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Drug Sensitivity Assay of Xanthomonas citri subsp. citri Using REMA Plate Method
采用REMA孔板法进行柑桔溃疡病亚型的药敏试验   

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Abstract

Resazurin Microtiter Assay (REMA) is a simple, rapid, reliable, sensitive, safe and cost-effective measurement of cell viability. Resazurin detects cell viability by converting from a nonfluorescent dye to the highly red fluorescent dye resorufin in response to chemical reduction of growth medium resulting from cell growth (Palomino et al., 2002). The REMA assay can be used as a fluorogenic oxidation-reduction indicator in a variety of cells, including bacteria, yeast and eukaryotes (Silva et al., 2013).

Keywords: Citrus canker(柑橘溃疡病), Plant pathogen(植物病原菌), Antibacterial(抗菌)

Materials and Reagents

  1. Chemicals: Synthetic esters of gallic acids (Ximenes et al., 2010)
  2. Bacterial strain: Wild type Xanthomonas citri subsp citri strain 306 (Schaad et al., 2005)
  3. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  4. Kanamycin (Sigma-Aldrich, catalog number: K4000 )
  5. Luria-Bertani broth (LB) culture medium
  6. Resazurin sodium salt (Sigma-Aldrich, catalog number: R7017 )

Equipment

  1. 96-well plate, polystyrene, with clear flat bottom wells (Greiner Bio-one, catalog number: 655101 )
  2. SPECTRAfluor Plus (Tecan) microfluorimeter
  3. Multichannel pipetman (Eppendorf)

Procedure

  1. Prepare stock solutions of chemicals (dried-powder samples) dissolving in 10% in DMSO (diluted in sterile water).
  2. Add 100 μl of water to columns 1 and 12 to avoid evaporation (Table 1).
  3. Dilute the stock solutions in LB medium directly in a 96-well plates using a 2-fold scheme (final volume of 100 μl per a well); after serial dilution, the most concentrated sample should have maximum 1% DMSO.
  4. Cells were grown in LB medium at 30 °C under rotation (200 rpm) until OD600 0.6 ( log phase).
  5. Add 10 μl of bacterial inoculum (standardized to 105 CFU/well).
    1. Negative control: 1% DMSO dissolved in LB.
    2. Positive control: Kanamycin at 15.6 μg/ml.

      Table 1. Example for setup of REMA 96-well assay plate



  6. Incubate the test plates at 30 °C for 6 h.
  7. Add 15 μl of a 0.01% (w/v) resazurin solution, and incubate at 30 °C for 2 h.
  8. Measure fluorescence at 530 nm (excitation) and 590 nm (emission) using a fluorescence scanning.
  9. Percentage of inhibition is defined as:
    [(average FU negative control) - (average FU test)]/(average FU negative control) x 100
    FU: Fluorescence Units


    Figure 1. Example for calculation of growth inhibition

    Note: Three independent experiments should be conducted, and the data is used to construct plots of chemical concentration versus cell growth inhibition in order to determine the MIC* (Figure 1).

    *The minimum inhibitory concentration (MIC) is defined as the lowest concentration of the antibiotic able to inhibit the growth of 90% of organisms.

Acknowledgments

This work was supported by FAPESP research grants 2004/09173-6, 2010/05099-7, and 2011/07458-7. This protocol was adapted from a previous work by Palomino et al. (2002).

References

  1. Palomino, J. C., Martin, A., Camacho, M., Guerra, H., Swings, J. and Portaels, F. (2002). Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46(8): 2720-2722.
  2. Schaad, N. W., Postnikova, E., Lacy, G. H., Sechler, A., Agarkova, I., Stromberg, P. E., Stromberg, V. K. and Vidaver, A. K. (2005). Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov. Syst Appl Microbiol 28(6): 494-518. 
  3. Silva, I. C., Regasini, L. O., Petronio, M. S., Silva, D. H., Bolzani, V. S., Belasque, J., Jr., Sacramento, L. V. and Ferreira, H. (2013). Antibacterial activity of alkyl gallates against Xanthomonas citri subsp. citri. J Bacteriol 195(1): 85-94.
  4. Ximenes, V. F., Lopes, M. G., Petronio, M. S., Regasini, L. O., Silva, D. H. and da Fonseca, L. M. (2010). Inhibitory effect of gallic acid and its esters on 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione in erythrocytes. J Agric Food Chem 58(9): 5355-5362. 

