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Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells
利用四唑红定量分析细菌细胞内的还原活性   

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Abstract

This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol allows the production of reproducible results in a relatively short time for Sinorhizobium meliloti 1021 cells grown both in exponential and stationary phases. The results here presented show that the S. meliloti cells deriving from exponential-phase cultures had increased cell redox potential as compared to the ones deriving from stationary-phase cultures. This means that under exponential growth conditions the S. meliloti cells generate higher amount of reducing equivalents needed for TTC reduction.

Keywords: Sinorhizobium meliloti 1021(苜蓿中华根瘤菌1021), Cell redox potential(细胞氧化还原电位), 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)(2,3,5-三苯基-2H-四唑氯化物(TTC)), 1,3,5-Triphenyltetrazolium formazan(1,3,5-三苯基四氮唑甲), Bacterial cells(细菌细胞)

Background

The TTC salt is a water-soluble and colorless compound that can be reduced to formazan, a highly colored compound. The irreversible formation of formazan can be quantified using a spectrophotometer. Owing to its property and its low reduction potential, this tetrazolium salt is widely used in both eukaryotes and prokaryotes as an indicator of cell redox activity, viability, drug susceptibility and substrate utilization assays (Byth et al., 2001; Hayashi et al., 2003; Raut et al., 2008; Lin et al., 2008). The net positive charge on tetrazolium salts facilitates cellular uptake due to the membrane potential, allowing their intracellular reduction (Berridge et al., 2005). In prokaryotes, the main studies of TTC reduction have concerned the Gram-negative respiring bacterium Escherichia coli, while only a few studies have been reported for members of the Rhizobiacea family. In this protocol, one of the best genetically characterized members of this family, the S. meliloti 1021 rhizobium strain, was used. The respiratory activity, expression of cytochrome terminal oxidases, of this strain was analysed using TTC as an indicator of cell redox potential.

To enable the development of a measurable color intensity and, at the same time, to avoid any possible inhibition of bacterial growth, the bacteria were incubated in the presence of TTC for an appropriate period of time compared to those described by other authors (Tengerdy et al., 1967; Byth et al., 2001; Tachon et al., 2009).

Materials and Reagents

  1. Sterile inoculation loop with incorporated needle (NUOVA APTACA, catalog number: 6001/SG/CS )
  2. 14-ml polypropylene round-bottom tubes (Corning, Falcon®, catalog number: 352059 )
  3. 50-ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  4. Safe-lock 2-ml tubes (Eppendorf, catalog number: 0030120094 )
  5. Sinorhizobium meliloti 1021
  6. 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Sigma-Aldrich, catalog number: T8877 )
  7. 1,3,5-Triphenyltetrazolium formazan (Sigma-Aldrich, catalog number: 93145 )
  8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D5879 )
  9. Tryptone (Sigma-Aldrich, catalog number: T9410 )
  10. Yeast extract (Sigma-Aldrich, catalog number: Y0375 )
  11. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C3306 )
  12. Na2HPO4 (Acantor® Performance Materials, J.T. Baker, catalog number: 4062-01 )
  13. NaH2PO4 (Acantor® Performance Materials, J.T. Baker, catalog number: 3818-05 )
  14. TYR broth medium (5 g/L tryptone, 3 g/L yeast extract, 6 mM CaCl2) (see Recipes)
  15. Sodium phosphate buffer (pH 7.5) (see Recipes)

Equipment

  1. Incubator room (at 30 °C)
  2. Rotary shaker
  3. Spectrophotometer (Beckman Coulter, catalog number: DU800 )
  4. Cuvettes for spectrophotometry application in the visible spectrum (Kartell, catalog number: 1938 )
  5. Micro-centrifuge (SCILOGEX D3024 High Speed Micro-Centrifuge) (Scilogex, catalog number: 912015139999 )
  6. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MegafugeTM 16R )
  7. Oven (at 65 °C)
  8. Balance (Mettler Toledo, catalog number: B204-S )

Procedure

  1. Growth of bacterial cells
    1. Pure cultures of S. meliloti 1021 stored at -80 °C were taken by sterile loops, inoculated in 15-ml round-botton tubes containing 2.0 ml of TYR medium and incubated for 24 h at 30 °C on a rotary shaker (200 rpm).
    2. Aliquots of the cultures (0.15 ml) were transferred into 50-ml conical centrifuge tubes containing 15 ml of fresh TYR medium (OD600 = 0.2) and the optical density measured at 600 nm using a spectrophotometer and cuvettes for spectrophotometry application in the visible spectrum.
    3. When the cultures reach the exponential growth phase (OD600 = 0.7), which requires about 4 h of incubation, stop the growth and split the cultures in 10 ml for biomass evaluation and 1.5 ml for TTC reduction measurement.

