Quantification of Bacterial Attachment to Tissue Sections

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Cell Reports
Jul 2017



Here we describe a method to test bacterial adhesion to paraffin embedded tissue sections. This method allows examining binding of different bacterial strains, transfected with a fluorescent protein reporter plasmid to various tissues, to better understand different mechanisms such as colonization. This assay provides a more physiological context to bacterial binding, than would have been achieved using adhesion assays to cell lines. The sections can be imaged using fluorescent microscopy and adhesion of various bacterial strains can be quantified and tested, simultaneously.

Keywords: Host-pathogen interactions (宿主-病原体相互作用), Bacterial attachment (细菌附着), Bacterial colonization (细菌定植)


Many types of bacteria, both commensal and pathogenic, express various adhesion molecules, allowing them binding to different surfaces of the host (Gur et al., 2015; Abed et al., 2016; Isaacson et al., 2017). This adhesion is crucial, as it is the first step of colonization and plays a role in both competition and survival, in different environments (Schilling et al., 2001). Many of these adhesins are lectins, binding sugar moieties on glycoproteins on various kinds of cells, such as epithelial cells and others (Abed et al., 2016; Isaacson et al., 2016). Over the years, many groups studying host-pathogen interactions used cell lines and tissue culture in order to try to understand bacterial adhesion to cells. Tissue sections give a more physiological context to the colonization study, as they provide organization and structures that are almost impossible to obtain using in vitro tissue culture. Furthermore, in immortalized or cancerous cells, the expression pattern of surface molecules, to which bacteria can bind, might be altered. In order to better understand physiological context of bacterial adherence, in both normal and pathological conditions, we chose to employ bacterial attachment to tissue sections.

Materials and Reagents

  1. Plastic 50 ml tubes for centrifugation (Greiner Bio One International, catalog number: 227270 )
  2. 1.5 ml tubes for transformation
  3. Petri dishes for bacteria (FL MEDICAL, catalog number: 29052 )
  4. Inoculation loop, 10 μl (Greiner Bio One International, catalog number: 731171 )
  5. Ice box with ice
  6. Slide jars for washing
  7. Superfrost Plus glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: J1800AMNT )
  8. Coverslip (Bar Naor, catalog number: BNBB024050A1 )
  9. Pipette tips (20-200 μl, 100-1,000 μl)
  10. Escherichia coli strain of interest (for example CFT073)
  11. Plasmids for fluorescent protein reporter expression (see references for examples)
  12. Calcium chloride (Sigma-Aldrich, catalog number: C5670 )
  13. Glycerol anhydrous (Avantor Performance Materials, J.T. Baker, catalog number: 2136 )
  14. Phospho-buffered saline (PBS 10x) (HyLabs, catalog number: BP-507/1Ld )
  15. Paraformaldehyde (PFA) (Bar Naor, catalog number: BN15711 )
  16. Xylene (Sigma-Aldrich, catalog number: 534056 )
  17. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  18. ProLongTM Glass Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36980 )
  19. Hoechst 33258 (Sigma-Aldrich, catalog number: 94403 )
  20. Dehydrated culture media, LB Broth (BD, DifcoTM, catalog number: 244620 )
  21. Agar purified for microbiology (Sigma-Aldrich, catalog number: 05038 )
  22. Erythromycin (Sigma-Aldrich, catalog number: E6376 )
  23. Ampicillin (Bio Basic, catalog number: AB0028 )
  24. Tris (Avantor Performance Materials, J.T. Baker, catalog number: 4109-1 )
  25. Sodium chloride (Avantor Performance Materials, J.T. Baker, catalog number: 3624-19 )
  26. Polyoxyethylene 20 sorbitan monolaurate (Tween 20) (Sigma-Aldrich, catalog number: 93774 )
  27. Bovine serum albumin (BSA) (VWR, Ameresco, catalog number: 97061-420 )
  28. Fetal bovine serum (FBS) (Biological Industries, catalog number: 04-0071A )
  29. Triton X-100 (Avantor Performance Materials, J.T. Baker, catalog number: X198-07 )
  30. LB medium (see Recipes)
  31. LB agar plates with antibiotics (see Recipes)
  32. TBSS solution (10x) (see Recipes)
  33. Blocking solution (see Recipes)


  1. Pipettes
  2. Autoclave
  3. Spectrophotometer (600 nm wavelength)
  4. Shaker
  5. Micro centrifuge
  6. Incubator
  7. Thermoblock
  8. Chemical hood
  9. Fluorescence microscope (TL-Nikon)


  1. ImagePro Analyzer 7.0 software
  2. Software for statistical analysis (GraphPad Prism software version 6.0 or later, for example)


The procedure outline is described in Figure 1.

