Quantification of Respiratory Activity in Biofilms

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Applied and Environmental Microbiology
Nov 2014



Bacteria live mostly as biofilms, not as planktonic cell populations. Bacterial cells living as biofilms are known to be in different physiological status. Persister cells are one of such physiological conditions and they are recognized as to be a stochastically produced sub-population of non-growing bacterial cells. The following protocol describes a method to determine the respiratory activity of cells within biofilms.

Materials and Reagents

  1. Bacterial culture [Pseudomonas aeruginosa (P. aeruginosa) PA14]. This method has also been performed for Escherichia coli (BW25113)
  2. LB broth Lennox (BD, catalog number: 240230 )
  3. CorningTM cellgroTM Ciprofloxacin Hydrochloride (Thermo Fisher Scientific, catalog number: 61-277-RF )
  4. BacLightTM RedoxSensorTM CTC Vitality Kit (Thermo Fisher Scientific, catalog number: B34956 )
  5. SYTO® 40 Blue Fluorescent Nucleic Acid Stain-5 mM Solution in DMSO (Thermo Fisher Scientific, catalog number: S11351 )
  6. Sodium chloride (VWR International, BDH®, catalog number: 0241-VBD )
  7. Aluminum Foil
  8. Spooled Masterflex peroxide-cured silicone tubing, L/S 14, 250 ft. (Cole-Parmer, catalog number: UX-96407-14 )
  9. Masterflex Norprene tubing (A60 G), L/S 13, 50 ft. (Cole-Parmer, catalog number: UX-06404-13 )
  10. PVDF barbed Y connector, 3/8" ID, 1/4", 2-3/8", 1-3/4"; Pack Of 10 (Cole-Parmer, catalog number: WU-30703-93 )
  11. Barbed fittings, Straight Connector, Clear PP,1/16" ID, 1/32", 25/32", 1/4" (Cole-Parmer, catalog number: WU-30506-00 )
  12. Barbed fittings, Reducing Connector, Clear PP,1/8" x 1/16" ID, 3/32", 13/16", 1/8" (Cole-Parmer, catalog number: WU-30506-06 )
  13. Barbed fittings, T connector, Kynar, 1/4" ID, 1/8", 1-15/16", 1-5/16"; 10/pack (Cole-Parmer, catalog number: WU-30703-75 )
  14. Micropipettes (P1000, P200, P20)
  15. Syringe 10 ml
  16. Syringe needle
  17. Glass microscope slides (25 x 75 x 1 mm)
  18. Glass microscope coverslips (60 x 24 mm; no 2)
  19. Saline (see Recipes)


  1. Peristaltic pump (Cole-Parmer, catalog number: EW-07553-80) with an 8 channel, 6 rollers, 3-stop Ismatec minicartridge pump head (Cole-Parmer, catalog number: EW-78002-50 )
  2. Inoculation ports (VWR® Sleeve Stoppers) (VWR International, catalog number: 89097-534 )
  3. GeneMate Incubated Shakers (BioExpress, catalog number: H-2000-M )
  4. Nalgene® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
  5. Nalgene® Top WorksTM Aseptic Closure System, Silicone, for Bottles and Carboys, Thermo Scientific (VWR International, catalog number: 2135-8303 )
  6. Acro® 50 Vent Filters, Pall Laboratory (VWR International, catalog number: 28143-616 )
  7. Anodized Transmission flow cell reactors (Biosurface technologies, catalog number: FC 81-Al )
  8. Confocal Scanning Microscope and Analysis software (e. g. Leica Confocal SP5 Imaging system and Software)


  1. COMSTAT software (http://www.comstat.dk)
  2. Intensity Luminance V1 software (http://bingweb.binghamton.edu/~scraver/IL.html)
  3. ImageJ software (http://imagej.nih.gov/ij/)
  4. Leica LAS AF software (Leica)


The procedure below, describes how to assess the respiratory activity of persister cells present within biofilms. However, the method can be adapted to overall biofilm populations. In this method, differential staining is used where respiratory activity (metabolic activity is assessed) using the monotetrazolium redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) that produces a fluorescent formazan (CTF, indicated by cells stained in red) when reduced. All cells are stained with the nucleic acid stain SYTO 40.

