H2O2 Kill Assays of Biofilm Bacteria

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Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized by marked tolerance to many biocides, including oxidants such as hydrogen peroxide. Hydrogen peroxide is both produced by host phagocytic cells, and used as an antimicrobial compound. Understanding biofilm tolerance to hydrogen peroxide is therefore relevant to the persistence of Pseudomonas aeruginosa in human infections (such as chronic Pseudomonas aeruginosa infections in cystic fibrosis airways) as well as in environmental settings (such as water pipes).
This protocol was developed to determine the tolerance of Pseudomonas aeruginosa biofilms to hydrogen peroxide (H2O2) killing. The bacteria are grown as colony biofilms on polycarbonate membranes, as previously described in Walters et al. 2003. The protocol may be adapted for other bacterial, with appropriate changes in H2O2 concentrations, since different bacterial species may be more or less susceptible to H2O2 than Pseudomonas aeruginosa.

Keywords: Pseudomonas aeruginosa(铜绿假单胞菌), Biofilms(生物膜), Oxidative stress(氧化应激), Hydrogen peroxide(过氧化氢), Tolerance(容忍)

Materials and Reagents

  1. Phosphate buffered saline (PBS) solution (Sigma-Aldrich, catalog number: P4417-100TAB )
  2. 30% w/w Hydrogen peroxide solution (undiluted, as sold commercially) (RICCA Chemical,  catalog number: 3821.7-32 )
  3. Sodium thiosulfate solution (dissolved in ddH2O) (Sigma-Aldrich,  catalog number: S8503 )
  4. 0.2 μM Polycarbonate 25 mm membranes (General Electric Company, catalog number: K02BP02500 )
  5. P.aeruginosa strains in freezer stock
  6. 25% Lennox broth (LB) medium (Becton Dickinson and Company, DifcoTM, catalog number: 240230 ) (see Recipes)
  7. 25% LB agar plates (see Recipes)


  1. 6-well and 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, model: 680 )
  5. Cuvettes for OD600 reading
  6. Shaking incubator at 37 °C, 250 rpm
  7. Static incubator at 37 °C
  8. Sterile glassware: 150 ml Erlenmeyer flasks, capped or foiled
  9. 1.5 ml and 2 ml microcentrifuge tubes
  10. Source of UV irradiation
  11. Sterile wire-loops (sterilized with 70% ethanol and flame)
  12. Stainless steel forceps (sterilized with 70% ethanol and flame)


  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. Gently place polycarbonate membranes on agar surface of fresh sterile 25% LB agar plates and sterilize the membranes by placing them under UV irradiation for 1 hour. Handle membranes carefully with sterile forceps and use the membranes immediately after sterilization. Use eyes and skin UV protective equipment. Use at least 3 membranes per strain per condition for adequate biological replicates, and up to 6 membranes may be placed on each agar plate.  
  4. Measure the OD600 of the overnight bacterial culture and dilute the bacterial suspension in LB medium to a starting concentration of 108 cells/ml. 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. Spot 5 μl (5 x 105 cells) onto the sterile membranes and allow the liquid to be absorbed (10-20 minutes).
  6. Incubate the colony biofilm on agar plates for 24 h at 37 °C.
  7. Day 3. Using sterile forceps, gently lift the membranes off the agar surface and transfer them (cells side down) into 6-well plates filled with 2 ml of 25% LB liquid medium in each well. Make sure the membranes are spread flat (i.e. not rolled up) and biofilm cells are remain on the membrane.
  8. For the H2O2 treated biofilms, add 30 μl H2O2 (150 mM) to each well in pulses every 10 minutes for 30 minutes (for a final concentration of 450 mM H2O2 per challenge). The pulsing is done to mimic a continuous exposure of cells to H2O2. In between H2O2 pulses, incubate cells at 37 °C without shaking. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates.  
  9. 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.
  10. To determine the viable cell count in H2O2 or PBS treated biofilms, collect biofilm cells by transferring the membranes and 2 ml of liquid from each well into 2 ml microcentrifuge tubes. Membranes are moved by gently lifting and rolling them using sterile forceps, with biofilm cells facing inward. The entire membrane should be submerged in liquid. Ensure to sterilize forceps between membrane transfers. Vortex biofilms in microcentrifuge tubes at maximal speed for at least 1 minute to detach and resuspend cells. Additional pipetting up and down and vortexing may also necessary to make sure there are no cell clumps visible in the bacterial suspension.
  11. Aliquot 100 μl of bacterial suspension 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.
  12. Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
  13. Determine hydrogen peroxide killing by comparing the viable CFU count in the PBS treated and the H2O2 treated conditions.


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


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).


  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.    
  2. Walters, M. C., 3rd, Roe, F., Bugnicourt, A., Franklin, M. J. and Stewart, P. S. (2003). Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 47(1): 317-323.    


