搜索

H2O2 Kill Assays of Planktonic Stationary Phase Bacteria
H2O2杀灭浮游生物静止期细菌的分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing of stationary phase bacterial cells to hydrogen peroxide and can be adapted to test other oxidants.

Materials and Reagents

  1. Phosphate buffered saline (PBS) solution (Sigma-Aldrich, catalog number: P4417-100TAB )
  2. 30% w/w Hydrogen peroxide stock solution (RICCA Chemical, catalog number: 3821.7-32 )
  3. Sodium thiosulfate solution (dissolved in ddH2O) (Sigma-Aldrich, catalog number: S8503 )
  4. P.aeruginosa strains in freezer stock
  5. 25% Lennox broth (LB) medium (Becton Dickinson and Company, DifcoTM, catalog number: 240230 ) (see Recipes)
  6. LB agar plates (see Recipes)

Equipment

  1. 96-well plates
  2. Standard petri plates
  3. Spectrophotometer (cuvette) (Thermo Fisher Scientific, model: GENESYS 10S UV-Vis )
  4. Spectrophotometer (96-well plate) (Bio-Rad Laboratories, model: 680 )
  5. Cuvettes for OD600 reading
  6. Shaker-incubator at 37 °C, 250 rpm
  7. Static incubator at 37 °C
  8. Sterile glassware: 150 ml Erlenmeyer flask, capped or foiled
  9. Sterile 15 mm glass test tubes and plastic caps
  10. Sterile wire-loops (sterilized with 70% ethanol and flame)

Procedure

  1. Day 0. Streak P.aeruginosa cells from the freezer stock onto a LB agar place and incubate statically overnight at 37 °C.
  2. Day 1. Pick 4-5 single colonies from the P.aeruginosa agar plate with a sterile wired-loop and inoculate 15 ml liquid LB medium in a 150 ml Erlenmeyer flask. Grow liquid bacterial cultures overnight for 16-18 hours at 37 °C, with shaking at 250 rpm.
  3. Day 2. Inoculate 15 ml liquid LB medium in a 150 ml flask with 1:100 of overnight bacterial culture. Grow cells to for 16-18 hours at 37 °C, with shaking at 250 rpm.
  4. Day 3. Determine the OD600 of the culture and dilute the bacterial suspension to a starting concentration of ~2.5 x 106 cells/ml (in total volume 1 ml LB). Depending on the bacterial strain used, the OD600 to CFU ratio will differ and needs to be determined for each strain: for example, for the PAO1 wild type strain, 108 cells/ml = ~OD600 0.1.
  5. To confirm the correct starting bacterial density (at ~2.5 x 106 cells/ml), aliquot 100 μl of the above bacterial suspension into 96-well plate. Serially dilute cells 1:10 in PBS to approximately ~2.5 x 102 cells/ml, then plate 100 μl on LB agar plates for CFU count. This will also be the CFU count for time zero measurement.
  6. Set up ~2.5 x 106 cells/ml x 1 ml per sample in sterile glass tubes, with at least 3 replicates per strain per condition. For H2O2 treated samples, add H2O2 (1 mM (2 μl) to 5 mM (10 μl) or other desired final concentration) to each samples in test tubes. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates. Incubate cells for 2 hours with shaking at 250 rpm at 37 °C.
  7. After H2O2 or PBS challenge, add 0.2% sodium thiosulfate to all samples to neutralize any remaining H2O2. Add even when samples are only challenges with PBS as a control.
  8. To determine the viable cell count in H2O2 or PBS treated samples, aliquot 100 μl of bacterial samples into 96-well plate, serially dilute cells 1:10, then plate 100 μl of each dilution on LB agar plates for CFU count. Incubate CFU count plates at 37 °C overnight.
  9. Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
  10. Determine hydrogen peroxide killing by comparing the viable CFU count in the PBS treated and the H2O2 treated conditions.

Recipes

  1. 25%  LB medium
    5 g LB powder medium per L
    Dissolved in ddH2O and autoclaved
  2. LB agar plates
    LB medium with 1.5% agar
    Dissolved in ddH2O and autoclaved

Acknowledgments

We would like to acknowledge CIHR (MOP-102727 to DN) and the Burroughs Wellcome Fund (1006827.01 to DN) for funding. This protocol was adapted from the previously published paper Khakimova et al. (2013).

References

  1. Khakimova, M., Ahlgren, H. G., Harrison, J. J., English, A. M. and Nguyen, D. (2013). The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol 195(9): 2011-2020.   