  1. Biofilm development in flow cell reactors.
    1. Prepare a streak plate on LB agar of a frozen stock of the bacteria of interest (in this case Pseudomonas aeruginosa PA14) and incubate under static conditions at 37 °C, for 24 h.
    2. Remove 2 colonies from the streak plate and inoculate a broth culture of 5 ml LB broth.
    3. Incubate at 37 °C with agitation (220 rpm) for a period of 12-24 h.
    4. Dilute the overnight culture to 1% in 50 ml of fresh LB medium in an Erlenmeyer flask.
    5. Incubate at 37 °C with agitation (220 rpm) for a period of 24 h.
    6. Measure the OD600 of the overnight culture and standardize the culture to have an OD600 of 0.8 in 10 ml of LB.
    7. Aspirate the culture with a 10 ml syringe with a needle and then cap the needle.
    8. Inoculate each flow cell reactor.
      1. In this example, biofilms were cultured in flow cell reactors (Figure 1), as described previously (Sauer et al., 2002; Davies and Marques, 2009; Marques et al., 2014). Each flow cell reactor was inoculated with 3 ml of a standardized overnight culture (OD600 of 0.8) and incubated, under static conditions, for a period of 1 h, to facilitate cell attachment.

        Figure 1. Flow cell reactor system. This is a once through continuous culture system. Once the bacterial culture is inoculated into the inoculation port, biofilms develop on the coverslip of the flow cell reactor. Only the bottom reactor is labeled, the top reactor is set in an identical manner. One of the flow cell reactor sets is labeled. A. Schematic version of a flow cell system where 2 flow cells are run together. B. Flow cell reactor.

    9. Following 1 h incubation at room temperature - to allow the bacteria to attach to the coverslip (this time can vary, depending on the bacterial strain) - initiate the flow.
    10. Allow biofilms to develop for a period of 6 days at room temperature (time and temperature may vary, depending on bacterial species used).
    11. At day 6 expose the P. aeruginosa PA14 biofilms to ciprofloxacin (150 x MIC) or saline (control) for a period of 18 h.
      Note: When assessing overall mature biofilm populations, not persister cell populations, at day 6 perform step 12. Respiratory activity can also be assessed at different days, during biofilm development.
    12. Expose the remaining biofilms to saline containing SYTO 40 (5 μM) and CTC (5 mM), for a period of 60 min. CTC has previously been used to determine the respiratory activity of bacteria within biofilms (Schaule et al., 1993).
      1. When attempting to determine the effects of certain compounds on persister cell activity, perform this step for 30 min only. The treatment to be performed should be initiated following this step.
    13. Take several z-stack images of biofilm cell clusters that have developed on the flow cell coverslip by confocal scanning laser microscopy (CSLM) using the following excitation/emission parameters:
      1. SYTO 40: ex 405, em 450-455
      2. CTC: ex 476, em 650-680.
      3. If performing step 12a, then following 30 min, expose the biofilm population to saline or the testing agent, together with SYTO 40 (5 μM) and CTC (5 mM) for further 60 min.
    14. Quantify relative fluorescence with the program Intensity Luminance V1 software. This program determines the relative fluorescence present on the images by quantifying the green, blue and red fluorescence. We have provided a link for the software, which contains the instructions of how to use it; we developed the program and made it available to other researchers as freeware.
      1. SYTO 40 (blue) will stain all cells while CTC stain (red) will stain only respiratory active cells. Thus, 100% of the cells will stain blue, making it possible to determine which percentage of the population is undergoing respiratory activity (red cells) during a 60 min period. If steps 12a and 13c were performed then, the results obtained for the initial 30 min should be used as baseline. In the case of persister cells, the difference between persister cells and overall population would be assessed.
    15. Perform quantitative analysis of biofilms using the COMSTAT software (Heydorn et al., 2000). A newer version of the COMSTAT software is now available, and has a plugin to enable its use with ImageJ.
      1. This program is widely used by biofilm researchers to determine and quantify: total biofilm biomass, biofilm cluster thickness, portion of slice occupied by the bacteria, surface area of biomass and surface to biovolume ratio. Blue channel (total) and red channel (respiratory active) images can be analyzed, and differences in respiratory activity between controls and treatments can be performed. In the case of persister cells, the difference between persister cells and overall population would be assessed.
    16. To assess the respiratory activity, compare between total cells (SYTO 40) and CTC stained cells (respiratory active cells).
    17. Repeat the experiment 3-4 times to ensure reproducible data and to allow for calculation of whether the results obtained are statistical significant when comparing controls to treated samples using ANOVA, followed by the Tukey’s comparison test.