Figure 1. Protocol outline. General outline of the protocol describing the three main stages of the assay. The left panel shows preparation of fluorescent protein expressing E. coli. The middle panel outlines the tissue adhesion test and the right panel shows an output of analysis of data acquired during imaging.

  1. Preparation of competent bacteria
    1. Grow E. coli strain of interest in 5 ml of sterile LB medium (see Recipes) in a 50 ml tube, overnight (12-18 h) at 220 rpm shaking at 37 °C.
    2. Inoculate 500 μl of the overnight starter culture into 50 ml of preheated LB (37 °C) and grow for two hours, at 220 rpm shaking at 37 °C until OD600 of 0.3 to 0.4.
    3. Centrifuge at 4 °C, 3,220 x g for 10 min.
    4. Discard supernatant, keep pellet on ice for 10 min.
    5. Suspend pellet in 20 ml of 0.1 M cold CaCl2.
    6. Leave for 25 min on ice.
    7. Centrifuge again as indicated in Step A3, discard supernatant and suspend pellet in 2 ml of 0.1 M CaCl2 + 15% glycerol.
      Note: Glycerol should be autoclaved and the CaCl2 solution should be filtered prior to use.
    8. Incubate on ice for 90 min.

  2. Bacterial transformation
    1. Take 100 μl of competent bacteria into a 1.5 ml tube and add 30 ng of the plasmid of choice encoding for–either GFP (Hansen et al., 2001) or mCherry (Sason et al., 2009).
    2. Incubate for 20 min on ice.
    3. Transfer tubes to a thermoblock heated to 42 °C for 90 sec.
    4. Move tubes to ice for 5 min.
    5. Add 1 ml sterile LB and shake for 1 h at 37 °C, 220 rpm.
    6. Centrifuge at 4,830 x g for 5 min.
    7. Resuspend pellet in 150 μl of fresh LB and seed on an LB agar plate supplemented with appropriate antibiotic for selection, according to resistance encoded on the plasmid of choice (here ampicillin and erythromycin, see Recipes).
    8. Incubate plate overnight at 37 °C.
    9. The next day–pick a single colony, grow in 5 ml LB (supplemented with appropriate antibiotics, see Recipes section) overnight (12-18 h) at 220 rpm shaking at 37 °C.
      Note: In order to avoid loss of fluorescent signal, it is strongly recommended that bacteria expressing fluorescent proteins should be protected from light at all times.
    10. Bring bacterial culture to OD600 of 1 (dilute in sterile 1x PBS).

  3. Tissue binding assay
    This assay uses 4 μm thick paraffin embedded section of tissues fixated in PFA, mounted on glass slides (see Materials and Reagents).
    1. Fill three staining jars with these three solutions and perform deparaffinization as described (Figure 2A):
      1. Xylene–5 min, 5 min, 2 min.
      2. Ethanol 100%–5 min, 5 min, 2 min.
      3. Ethanol 96%–3 min, 2 min, 2 min.
      Note: Deparaffinization is done in a chemical hood.
    2. Cover sections with blocking solution (see Recipes) and incubate at room temperature for 6 h.
    3. Suspend 50 μl of bacteria at OD600 = 1 in 950 μl blocking solution, after discarding blocking solution, lay the bacterial suspension gently on slide.
    4. Incubate overnight at 4 °C in a wet chamber (line chamber with wet tissues), protected from light.
    Next day washing:
    1. Prepare 2 staining jars filled with 1x PBS and another staining jar containing PBS with 0.05% Tween 20 (PBST, Figure 2B).
    2. Wash twice with PBS for 5 min per wash. Transfer the slides to PBST and wash for 10 min (Figure 2B).