简介

固定相细菌对过氧化氢具有高度耐受性。 该方案被开发用于测试不同铜绿假单胞菌菌株对过氧化氢杀伤的易感性。 该测定提供了一种可靠的方法来测量固定相细菌细胞杀死过氧化氢并可适用于测试其他氧化剂。

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)溶液(Sigma-Aldrich,目录号:P4417-100TAB)
  2. 30%w/w过氧化氢储备溶液(RICCA Chemical,目录号:3821.7-32)
  3. 硫代硫酸钠溶液(溶解于ddH 2 O)(Sigma-Aldrich,目录号:S8503)
  4. 菌株
  5. 25%Lennox肉汤(LB)培养基(Becton Dickinson and Company,Difco TM ,目录号:240230)(参见Recipes)
  6. LB琼脂平板(见Recipes)

设备

  1. 96孔板
  2. 标准培养皿
  3. 分光光度计(比色杯)(Thermo Fisher Scientific,型号:GENESYS 10S UV-Vis)
  4. 分光光度计(96孔板)(Bio-Rad Laboratories,型号:680)
  5. 用于OD <600> 读数的比色皿
  6. 摇床培养箱中,37℃,250rpm,
  7. 在37℃下静置培养箱
  8. 灭菌玻璃器皿:150 ml锥形瓶,盖上或箔片
  9. 无菌15 mm玻璃试管和塑料盖
  10. 无菌线环(用70%乙醇和火焰灭菌)

程序

  1. 第0天将来自冷冻库的条纹的绿脓杆菌细胞在LB琼脂位置上并在37℃静态过夜温育。
  2. 第1天。用无菌有线环从洋葱琼脂平板上挑取4-5个单菌落,并在150ml锥形瓶中接种15ml液体LB培养基。使液体细菌培养物在37℃下在250rpm振荡下生长16-18小时。
  3. 第2天。在具有1:100过夜细菌培养物的150ml烧瓶中接种15ml液体LB培养基。生长细胞在37℃下16-18小时,以250rpm振荡。
  4. 第3天。测定培养物的OD 600,并将细菌悬浮液稀释至约2.5×10 6个细胞/ml的起始浓度(总体积为1ml LB) 。取决于所使用的细菌菌株,OD 600与CFU的比率将不同,并且需要对每种菌株确定:例如,对于PAO1野生型菌株,10 8 细胞/ml =〜OD 600 = 0.1
  5. 为了确认正确的起始细菌密度(〜2.5×10 6个细胞/ml),将100μl的上述细菌悬浮液等分到96孔板中。在PBS中以1:10的比例连续稀释细胞至约〜2.5×10 2个细胞/ml,然后在LB琼脂平板上平板100μl用于CFU计数。这也是时间零测量的CFU计数。
  6. 在无菌玻璃管中设置〜2.5×10 6个细胞/ml×1ml每个样品,每个条件每个菌株至少3个重复。对于H 2 O 2 O 2处理的样品,将H 2 O 2 O 2(1mM(2μl)加入到5μl mM(10μl)或其它所需的终浓度)。包括用PBS攻击的未处理对照。每个条件应至少进行三次重复。孵育细胞2小时,在250 rpm,37℃下摇动
  7. 在H 2 O 2 O 2或PBS攻击后,向所有样品中加入0.2%硫代硫酸钠以中和任何剩余的H 2 O 2 O 2,/sub>。即使当样品仅作为对照的PBS时,也添加。
  8. 为了测定H 2 O 2 Sub或PBS处理的样品中的活细胞计数,将100μl细菌样品等分成96孔板,连续稀释细胞1:10,然后板100μl每种稀释液在LB琼脂平板上用于CFU计数。将CFU计数板在37℃孵育过夜
  9. 第4天在LB琼脂平板上计数CFU,并基于所应用的稀释因子计算每个生物膜的活CFU。
  10. 通过比较PBS处理的条件和H 2 O 2 O 2处理条件中的活CFU计数来确定过氧化氢杀伤。

食谱

  1. 25%  LB培养基
    5 g LB粉末培养基/L
    溶于ddH 2 O中并高压灭菌
  2. LB琼脂平板上 含1.5%琼脂的LB培养基 溶于ddH 2 O中并高压灭菌

致谢

我们要感谢CIHR(MOP-102727到DN)和Burroughs Wellcome基金(1006827.01到DN)的资金。 该协议改编自以前发表的论文Khakimova等人(2013)。

参考文献

  1. Khakimova,M.,Ahlgren,H. G.,Harrison,J. J.,English,A. M. and Nguyen,D.(2013)。 严格的反应控制铜绿假单胞菌中的过氧化氢酶,是过氧化氢所必需的 和抗生素耐受性。细菌 195(9):2011-2020。   
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Khakimova, M. and Nguyen, D. (2013). H2O2 Kill Assays of Planktonic Stationary Phase Bacteria. Bio-protocol 3(21): e953. DOI: 10.21769/BioProtoc.953.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。