  2. TTC assay
    1. Centrifuge the 10 ml bacterial cultures in 15-ml polypropylene round-bottom at 5,000 x g for 20 min (at room temperature) and discard the supernatant.
    2. Incubate the cells at 65 °C in an oven for 4 h until dryness and weigh the cells with a balance.
    3. Centrifuge the 1.5 ml bacterial cultures in 2-ml Eppendorf tubes at 8,000 x g for 5 min (at room temperature) and discard the supernatant.
    4. Wash the bacteria with 1 ml of 50 mM sodium phosphate buffer (pH 7.5), centrifuge at 8,000 x g for 5 min (at room temperature) and discard the supernatant.
    5. Resuspend the cells in 1 ml of 50 mM sodium phosphate buffer (pH 7.5) containing 24 mM 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) and incubate at 30 °C for 1 h on a rotary shaker at 200 rpm (Figure 1).


      Figure 1. Picture of the cell suspensions after 1 h of incubation at 30 °C

    6. Collect the cells by centrifugation at 8,000 x g for 5 min (at room temperature) and discard the supernatant (Figure 2).


      Figure 2. Picture of the bacterial cells after the centrifugation step

    7. Resuspend the cells thoroughly in 1 ml 99.5 % DMSO (at room temperature) to dissolve the produced formazan.
    8. Spin down the bacterial cells by one-minute centrifugation at 13,000 x g (at room temperature) and collect the supernatants.
    9. Prepare formazan standard solutions at different known concentrations dissolving the powder in 99.5 % DMSO.
    10. Read the absorbance of the samples (supernatants) and formazan standards at 510 nm using a spectrophotometer. Use the 99.5 % DMSO as blank control.
    11. Plot the calibration curve of the formazan standards (mg/ml) against the absorbance of the solutions at 510 nm.
    12. Determinate the amount of formazan produced from reactions with the bacterial cells directly from the formazan calibration curve.
    13. Normalize the data to cell biomass (g) corresponding to 1.5 ml bacterial cells and determine the amount of formazan produced per g cells.

Data analysis

  1. Perform at least five biological replicates, each conducted at different times.
  2. An example of the excel Formazan standard curve is described in Table 1.
  3. The readings of some experimental samples and the amount of formazan they produced are reported in Table 2 and Table 3.

    Table 1. Formazan standards and their relative absorbance at 510 nmv


    Optical densities were determined using a spectrophotometer and data were plotted as shown in Figure 3. 


    Figure 3. Formazan standard curve. The optical density at 510 nm was determined for a range of Formazan standards from 0.0075-0.3 mg/ml. The linear regression line was added by using the Excel 2011 for Mac (14.1.0 version). The equation for the regression line and the correlation coefficient are shown on the graph. 

    Table 2. Amount of formazan produced by S. meliloti 1021 cells grown up to exponential phase


    The amount of formazan reported in this table was calculated by interpolating the standard solution points reported in Table 1 and elaborated in Figure 1. 

    Table 3. Amount of formazan produced by S. meliloti 1021 cells grown up to stationary phase


    The amount of formazan reported in this Table was calculated by interpolating the standard solution points reported in Table 1 and elaborated in Figure1. 