该方案用于确定绿脓杆菌生物膜对过氧化氢(H 2 O 2 O 2)杀伤的耐受性。细菌作为菌落生物膜在聚碳酸酯膜上生长,如先前在Walters等人2003中所述。该方案可以适用于其它细菌,在H 2亚型中具有适当的变化, O 2浓度,因为不同的细菌物种可能比H 2 O 2比对绿脓杆菌更敏感或更不敏感。

关键字:铜绿假单胞菌, 生物膜, 氧化应激, 过氧化氢, 容忍


  1. 磷酸盐缓冲盐水(PBS)溶液(Sigma-Aldrich,目录号:P4417-100TAB)
  2. 30%w/w过氧化氢溶液(未稀释,商业销售)(RICCA Chemical,目录号:3821.7-32)
  3. 硫代硫酸钠溶液(溶于ddH 2 O)(Sigma-Aldrich,目录号:S8503)
  4. 0.2μM聚碳酸酯25mm膜(通用电气公司,目录号:K02BP02500)
  5. 菌株
  6. 25%Lennox肉汤(LB)培养基(Becton Dickinson and Company,Difco TM ,目录号:240230)(参见Recipes)
  7. 25%LB琼脂平板(见配方)


  1. 6孔和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,型号:680)
  5. 用于OD <600> 读数的比色皿
  6. 在37℃,250rpm下振荡孵育器
  7. 在37℃下静置培养箱
  8. 无菌玻璃器皿:150 ml锥形瓶,盖上或箔片
  9. 1.5 ml和2 ml微量离心管
  10. 紫外线辐射源
  11. 无菌线环(用70%乙醇和火焰灭菌)
  12. 不锈钢钳(用70%乙醇和火焰灭菌)


  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。 生长液体细菌培养过夜16-18 小时,在250rpm振荡。
  3. 第2天。将聚碳酸酯膜轻轻地放置在新鲜无菌25%LB琼脂板的琼脂表面上,并通过将膜置于UV照射下1小时对膜进行灭菌。用无菌镊子小心处理膜,灭菌后立即使用膜。使用眼睛和皮肤的紫外线防护设备。每个条件每个条件使用至少3个膜进行足够的生物复制,并且可以在每个琼脂平板上放置多达6个膜。  
  4. 测量过夜细菌培养物的OD 600,并将LB培养基中的细菌悬浮液稀释至10 8细胞/ml的起始浓度。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. 点5μl(5×10 5个细胞)到无菌膜上,并允许液体被吸收(10-20分钟)。
  6. 孵育菌落生物膜在琼脂板上在37℃下24小时
  7. 第3天。使用无菌镊子,轻轻地将膜从琼脂表面上提起,并将它们(细胞侧向下)转移到每孔中装有2ml 25%LB液体培养基的6孔板中。确保膜扩散 平面( 不卷起),并且生物膜细胞保留在膜上
  8. 对于H 2 O 2 O 2处理的生物膜,向每个孔中加入30μlH 2 O 2 O 2(150mM)以每10分钟脉冲,持续30分钟(对于每次攻击的最终浓度为450mM H 2 O 2 2次)。进行脉冲以模拟细胞连续暴露于H 2 O 2 O 2。在H 2 O 2 O 2脉冲之间,在37℃温育细胞而不摇动。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。  
  9. 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。
  10. 为了测定H 2 O 2或PBS处理的生物膜中的活细胞计数,通过将膜和2ml液体从每个孔转移到2ml微量离心管中收集生物膜细胞。通过轻轻地举起并使用无菌镊子使膜滚动,使生物膜细胞面向内部。整个膜应浸没在液体中。确保膜传输之间的镊子消毒。在微量离心管中以最大速度涡旋生物膜至少1分钟以分离和重悬细胞。另外的上下移动和涡旋也可能是必要的,以确保在细菌悬浮液中没有可见的细胞团块。
  11. 等分100微升细菌悬浮液96孔板,连续稀释细胞1:10,然后板100微升的每个稀释在LB琼脂板上的CFU计数。将CFU计数板在37℃孵育过夜
  12. 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。
  13. 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。


  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. 25%LB琼脂平板上 含1.5%琼脂的25%LB培养基 溶解在ddH 2 O中并高压釜


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)。


  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,是过氧化氢和抗生素耐受性所必需的。 195(9):2011-2020。    
  2. Walters,M.C.,3rd,Roe,F.,Bugnicourt,A.,Franklin,M.J.and Stewart,P.S.(2003)。 抗生素渗透,氧气限制, 和低代谢活性对铜绿假单胞菌生物膜对环丙沙星和妥布霉素的耐受性。 Antimicrob Agents Chemother 47(1):317-323。    
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Khakimova, M. and Nguyen, D. (2013). H2O2 Kill Assays of Biofilm Bacteria. Bio-protocol 3(21): e952. DOI: 10.21769/BioProtoc.952.

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