      Figure 2. Washing/deparaffinization illustration. Prepare three washing jars filled with the solution indicated on the jar in the illustration and keep the slides in the jar for the indicated amount of time. Figure 2A illustrates the first round of slide deparaffinization in preparation for the binding assay. Figure 2B illustrates washing after overnight incubation to wash off unbound bacteria, dark purple jar contains PBS supplemented with tween (PBST).

    3. Dilute Hoechst 33258 1:5,000 in 1x PBS at approximately 200 μl per slide, apply and incubate for 20 min at room temperature. Protect from light.
    4. Apply mounting medium to slide and cover with a coverslip.
    5. Imagining can be done under a fluorescence microscope using a 60x magnification.
      Note: Scan at least four fields per slide.

Data analysis

  1. Images obtained from the fluorescence microscope are converted to 8 bit images by fluorescence microscopy image analysis software (see Figure 3, for example).
  2. Fluorescent bacteria should be quantified (for each field) by two different experimenters for a total tissue area of 1,600 µm2.
  3. Convert field to mm2. Each fluorescent bacterium counted represents a colony forming unit (CFU), data are represented as Log10CFU/mm2.
    Example: For convenience purposes, this example will refer to a field of 100 µm2.

                                        (CFU in 100 µm2) = (CFU in 0.1 mm2)
                                       10x (CFU in 100 µm2) = CFU in 1 mm2

    If the counted CFU in 100 µm2 is 1,000, the CFU in mm2 will be 10,000 (or, 104) and therefore the log10CFU/mm2 is 4.
  4. Each spot of a single bacterium is referred to as a CFU.

    Figure 3. Fluorescent microscope image of GFP expressing uropathogenic E. coli (UPEC) adhesion paraffin embedded bladder tissue section. Scale bar = 50 µm.


  1. LB medium
    1 L of double distilled water (DDW)
    20 g LB dehydrated culture media
    Mix until dissolved
    Autoclave at 121 °C for 30 min and aliquot
  2. LB agar plates with antibiotics
    1 L of DDW
    20 g LB dehydrated culture media
    15 g of purified agar
    Mix and autoclave. Agar will dissolve during autoclave heating
    Let cool until LB agar can be handled, before it gets solidified
    Add antibiotics (erythromycin at 6 mg/ml and ampicillin at 1 mg/ml) and pour plates
  3. TBSS solution (10x)
    500 ml 0.5 M Tris (pH 7.4)
    800 ml 2 M NaCl
    2 ml Tween 20
    Add DDW up to 2 L
    Mix well
  4. Blocking solution
    100 ml 1x TBSS
    15 g BSA
    15 ml FBS
    5.75 ml 5% Triton X-100


This study was supported by the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP/2007-2013) ERC grant 320473-BacNK. Further support came from the I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation and by the I-Core on Chromatin and RNA in Gene Regulation, the GIF Foundation, the Lewis Family Foundation, an ICRF professorship grant, a Helmholtz Israel grant, a Kamin grant, and the Rosetrees Trust (all to O.M.). O.M is a Crown professor of Molecular Immunology. The authors declare no conflict of interests.


  1. Abed, J., Emgard, J. E., Zamir, G., Faroja, M., Almogy, G., Grenov, A., Sol, A., Naor, R., Pikarsky, E., Atlan, K. A., Mellul, A., Chaushu, S., Manson, A. L., Earl, A. M., Ou, N., Brennan, C. A., Garrett, W. S. and Bachrach, G. (2016). Fap2 mediates fusobacterium nucleatum colorectal adenocarcinoma enrichment by binding to tumor-expressed gal-GalNAc. Cell Host Microbe 20(2): 215-225.
  2. Gur, C., Ibrahim, Y., Isaacson, B., Yamin, R., Abed, J., Gaml iel, M., Enk, J., Bar-On, Y., Stanietsky-Kaynan, N., Coppenhagen-Glazer, S., Shussman, N., Almogy, G., Cuapio, A., Hofer, E., Mevorach, D., Tabib, A., Ortenberg, R., Markel, G., Miklic, K., Jonjic, S., Brennan, C. A., Garrett, W. S., Bachrach, G. and Mandelboim, O. (2015). Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack. Immunity 42(2): 344-355.
  3. Hansen, M. C., Palmer, R. J., Jr., Udsen, C., White, D. C. and Molin, S. (2001). Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration. Microbiology 147(Pt 5): 1383-1391.
  4. Isaacson, B., Hadad, T., Glasner, A., Gur, C., Granot, Z., Bachrach, G. and Mandelboim, O. (2017). Stromal cell-derived factor 1 mediates immune cell attraction upon urinary tract infection. Cell Rep 20(1): 40-47.
  5. Sason, H., Milgrom, M., Weiss, A. M., Melamed-Book, N., Balla, T., Grinstein, S., Backert, S., Rosenshine, I. and Aroeti, B. (2009). Enteropathogenic Escherichia coli subverts phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon epithelial cell infection. Mol Biol Cell 20(1): 544-555.
  6. Schilling, J. D., Mulvey, M. A. and Hultgren, S. J. (2001). Structure and function of Escherichia coli type 1 pili: new insight into the pathogenesis of urinary tract infections. J Infect Dis 183 (Suppl 1): S36-S40.