Recipes

  1. TYR broth medium (1 L)
    5 g tryptone
    3 g yeast extract
    Add ddH2O to 1 L. Sterilize by autoclaving
    After the solution has cooled add 12 ml sterile 0.5 M CaCl2
  2. Sodium phosphate buffer (pH 7.5)
    1. Prepare 1 M NaH2PO4 solution by dissolving 1.2 g in a final volume of 10 ml double distilled water
    2. Prepare 1 M Na2HPO4 solution by dissolving 1.42 g in a final volume of 10 ml double distilled water
    3. Mix 1.6 ml 1 M NaH2PO4 solution and 8.4 ml 1 M Na2HPO4 solution and dilute to 200 ml with double distilled water to prepare 50 mM sodium phosphate buffer, pH 7.5

Acknowledgments

The method for the TTC assay was adapted from Tachon et al. (2009). This work was partially supported by a dedicated grant from the Italian Ministry of Economy and Finance to the National Research Council for the project CISIA ‘Innovazione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare - Legge No. 191/2009’. This work was also partially supported by the European Commission for funding the ABSTRESS project (FP7 KBBE-2011-289562). The authors declare that they have no conflict of interests.

References

  1. Berridge, M. V., Herst, P. M. and Tan, A. S. (2005). Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11: 127-152.
  2. Byth, H. A., Mchunu, B. I., Dubery, I. A. and Bornman, L. (2001). Assesment of a simple, non-toxic almar blue cell survival assay to monitor tomato cell viability. Phytochem Anal 12: 340-346.
  3. Hayashi, S., Kobayashi, T. and Honda, H. (2003). Simple and rapid cell growth assay using tetrazolium violet coloring method for screening of organic solvent tolerant bacteria. J Biosci Bioeng 96(4): 360-363.
  4. Lin, Y. C., Agbanyim, C. N., Miles, R. J., Nicholas, R. A., Kelly, D. P. and Wood, A. P. (2008). Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster. J Appl Microbiol 105(2): 492-501.
  5. Raut, U., Narang, P., Mendiratta, D. K., Narang, R. and Deotale, V. (2008). Evaluation of rapid MTT tube method for detection of drug susceptibility of Mycobacterium tuberculosis to rifampicin and isoniazid. Indian J Med Microbiol 26(3): 222-227.
  6. Tachon, S., Michelon, D., Chambellon, E., Cantonnet, M., Mezange, C., Henno, L., Cachon, R. and Yvon, M. (2009). Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis. Microbiology 155(Pt 9): 2941-2948.
  7. Tengerdy, R. P., Nagy, J. G. and Martin, B. (1967). Quantitative measurement of bacterial growth by the reduction of tetrazolium salts. Appl Microbiol 15(4): 954-955.

简介

该方案描述了使用2,3,5-三苯基-2H-四唑氯化物(TTC)盐来评估根瘤细胞的细胞氧化还原电位。由TTC还原产生的鲜色不溶性1,3,5-三苯基四唑甲The的生产是不可逆的,可以使用分光光度计轻松地定量。该方案允许在相对较短的时间内在指数阶段和固定阶段生长的中华根瘤菌中华根瘤菌1021细胞产生可重复的结果。这里提出的结果表明,与来自固定相培养物的细胞相比,从指数阶段培养物得到的猕猴桃细胞具有增加的细胞氧化还原电位。这意味着在指数增长条件下, meliloti 细胞产生更大量的TTC减少所需的还原等同物。

背景 TTC盐是一种水溶性和无色的化合物,可以还原成甲an,高度着色的化合物。甲醛的不可逆形成可以使用分光光度计进行定量。由于其性质和其降低的潜力,该四唑盐广泛用于真核生物和原核生物两者,作为细胞氧化还原活性,活力,药物敏感性和底物利用测定的指标(Byth等人, 2001; Hayashi等人,2003; Raut等人,2008; Lin等人,2008)。由于膜电位,四唑鎓盐的净正电荷有助于细胞摄取,从而允许其细胞内还原(Berridge等人,2005)。在原核生物中,TTC降低的主要研究涉及革兰氏阴性呼吸细菌大肠杆菌,而对Rhizobiacea 家族的成员只有少数研究报道。在这个协议中,这个家族最好的遗传特征成员之一, meliloti 1021 根瘤菌菌株。使用TTC作为细胞氧化还原电位的指标来分析该菌株的呼吸活性,细胞色素末端氧化酶的表达。
 为了实现可测量的色彩强度的开发,并且同时为了避免细菌生长的任何可能的抑制,在TTC的存在下将细菌与其他作者所描述的相比适当的时间段( Tengerdy等人,1967; Byth等人,2001; Tachon等人,2009)。