Representative data

Our laboratory has made use of this type of assay to assess whether the fatty acid signaling molecule cis-2-decenoic acid (cis-DA) would increase the respiratory activity of P. aeruginosa persister cells present in biofilms (Marques et al., 2014). Biofilms were cultured as described in the procedure above, followed by exposure to ciprofloxacin (150 mg/L) for a period of 24 h. Subsequently, the remaining biofilm population, consisting of solely persister cells, was exposed to saline containing SYTO 40 and CTC for a period of 30 min (to obtain a baseline for the stain measurements) followed by a 1 h exposure to either saline or cis-DA (100 nM) in saline. Biofilms were visualized by confocal scanning laser microscopy (Leica Confocal TCS SP5 Imaging System with a DMI 6000 inverted microscope) using the parameters described in the procedure above. Images were acquired with the Leica LAS AF software. In the presence of cis-DA, a significant increase of CTC stain (red panel) was observed compared to exposure to saline alone (Figure 2A) consisting of a 10% increase (P<0.0001). We also observed that persister cells display a respiratory activity, which is indicative of an active metabolic status, albeit being 2.5% (Figure 2B), a low level not visually observed (Figure 2A).

Figure 2. Respiratory activity of P. aeruginosa PA14 persister cells upon exposure to cis-DA. A. Respiratory activity was evaluated by the cell’s ability to metabolize CTC into a fluorescent formazan, following 1 h incubation with saline or cis-DA in saline. SYTO 40 was used to stain overall population; bar equals 25 μm. B. The percentage of CTC stained population of persister cells compared to SYTO 40 stained population was calculated. Error bars indicate standard deviation. *, P<0.001 - Significantly different from persister cells treated with saline alone, as indicated by one way ANOVA. [Copyright© 2014, American Society for Microbiology (Marques et al., 2014)]


Images of background measurements, using confocal microscopy, should be acquired 25 min following the initial exposure of biofilms to CTC and SYTO 40. Second exposure of to CTC and SYTO 40 should only be initiated once all the background images were acquired.


  1. Saline
    8.5 g sodium chloride
    1 L of distilled water (DI)


The respiratory activity assay using CTC and SYTO 40 is a modification of previously published protocols (Schaule et al., 1993). COMSTAT analysis is based on previously published software (Heydorn et al., 2000). This work was supported by SUNY structural funds.


  1. Davies, D. G. and Marques, C. N. (2009). A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 191(5): 1393-1403.
  2. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. and Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146: 2395-2407.
  3. Marques, C. N., Morozov, A., Planzos, P. and Zelaya, H. M. (2014). The fatty acid signaling molecule cis-2-decenoic acid increases metabolic activity and reverts persister cells to an antimicrobial-susceptible state. Appl Environ Microbiol 80(22): 6976-6991.
  4. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. and Davies, D. G. (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184(4): 1140-1154.
  5. Schaule, G., Flemming, H. C. and Ridgway, H. F. (1993). Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl Environ Microbiol 59(11): 3850-3857.