简介

Resazurin Microtiter Assay(REMA)是一种简单,快速,可靠,灵敏,安全和经济有效的细胞活力测量。 刃天青通过响应于由细胞生长产生的生长培养基的化学还原而从非荧光染料转化为高度红色荧光染料试卤灵来检测细胞存活力(Palomino等人,2002)。 REMA测定法可用作各种细胞,包括细菌,酵母和真核生物中的荧光氧化 - 还原指示剂(Silva等人,2013)。

关键字:柑橘溃疡病, 植物病原菌, 抗菌

材料和试剂

  1. 化学品:没食子酸的合成酯(Ximenes等人,2010)
  2. 细菌菌株:野生型柑橘黄单胞菌亚种citri 菌株306(Schaad等人,2005)
  3. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  4. 卡那霉素(Sigma-Aldrich,目录号:K4000)
  5. Luria-Bertani肉汤(LB)培养基
  6. 刃天青钠盐(Sigma-Aldrich,目录号:R7017)

设备

  1. 96孔板,聚苯乙烯,具有透明平底孔(Greiner Bio-one,目录号:655101)
  2. SPECTRAfluor Plus(Tecan)显微荧光光度计
  3. 多通道移液器(Eppendorf)

程序

  1. 制备溶于10%DMSO(稀释于无菌水中)的化学品储存溶液(干粉样品)
  2. 在第1列和第12列中加入100μl水,以避免蒸发(表1)
  3. 使用2倍方案(每孔的终体积为100μl)将储备溶液在LB培养基中直接稀释在96孔板中; 在系列稀释后,最浓缩的样品应该含有最多1%的DMSO
  4. 细胞在30℃下在LB培养基中在旋转(200rpm)下生长直到OD 600 = 0.6(对数期)。
  5. 加入10μl细菌接种物(标准化为10 CFU /孔)
    1. 阴性对照:溶于LB中的1%DMSO
    2. 阳性对照:卡那霉素15.6μg/ml
      表1. REMA 96孔测定板设置示例


  6. 将测试板在30℃孵育6小时。
  7. 加入15μl的0.01%(w/v)刃天青溶液,并在30℃孵育2小时。
  8. 使用荧光扫描在530nm(激发)和590nm(发射)测量荧光
  9. 抑制百分比定义为:
    [(平均FU阴性对照) - (平均FU试验)] /(平均FU阴性对照)×100
    FU:荧光单位


    图1.生长抑制计算示例

    注意:应进行三个独立实验,并且数据用于构建化学浓度对细胞生长抑制的图,以确定MIC *(图1)。
    最小抑制浓度(MIC)定义为能够抑制90%生物生长的抗生素的最低浓度。

致谢

这项工作得到FAPESP研究资助2004/09173-6,2010/05099-7和2011/07458-7的支持。该协议改编自Palomino等人以前的工作。(2002)。

参考文献

  1. Palomino,J.C.,Martin,A.,Camacho,M.,Guerra,H.,Swings,J.and Portaels,F。(2002)。 Resazurin微量滴定板测定板:用于检测结核分枝杆菌中的耐药性的简单且便宜的方法。 Antimicrob Agents Chemother 46(8):2720-2722。
  2. Schaad,N.W.,Postnikova,E.,Lacy,G.H.,Sechler,A.,Agarkova,I.,Stromberg,P.E.,Stromberg,V.K.and Vidaver,A.K。(2005)。 野油菜黄单胞菌属citri(ex Hasse 1915)Dye 1978形式A,B/C/D和E作为X.smithii subsp。 citri(ex Hasse)sp。 nov。 nom。 rev。梳。 nov。,X.fuscans subsp。 aurantifolii(ex Gabriel 1989)sp。 nov。 nom。 rev。梳。苜蓿, citrumelo(ex Riker and Jones)Gabriel et al。,1989 sp。 nov。 nom。 rev。梳。 nov。野油菜(Avitestris pv malvacearum)(ex smith 1901)Dye 1978 as X.smithii subsp。史密斯梳。 nov。 nom。 nov。野油菜苜蓿(ex Riker and Jones,1935)dye 1978 as X. alfalfae subsp。苜蓿(ex Riker et al。,1935) nov。 nom。 rev。和"var.fuscans"的X.campestris pv。 Phaseoli(ex Smith,1987)Dye 1978 as X.fuscans subsp。弗洛斯坎斯nov。 Syst Appl Microbiol 28(6):494-518。 
  3. Silva,I.C.,Regasini,L.O.,Petronio,M.S.,Silva,D.H.,Bolzani,V.S.,Belasque,J.,Jr.,Sacramento,L.V.and Ferreira,H。(2013)。 没食子酸烷基酯对抗黄单胞菌的抗菌活性 195(1):85-94。
  4. Ximenes,V.F.,Lopes,M.G.,Petronio,M.S.,Regasini,L.O.,Silva,D.H.and da Fonseca,L.M。(2010)。 没食子酸及其酯对2,2'-偶氮二(2-脒基丙烷)盐酸盐的抑制作用 (AAPH)诱导的溶血和红细胞中细胞内谷胱甘肽的耗尽。 J Agric Food Chem 58(9):5355-5362。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Silva, I. C. and Ferreira, H. (2013). Drug Sensitivity Assay of Xanthomonas citri subsp. citri Using REMA Plate Method. Bio-protocol 3(16): e861. DOI: 10.21769/BioProtoc.861.
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