关键字:苜蓿中华根瘤菌1021, 细胞氧化还原电位, 2,3,5-三苯基-2H-四唑氯化物(TTC), 1,3,5-三苯基四氮唑甲, 细菌细胞

材料和试剂

  1. 带有针头的无菌接种环(NUOVA APTACA,目录号:6001/SG/CS)
  2. 14-ml聚丙烯圆底管(Corning,Falcon ®,目录号:352059)
  3. 50 ml锥形离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339652)
  4. 安全锁定2毫升管(Eppendorf,目录号:0030120094)
  5. 中华根瘤菌中华根瘤菌 1021
  6. 2,3,5-三苯基-2H-四唑氯化物(TTC)(Sigma-Aldrich,目录号:T8877)
  7. 1,3,5-三苯基四唑甲an(Sigma-Aldrich,目录号:93145)
  8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D5879)
  9. 胰蛋白胨(Sigma-Aldrich,目录号:T9410)
  10. 酵母提取物(Sigma-Aldrich,目录号:Y0375)
  11. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C3306)
  12. Na 2 HPO 4(AcantorPerformance Materials,J.T.Baker,目录号:4062-01)
  13. NaH 2 PO 4(Acantor,Performance Materials,J.T.Baker,目录号:3818-05)
  14. TYR肉汤培养基(5g/L胰蛋白胨,3g/L酵母提取物,6mM CaCl 2)(参见食谱)
  15. 磷酸钠缓冲液(pH 7.5)(参见食谱)

设备

  1. 孵化室(30°C)
  2. 旋转振动筛
  3. 分光光度计(Beckman Coulter,目录号:DU800)
  4. 分光光度法在可见光谱中应用的比色杯(Kartell,目录号:1938)
  5. 微离心机(SCILOGEX D3024高速微型离心机)(Scilogex,目录号:912015139999)
  6. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Megafuge TM 16R)
  7. 烤箱(65°C)
  8. 余量(Mettler Toledo,目录号:B204-S)

程序

  1. 细菌细胞生长
    1. 纯文化。在无菌环下取出储存在-80℃的蜜i子1021,接种于含有2.0ml TYR培养基的15ml圆形管中,并在旋转振荡器(200rpm)下于30℃温育24小时, 。
    2. 将培养物的等分试样(0.15ml)转移到含有15ml新鲜TYR培养基(OD 600)= 0.2的50ml锥形离心管中,并使用分光光度计和比色皿测量在600nm处的光密度用于可见光谱中的分光光度法。
    3. 当培养物达到需要约4小时孵育的指数生长期(OD 600)= 0.7时,停止生长并将培养物分成10ml,用于生物质评估,1.5ml用于TTC还原测量。

  2. TTC测定
    1. 离心10分钟的细菌培养物在15 ml聚丙烯圆底,5000 x g,20分钟(室温),弃去上清液。
    2. 将细胞在65℃下在烘箱中孵育4小时直到干燥,并用平衡称重细胞
    3. 将8ml xg的2ml Eppendorf管中的1.5ml细菌培养物离心5分钟(室温)并弃去上清液。
    4. 用1ml的50mM磷酸钠缓冲液(pH7.5)洗涤细菌,以8,000×g离心5分钟(室温)并弃去上清液。
    5. 将细胞重悬于含有24mM 2,3,5-三苯基-2H-四唑氯化物(TTC)的1ml 50mM磷酸钠缓冲液(pH7.5)中,并在旋转振荡器上以200rpm温育30℃1小时(图1)

      图1.在30℃孵育1小时后细胞悬液的图片

    6. 以8,000 x g离心收集细胞5分钟(室温),弃去上清液(图2)。


      图2.离心步骤后的细菌细胞的图片

    7. 将细胞悬浮于1ml 99.5%DMSO(室温)中以溶解生成的甲。
    8. 将细菌细胞以13,000 x g(室温)离心1分钟,收集上清液。
    9. 以不同的已知浓度制备甲an标准溶液,将粉末溶解在99.5%的DMSO中
    10. 使用分光光度计在510nm处读取样品(上清液)和甲an标准物的吸光度。使用99.5%DMSO作为空白对照
    11. 绘制甲an标准品的标准曲线(mg/ml)对溶液在510nm处的吸光度。
    12. 从甲an标准曲线中直接测定与细菌细胞反应产生的甲the的量
    13. 将数据归一化为对应于1.5ml细菌细胞的细胞生物量(g),并测定每g细胞产生的甲the的量。