细菌主要作为生物膜,而不是作为浮游细胞群。 作为生物膜存在的细菌细胞已知处于不同的生理状态。 持续细胞是这样的生理条件之一,并且它们被认为是非生长细菌细胞的随机产生的亚群。 以下方案描述了确定生物膜内细胞的呼吸活性的方法。


  1. 细菌培养物[铜绿假单胞菌(<铜绿假单胞菌] PA14]。 此方法也已在大肠杆菌(BW25113)
  2. LB肉汤Lennox(BD,目录号:240230)
  3. (Thermo Fisher Scientific,目录号:61-277-RF)的紫杉醇和/或盐酸吡咯烷
  4. BacLight TM RedoxSensor TM CTC Vitality Kit(Thermo Fisher Scientific,目录号:B34956)
  5. 在DMSO(Thermo Fisher Scientific,目录号:S11351)中的SYTO <40>蓝色荧光核酸染料-5mM溶液
  6. 氯化钠(VWR International,BDH ,目录号:0241-VBD)
  7. 铝箔
  8. 缠绕的Masterflex过氧化物固化的硅氧烷管,L/S 14,250英尺(Cole-Parmer,目录号:UX-96407-14)
  9. Masterflex Norprene管(A60G),L/S 13,50英尺(Cole-Parmer,目录号:UX-06404-13)
  10. PVDF有倒钩的Y型连接器,3/8"ID,1/4",2-3/8",1-3/4" Pack Of 10(Cole-Parmer,目录号:WU-30703-93)
  11. 直型连接器,清除PP,1/16"ID,1/32",25/32",1/4"(Cole-Parmer,目录号:WU-30506-00)
  12. 倒钩连接器,清除PP,1/8"x 1/16"ID,3/32",13/16",1/8"(Cole-Parmer,目录号:WU-30506-06) />
  13. 倒钩接头,T型连接器,Kynar,1/4"ID,1/8",1-15/16",1-5/16" 10/pack(Cole-Parmer,目录号:WU-30703-75)
  14. 微量移液器(P1000,P200,P20)
  15. 注射器10毫升
  16. 针头
  17. 玻璃显微镜载片(25×75×1mm)
  18. 玻璃显微镜盖玻片(60×24mm;无2)
  19. 盐水(见配方)


  1. 蠕动泵(Cole-Parmer,目录号:EW-07553-80),带有8通道,6个辊,3挡Ismatec小型泵头(Cole-Parmer,目录号:EW-78002-50)
  2. 接口(VWR ®套筒塞)(VWR International,目录号:89097-534)
  3. GeneMate Incubated Shakers(BioExpress,目录号:H-2000-M)
  4. Nalgene ® Carboys with Handles,Polypropylene,Thermo Scientific(VWR International,目录号:16101-084)
  5. Nalgene ® Top Works TM 无菌封闭系统,硅胶,用于瓶和瓶,Thermo Scientific(VWR国际,目录号:2135-8303)
  6. Acro ® 50通风过滤器,Pall Laboratory(VWR国际,目录号:28143-616)
  7. 阳极化传输流动池反应器(Biosurface technologies,目录号:FC81-A1)
  8. 共聚焦扫描显微镜和分析软件(例如 Leica Confocal SP5成像系统和软件)


  1. COMSTAT软件( http://www.comstat.dk
  2. 强度亮度V1软件( http://bingweb.binghamton.edu/~scraver/IL.html
  3. ImageJ软件( http://imagej.nih.gov/ij/
  4. Leica LAS AF软件(Leica)


以下程序描述如何评估存在于生物膜内的持续细胞的呼吸活性。 然而,该方法可以适应于总体生物膜群体。 在该方法中,使用差异染色,其中使用产生荧光甲an(CTF,由红色染色的细胞表示)的单四唑鎓氧化还原染料5-氰基-2,3-二甲苯基四唑氯化物(CTC)评价呼吸活性 )减少。 所有细胞用核酸染色SYTO 40染色。

