.6 –

Bacillus subtilis JCM1465 1.7 2.4 2.5 1.1 –

Enterococcus faecalis JCM5803 –

Listeria monocytogenes ATCC15313 4.3 3.6 3.3 4.8 –

Micrococcus luteus NBRC13867 3.5 2.1 1.6 1.5 –

Staphylococcus aureus subsp. aureus NBRC12732 2.7 2.4 2.3 2.3 –

Staphylococcus epidermidis NBRC12993 5.1 4.8 4.3 2.5 –

Yeast

Candida albicans JCM2085 1.4 0.5 0.4 0.2 –

Candida krusei NBRC1395 2.0 1.0 0.8 1.0 –

Candida parapsilosis NBRC1396 3.9 1.6 1.2 1.1 –

Saccharomyces cerevisiae NBRC2347 2.5 1.2 0.8 0.6 –

Note. Values are in hours (h).



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change of 0.5 by microbial cells at the density of 106 CFU/ml. It is

undesirable that the sensitivity is reduced by a further decrease of

the concentration of 2-methyl-1,4-NQ. Hence, we decided to use

2-methyl-1,4-NQ at the final concentration of 5 lM for gram-posi-

tive bacteria, yeast, and V. parahaemolyticus with regard to the tox-

icity and sensitivity.

From the results described above, it is thought that microorgan-

isms will be detected by changing the concentration of 2-methyl-

1,4-NQ depending on the species of microorganisms measured

when the cell density is relatively low or unknown.

Rapid determination of MIC

The broth microdilution method proposed by the CLSI requires

at least 18 to 22 h to obtain the final antimicrobial susceptibility

results. Thus, a rapid susceptibility test is thought to be useful.

To evaluate the applicability of the WST-8 colorimetric method

for a rapid susceptibility test, MICs determined by the current

method were compared with those obtained by the CLSI method.

To determine the incubation time of bacteria and antibiotics,

the MICs obtained by the current method at various incubation

times were compared with those obtained by the CLSI method

requiring 22 h. After incubation, the reaction with the detection re-

agent for 2 h was performed. E. coli and B. cereus were employed as

representative gram-negative and gram-positive bacteria, respec-

tively. The effects of the incubation time on susceptibility curves

of E. coli and B. cereus are shown in Fig. 5. The MICs obtained by

the broth microdilution method were 2.0 to 4.0 and 64 lg/ml for

AMP against E. coli and CFX against B. cereus, respectively. Above

the incubation time of 6 h, the MICs determined by the current

method were 2.0 and 64 lg/ml for AMP against E. coli and CFX

against B. cereus, respectively. At the incubation time of 4 h, the

MIC value for AMP against E. coli

was lower than that obtained

by the conventional method because AMP merely delayed the pro-

liferation of E. coli at the concentration of 1 lg/ml. At the incuba-

tion time of 2 h, a higher MIC was obtained because the

incubation period of bacteria and antibiotics might be insufficient.

Therefore, we decided to incubate bacteria and antibiotics for 6 h

with regard to rapidity and accuracy of measurement.

The MICs for5 kindsof antibiotics against 10 kindsof bacteria are

presented in Table 3. E. coli, K. pneumoniae, P. aeruginosa, S. marces-

cens, and S. enterica wereemployed as representative gram-negative

bacteria. B. cereus, E. faecalis, L. monocytogenes, S. aureus, and M. lu-

teus were usedas representative gram-positive bacteria. AMP, CPFX,

CFX, CP,and GMwere appliedas the representative penicillins, quin-

olones, cephalosporins, chloramphenicols, and ansamycins, respec-

tively. There was 94% agreement within one dilution between the

MICs obtained by the current method and the broth microdilution

method. The level of agreement within two dilutions between the

both MICs was 100%. These results suggest that the current method

0.0

1.0

2.0

3.0

4.0

0.1 1 10 100 1000

AMP concentration (µg/ml)

A b s o

r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

0.1 1 10 100 1000

CFX concentration (µg/ml)

A b s o r b a n c e ( 4 6 0 n m )

A B

Fig. 5. Effects of the incubation time of bacteria and antibiotics on susceptibility curves: (A) E. coli; (B) B. cereus. Microbial cells were incubated in cation-adjusted Mueller–

Hinton broth containing antibiotics at various concentrations for 2 to 8 h at 35 C. Then thereaction with the detection reagent for 2 h wasperformed. Formazan produced by

microorganisms was measured at 460 nm with a microplate reader. Incubation times (h): 2, j; 4, ; 6, N; 8, d.