数据分析

  1. 进行至少五次生物重复,每次在不同时间进行。
  2. Formazan标准曲线的示例在表1中描述。
  3. 一些实验样品的读数和它们产生的甲the的量在表2和表3中报告
    表1. Formazan标准品及其在510nmv下的相对吸光度


    使用分光光度计测定光密度,数据如图3所示。 


    图3. Formazan标准曲线在0.0075-0.3mg/ml范围内,测定510nm范围内的莫扎唑标准品的光密度。通过使用Excel 2011 for Mac(14.1.0版本)添加了线性回归线。回归线方程和相关系数显示在图表上。

    表2.由生成的甲2.的量 S。 meliloti 1021个细胞生长到指数期


    通过内插表1中报告并在图1中详细阐述的标准溶液点计算该表中报告的甲烷量。 

    表3.由生成的甲3.的量 S。 meliloti 1021细胞生长至固定相


    通过内插表1中报告的标准溶液点,并在图1中详细阐述,计算了该表中报告的甲The量。 

食谱

  1. TYR肉汤培养基(1升)
    5克胰蛋白胨
    3克酵母提取物
    将ddH 2 O加至1 L.用高压消毒灭菌 溶液冷却后,加入12 ml无菌0.5 M CaCl 2 2 /
  2. 磷酸钠缓冲液(pH7.5)
    1. 通过将1.2g溶解在最终体积的10ml双蒸水中制备1M NaH 2 PO 4溶液
    2. 通过将1.42g溶解在最终体积的10ml双蒸水中来制备1M Na 2 HPO 4溶液
    3. 混合1.6ml 1M NaH 2 PO 4溶液和8.4ml 1M Na 2 HPO 4溶液并稀释至200ml,用双蒸水制备50mM磷酸钠缓冲液,pH7.5

致谢

TTC测定的方法由Tachon等人(2009)改编。这项工作得到了意大利经济和财政部向国家研究委员会提供的专项资金的部分支持,该项目是CISIA"Innovazione e Sviluppo del Mezzogiorno" - Conoscenze Integrate perSostenibilitàed Innovazione del Made in Italy Agroalimentare - Legge No. 191/2009"。这项工作也得到了欧盟委员会资助ABSTRESS项目(FP7 KBBE-2011-289562)的部分支持。作者宣称他们没有利益冲突。

参考文献

  1. Berridge,MV,Herst,PM and Tan,AS(2005)。  评估一种简单,无毒的阿尔玛蓝细胞存活实验来监测番茄细胞的活力。 Phytochem Anal 12:340-346。
  2. Hayashi,S.,Kobayashi,T。和Honda,H。(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16233537"目标="_ blank">使用四唑紫色染色方法进行简单快速的细胞生长测定,用于筛选有机溶剂耐受细菌。 J Biosci Bioeng 96(4):360-363。 />
  3. Lin,YC,Agbanyim,CN,Miles,RJ,Nicholas,RA,Kelly,DP and Wood,AP(2008)。  用于评估支原体中底物氧化和菌株分化的四氮唑还原方法,特别是参考支原体支原体和 Mycoplasma mycoides cluster。 J Appl Microbiol 105(2):492-501。
  4. Raut,U.,Narang,P.,Mendiratta,DK,Narang,R。和Deotale,V.(2008)。用于检测结核分枝杆菌对利福平和异烟肼的药物敏感性的快速MTT管法的评估印度J Med Microbiol 26(3):222-227。
  5. Tachon,S.,Michelon,D.,Chambellon,E.,Cantonnet,M.,Mezange,C.,Henno,L.,Cachon,R.and Yvon,M。(2009)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19520722"target ="_ blank">实验条件影响乳酸杆菌电子传递链中四唑紫还原的位点乳酸菌。 微生物学 155(Pt 9):2941-2948。
  6. Tengerdy,RP,Nagy,JG和Martin,B.(1967)。  通过还原四唑盐进行细菌生长的定量测量。应用微生物15(4):954-955。
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引用:Defez, R., Andreozzi, A. and Bianco, C. (2017). Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells. Bio-protocol 7(2): e2115. DOI: 10.21769/BioProtoc.2115.
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