  1. 流动池反应器中的生物膜发展
    1. 在细菌的冷冻原液的LB琼脂上制备条纹板 感兴趣(在这种情况下为绿脓假单胞菌 PA14)并孵育 静置条件在37℃,24小时
    2. 从条纹板上除去2个菌落,并接种5ml LB肉汤的肉汤培养物
    3. 在37℃下搅拌(220rpm)孵育12-24小时
    4. 在锥形烧瓶中的50ml新鲜LB培养基中稀释过夜培养物至1%
    5. 在37℃下搅拌(220rpm)孵育24小时
    6. 测量过夜培养物的OD 600,并将培养物标准化,使其在10ml LB中具有OD 600 = 0.8。
    7. 用10ml注射器用针头吸出培养物,然后盖上针
    8. 接种每个流动池反应器。
      1. 在该实施例中,在流动池反应器中培养生物膜 (图1),如先前所述(Sauer等人,2002; Davies and Marques,2009; Marques等人,2014)。 每个流动池反应器 用3ml标准化的过夜培养物(OD 600为0.8)接种, 并在静态条件下温育1小时至1小时 促进细胞附着。

        图1.流动池反应器系统。这是一个一次性通过连续培养系统。 一旦细菌 培养物接种到接种口,生物膜上发育   盖玻片的流动池反应器。 只有底部反应器被标记,   顶部反应器以相同的方式设置。 一个流动池 反应器组被标记。 A.流动池系统的示意图 其中2个流动池一起运行。 B.流动池反应器。

    9. 在室温下孵育1小时 - 以允许细菌 附着到盖玻片(这一次可以变化,取决于细菌 应变) - 启动流动。
    10. 允许生物膜发展为 在室温下6天的时间(时间和温度可以变化, 取决于使用的细菌种类)
    11. 在第6天暴露P。铜绿假单胞菌PA14生物膜与环丙沙星(150×MIC)或盐水(对照)一起培养18小时。
      注意:当评估整体成熟生物膜群体时,不持续 细胞群,在第6天进行步骤12 也可以在生物膜发育的不同日期进行评估。
    12. 将剩余的生物膜暴露于含有SYTO 40(5μM)和的盐水中 CTC(5mM),持续60分钟。 CTC以前已经用过 确定生物膜内细菌的呼吸活性(Schaule等人,1993)。
      1. 当试图确定的影响 某些化合物对持续细胞活性,执行此步骤30 分钟。应该开始进行的治疗 这一步。
    13. 采取生物膜细胞簇的几个z堆栈图像 已通过共聚焦扫描在流动池盖玻片上发展 激光显微镜(CSLM)使用以下激发/发射 参数:
      1. SYTO 40:ex 405,em 450-455
      2. CTC:ex 476,em 650-680。
      3. 如果进行步骤12a,则在30分钟后,暴露生物膜 群体对盐水或测试剂,与SYTO 40(5μM) 和CTC(5mM)处理另外60分钟。
    14. 量化相对 荧光与程序强度亮度V1软件。 这个 程序通过确定图像上存在的相对荧光 量化绿色,蓝色和红色荧光。 我们提供了一个 链接的软件,其中包含如何使用它的说明;   我们开发了该程序,并将其提供给其他研究人员 免费软件
      1. SYTO 40(蓝色)将染色所有细胞,而CTC染色 (红色)将仅染色呼吸活性细胞。 因此,100%的细胞 将染成蓝色,使得可以确定哪个百分比   人口在60年期间经历呼吸活动(红细胞) min。 如果然后执行步骤12a和13c,则结果 获得初始30分钟应作为基线。 在这种情况下 的持续细胞,持续细胞和整体之间的差异 人口将被评估。
    15. 进行定量分析 生物膜,使用COMSTAT软件(Heydorn等人,2000)。 更新 版本的COMSTAT软件现在可用,并且有一个插件 启用它与ImageJ的使用。
      1. 这个程序被广泛使用 生物膜研究人员确定和量化:总生物膜生物量, 生物膜簇厚度,细菌占据的切片部分, 生物量的表面积和表面与生物体积的比率。 蓝色通道 (总)和红色通道(呼吸活动)图像,并且   控制和治疗之间的呼吸活性的差异 执行。 在持续细胞的情况下,两者之间的差异 持续细胞和总体人群。
    16. 至 评估呼吸活性,比较总细胞(SYTO 40) 和CTC染色的细胞(呼吸活性细胞)。
    17. 重复 实验3-4次,以确保可重复的数据并允许 计算所得结果是否具有统计显着性 当使用ANOVA将对照与处理的样品进行比较时, Tukey的比较测试。