Table 3

MICs determined by the current method versus the broth microdilution method.

Antibiotic Esc herichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Serratia marcescens Salmonella enterica

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

AMP 2 2–4 32 128 >256 >256 64 128 1 1–2

CFX 0.062–0.125 0.031–0.125 0.007 0.007–0.015 8 8 0.5 0.5 0.125 0.062–0.125

CP 8 16 2 2 32 64 32 32 16 16

GM 0.5–1 1 0.125 0.125–0.25 1 2–8 0.5 1 0.25 0.25–0.5

CPFX 0.062 0.031–0.062 0.062 0.062–0.125 0.062 0.062–0.125 0.062 0.062 0.031 0.015

Antibiotic Bacillus cereus Enterococc us faecalis Listeria monoc ytogenes Staphylococcus aureus Micrococcus luteus

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

Current

method

Microdilution

method

AMP >256 >256 1 0.5–1 0.25 0.125 0.125 0.125–0.25 0.007 0.003–0.007

CFX 64 64 0.5–1 1 16 16 4 4 0.125 0.125

CP 4 4 8 8 8 4 8 8 2 4

GM 0.25 0.25 16 8–16 0.125 0.062–0.125 0.031 0.031–0.062 0.5 0.25–0.5

CPFX 0.125 0.125–0.25 2 2 16 2–4 1 0.25 4 2-4

Note. AMP, ampicillin; CFX, cefotaxime; CP, chloramphenicol; GM, gentamicin; CPFX, ciprofloxacin. Values are in concentrations (lg/ml).



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provides a useful method for the rapid determination of antimicro-

bial susceptibility of bacteria.

Advantage of WST-8 as compared with XTT in antimicrobial

susceptibility testing

WST-8 and XTT are similar sulfonated tetrazolium salts, as

shown in Fig. 1. XTT has been used in rapid colorimetric assays

for antimicrobial susceptibility testing of both bacteria and fungi

[2,3]. However, culture media used for microbial cultivation con-

tain various components that may affect noncellular reduction of

tetrazolium salts. The noncellular reduction of tetrazolium salts

leads to an underestimation of the activity of antimicrobial sub-

stances. Therefore, the influences of Mueller–Hinton broth, which

is usually used for the antimicrobial susceptibility testing and

screening of antimicrobial substances, on the reduction of WST-8

and XTT in the presence of 2-methyl-1,4-NQ were compared

(Fig. 6). When XTT was employed, the noncellular reductions in

Mueller–Hinton broth in the absence of microorganisms were

marked. Furthermore, the noncellular reduction of XTT was pro-

moted by the addition of antibiotics such as CFX. The absorbance

increase of approximately 0.05 was observed for 2 h. Therefore,

the XTT colorimetric method would give an inaccurate result be-

cause the MIC is read as the lowest concentration of antimicrobial

substance at which absorbance change is less than 0.05 versus the

blank value that was obtained without microorganisms. On the

other hand, Mueller–Hinton broth gave minimal rise to the noncel-

lular reduction of WST-8. From the results described above, we be-

lieve that WST-8 is superior to XTT for antimicrobial susceptibility

testing.

Rapid screening for bacteriocin-producing lactic acid bacteria

The spot-on-lawn method is usually used for determining bac-

teriocin activity of lactic acid bacteria [12,13]. However, it is diffi-cult to screen a large number of samples of lactic acid bacteria by

the conventional method because spotting samples on solid cul-

ture medium requires a great deal of labor and the formation

and measurement of inhibition zone (halos) is time-consuming.

Therefore, a high sampling frequency is required for screening of

bacteriocin-producing lactic acid bacteria. The current method

using a microtiter plate is a suitable method to meet this demand.

To determine the measurement conditions, the effects of the

incubation time of bacteria and bacteriocin were studied. As test

organisms, B. cereus, S. aureus, L. monocytogenes, and M. luteus were

used. Fig. 7 shows the effect of the incubation times on susceptibil-

0.0

0.1

0.2

0.3

0.4

0.5

0 1 2 3 4

Time (h)

A b s o r b a n c e

Fig. 6. Influences of medium components on the noncellular reduction of tetrazo-

lium salts. Tetrazolium salts (0.5 mM) were incubated in cation-adjusted Mueller–

Hinton broth containing various antibiotics (64 lg/ml) with 2-methyl-1,4-NQ

(5lM) at 37 C. The formazans produced were temporally measured at 460 or

470 nm for WST-8 or XTT, respectively, with a microplate reader. WST-8, open

symbols; XTT, closedsymbols. Antibiotics: CFX,h andj; CPFX,e and; GM,D and

N; none, s andd.

0.0

1.0

2.0

3.0

4.0

10 100 1000

Nisin concentration (IU/ml)

A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

10 100 1000


A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

10 100 1000


A b s o r b a n c e ( 4 6 0 n m )

0.0

1.0

2.0

3.0

4.0

10 100 1000


A b s o r b a n c e ( 4 6 0 n m )

A B

DC

Fig. 7. Effects of incubation time of bacteria andnisin on susceptibility curves: (A) B. cereus; (B) S. aureus; (C) L. monocytogenes; (D) M. luteus. Microbial cells were incubated in

cation-adjusted Mueller–Hinton broth containing nisin at various concentrations for 2 to 8 h at 37 C. Then the reaction with the detection reagent for 2 h was performed.Formazan produced by microorganisms was measured at 460 nm with a microplate reader. Incubation times (h): 2, j; 4, ; 6, N; 8, d.



8/9

ity curves of test organisms against nisin. After a certain incubation

time, the reaction with the detection reagent for 2 h was per-

formed. The MIC was read as the lowest concentration of nisin at

which absorbance change was less than 0.05 versus the blank va-

lue obtained without test organisms. Above the incubation time

of 4 h, the MICs determined by the current method became con-

stant. However, the absorbance changes versus the blank value

were relatively small at the incubation time of 4 h. Therefore, 6 h

was selected as the incubation time for the screening of bacterio-

cin-producing lactic acid bacteria.

The ability of 51 strains of lactic acid bacteria isolated from the

natural world and various foods to produce bacteriocin were eval-

uated. As a positive or negative control, Lactococcus lactis

NBRC12007 or NBRC100933 was used, respectively. L. lactis

NBRC12007, which is well known to have a high ability to produce

nisin, showed a strong inhibitory effect against gram-positive bac-

teria. Thus, we defined the antimicrobial activity of L. lactis

NBRC12007 as the threshold to identify lactic acid bacteria having

high bacteriocin productivity. As shown in Table 4, among the 51

strains tested, only four strains were capable of producing bacte-

Table 4

Antimicrobial activity determined by the current method versus the spot-on-lawn method.

Bacillus cereus Staphylococcus aureus Listeria monocytogenes Micrococcus luteus

Current

method

Spot-on-lawn

method

Current

method

Spot-on-lawn

method

Current

method

Spot-on-lawn

method

Current

method

Spot-on-lawn

method

Lactococcus lactis NBRC12007 + + + + + + + +

Lactococcus lactis NBRC100933

Sample

1 + + + + + + + +

2 + ± + + + ± + +

3

4

5 + ± + + + + + ±

6

7

8

9

10

11

12

13

14

15

16

17

18 + +

19 + +

20 21

22 ± + + + + + +

23

24

25

26

27

28 ± + + + + + +

29 + ± + + + + + ±

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

Note. Current method: +, complete inhibition of formazan formation after assay of 8 h; ±, delay of formazan formation (inhibition of formazan formation after assay of 8 h but

no inhibition of formazan formation after assay of 24 h); , no inhibition of formazan formation after assay of 8 h. Spot-on-lawn method: +, complete inhibition of test

organism’s growth after incubation of 24 h; ±, delay of indicator’s growth (inhibition of test organism’s growth after incubation of 24 h but no inhibition of test organism’sgrowth after incubation of 48 h); , no inhibition of test organism’s growth after incubation of 24 h.



9/9

riocin equal or superior to L. lactis NBRC12007. Antimicrobial spec-

tra obtained by the current method showed good agreement with

those obtained by the spot-on-lawn method.

Discussion

The WST-8 colorimetric method for microbial viability assay

was applied to antimicrobial susceptibility testing and screening

of bacteriocin-producing lactic acid bacteria, and its efficiency

was demonstrated.

The measurement conditions, in particular the effect of the con-

centration of 2-methyl-1,4-NQ, was studied for proliferation assays

of gram-negative bacteria, gram-positive bacteria, and pathogenic

yeast, respectively. The results showed that high concentrations

of 2-methyl-1,4-NQ suppressed proliferation of some microbes

(Table 1). Therefore, we decided to use 2-methyl-1,4-NQ at differ-

ent concentrations depending on the species of microorganism.