我们的实验室利用这种类型的测定法来评估脂肪酸信号分子顺式-2-癸烯酸(顺式-DA)是否会增加 em> P。铜绿色持续细胞存在于生物膜中(Marques等人,2014)。如上述方法所述培养生物膜,然后暴露于环丙沙星(150mg/L)24小时。随后,将仅由持续细胞组成的剩余生物膜群体暴露于含有SYTO 40和CTC的盐水中30分钟(以获得染色测量的基线),然后暴露于盐水或顺式-DDA(100nM)的盐水溶液。通过共聚焦扫描激光显微镜(Leica Confocal TCS SP5 Imaging System with DMI 6000 inverted microscope)使用上述方法中描述的参数显现生物膜。使用Leica LAS AF软件采集图像。在存在顺式-DA的情况下,与暴露于由10%增加(P <0.0001)组成的单独的盐水(图2A)相比,观察到CTC染色的显着增加(红色图)。我们还观察到持续细胞显示呼吸活性,这表明活性代谢状态,尽管是2.5%(图2B),低水平没有目视观察到(图2A)。

图2.P的呼吸活性。在暴露于顺式-DA时,绿脓杆菌PA14存留细胞。 A.呼吸活性通过细胞在用盐水孵育1小时后将CTC代谢为荧光甲's的能力来评价或顺式-dDA。 SYTO 40用于染色总体人群;条等于25μm。 B.计算持续细胞的CTC染色群体与SYTO 40染色群体相比的百分比。误差线表示标准偏差。 *,P <0.001 - 与单独用盐水处理的持续细胞显着不同,如通过单因素方差分析所示。 [版权© 2014,美国微生物学会(Marques ,2014年]]


背景测量的图像使用共聚焦显微镜,应该在生物膜初始暴露于CTC和SYTO 40后25分钟获取。对于CTC和SYTO 40的第二次暴露应当仅在获得所有背景图像时开始。


  1. 盐水
    8.5克氯化钠 1升蒸馏水(DI)


使用CTC和SYTO 40的呼吸活性测定是先前公开的方案的修改(Schaule等人,1993)。 COMSTAT分析基于先前公布的软件(Heydorn等人,2000)。这项工作得到了SUNY结构基金的支持。


  1. Davies,D.G.and Marques,C.N。(2009)。 脂肪酸信使负责诱导微生物生物膜中的分散。 J Bacteriol 191(5):1393-1403
  2. Heydorn,A.,Nielsen,A.T.,Hentzer,M.,Sternberg,C.,Givskov,M.,Ersboll,B.K.and Molin,S。(2000)。 新型计算机程序COMSTAT对生物膜结构的定量。微生物学 et al。,146:2395-2407。
  3. Marques,C.N.,Morozov,A.,Planzos,P.and Zelaya,H.M。(2014)。 脂肪酸信号分子顺式-2-癸烯酸增加代谢活性并将持续细胞恢复到抗微生物敏感状态。 Appl Environ Microbiol 80(22):6976-6991。
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引用:Marques, C. N. and Craver, S. A. (2015). Quantification of Respiratory Activity in Biofilms. Bio-protocol 5(18): e1591. DOI: 10.21769/BioProtoc.1591.



Chloe Mica
Creative Peptides
These references are extremely useful to me because I am preparing my paper about biofilms. I have no idea that there has saline in it. Maybe I have to look for related information to widen my horizon. Informative article.
10/21/2015 11:58:32 PM Reply