The final concentration of 40 lM was chosen for gram-negative

bacteria except for V. parahaemolyticus. For gram-positive bacteria,

yeast, and V. parahaemolyticus, we decided to use 2-methyl-1,4-NQ

at the final concentration of 5 lM. In the case of relatively high cell

density, good proliferations of all microorganisms examined were

obtained at the final concentration of 40 lM, as shown in Table

2. Therefore, the final concentration of 40 lM can be used when

it is known that the microbial cell density is relatively high. In

applications such as antimicrobial susceptibility testing and

screening of bacteriocin-producing lactic acid bacteria, 2-methyl-

1,4-NQ should be used at different concentrations depending on

the species of microorganisms because the density of living micro-

bial cells is unknown after the incubation of microorganisms and

antimicrobial substance.

In antimicrobial susceptibility testing, MICs for 5 kinds of anti-

biotics against 10 kinds of bacteria were measured. The agreement

within one dilution between the MICs obtained by the current

method and the broth microdilution method was 94%. The detec-

tion time with the current method was 8 h total. On the otherhand, the conventional method requires 22 h to determine the

MIC. In the case of L. monocytogenes, the MIC values obtained by

the proposed method were slightly higher than those determined

by the broth microdilution method. Therefore, it may require more

than 6 h to obtain more accurate MIC values in this case. However,

the differences between the MICs obtained by both methods were

within 2 dilutions, and this difference is acceptable for antimicro-

bial susceptibility testing. These results suggest that the WST-8

method could provide a useful means for accurate and rapid deter-

mination of antimicrobial susceptibility testing. The current meth-

od can substitute for the tedious and time-consuming conventional

protocols, particularly the broth microdilution method.

In screening of bacteriocin-producing lactic acid bacteria, the

abilities of 51 strains of lactic acid bacteria isolated from the natu-ral world and various foods to produce bacteriocin were evaluated.

Table 4 shows the antimicrobial activity of 51 isolates of lactic acid

bacteria against four test organisms. A slight disagreement was ob-

served between the results obtained by the proposed method and

those obtained by the spot-on-lawn method. In the conventional

method, some tests were judged to be positive for antimicrobial

activity after the incubation time of 24 h and then to be negative

after 48 h because the growths of test organisms were merely de-

layed by bacteriocin produced by lactic acid bacteria. In this case, it

is thought that the quantity and/or quality of bacteriocin produced

by lactic acid bacteria are insufficient to inhibit the growth of test

organisms completely. The current method judged these tests to be

positive for antimicrobial activity. This result shows that the pro-

posed method is a suitable means of screening of bacteriocin-pro-ducing lactic acid bacteria because this method can detect

antimicrobial activity more widely. Some tests were judged to be

positive for antimicrobial activity after 8 h assay and then to be

negative after 24 h in the proposed method. It is thought that for-

mazan formation is delayed in the case of the antimicrobial sub-

stances having a bacteriostatic effect. Therefore, this assay

system might be able to distinguish between bacteriocidal effects

and bacteriostatic effects of antimicrobial substances by two

observations at regular intervals.

Amongthe 51 strains, only4 were capable of producing bacterio-

cinequal or superiorto L. lactis NBRC12007.Therefore,it is necessary

to measure a large number of samples to find lactic acid bacteria

having high antimicrobial activity in the natural world. The WST-8

colorimetric method using a microtiter plate is an effective method

to meet this demand. In addition, the conventional method requires

a great deal of labor to spot samples on solid culture medium, and

the formation and measurement of inhibition zones (halos) is

time-consuming. The proposed procedure using a microtiter plate

anda micropipette is simpler, and the results canbe obtainedwithin

8 h. Furthermore, the current method needs only one microtiter

plate when antimicrobial activities of 24 samples against four test

organisms are measured. On the other hand, the spot-on-lawn

method requires 12 dishes when 8 samples are spotted on each dish

to measurethe same numberof samples. Thus, thecurrentmethod is

suitable for screening a large number of samples andis simpler and

quicker than the conventional method.

In conclusion, the WST-8 colorimetric method using a microti-

ter plate is a valuable method for antimicrobial susceptibility test-

ing and screening of antimicrobial substances.

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