在这里我们介绍一种方法来测试石蜡包埋组织切片的细菌粘附。 该方法允许检查用荧光蛋白报道质粒转染的不同细菌菌株与各种组织的结合,以更好地理解不同的机制,例如定殖。 该测定为细菌结合提供了更多的生理学背景,比使用细胞系的粘附测定法已经实现的更多。 可以使用荧光显微镜对切片进行成像,并且可以同时量化和测试各种细菌菌株的粘附。

【背景】许多类型的细菌(共生的和致病的)表达各种粘附分子,允许它们结合到宿主的不同表面(Gur等人,2015; Abed等人 ,2016年;艾萨克森等人,2017年)。这种粘附是至关重要的,因为它是殖民化的第一步,并在不同的环境中在竞争和生存中发挥作用(Schilling et al。,2001)。这些粘附素中的许多是凝集素,在各种细胞上的糖蛋白上的结合糖部分,如上皮细胞和其他细胞(Abed等人,2016; Isaacson等人 ,2016)。多年来,许多研究宿主 - 病原体相互作用的小组使用细胞系和组织培养来试图了解细菌对细胞的粘附。组织切片为定植研究提供了更多的生理学背景,因为它们提供了使用体外组织培养几乎不可能获得的组织和结构。此外,在永生化或癌细胞中,细菌可以结合的表面分子的表达模式可能会改变。为了更好地理解细菌粘附的生理学背景,在正常和病理条件下,我们选择使用细菌附着到组织切片。

关键字:宿主-病原体相互作用, 细菌附着, 细菌定植


  1. 塑料50毫升离心管(Greiner Bio One International,目录号:227270)
  2. 用于转化的1.5ml试管
  3. 细菌培养皿(FL MEDICAL,目录号:29052)
  4. 接种环,10μl(Greiner Bio One International,目录号:731171)
  5. 带冰的冰箱
  6. 滑动瓶子进行清洗
  7. Superfrost Plus玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:J1800AMNT)
  8. Coverslip(Bar Naor,产品目录号:BNBB024050A1)
  9. 移液器吸头(20-200μl,100-1,000μl)
  10. 感兴趣的大肠杆菌菌株(例如CFT073)
  11. 用于荧光蛋白报告子表达的质粒(参见实例)
  12. 氯化钙(Sigma-Aldrich,目录号:C5670)
  13. 无水甘油(Avantor Performance Materials,J.T.Baker,目录号:2136)
  14. 磷酸盐缓冲盐水(PBS 10x)(HyLabs,目录号:BP-507 / 1Ld)
  15. 多聚甲醛(PFA)(Bar Naor,目录号:BN15711)
  16. 二甲苯(Sigma-Aldrich,目录号:534056)
  17. 乙醇(Sigma-Aldrich,目录号:E7023)
  18. ProLong TM Glass Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM,目录号:P36980)
  19. Hoechst 33258(Sigma-Aldrich,目录号:94403)
  20. 脱水培养基,LB肉汤(BD,Difco TM,目录号:244620)
  21. 纯化用于微生物学的琼脂(Sigma-Aldrich,目录号:05038)
  22. 红霉素(Sigma-Aldrich,目录号:E6376)
  23. 氨苄青霉素(Bio Basic,目录号:AB0028)
  24. Tris(Avantor Performance Materials,J.T.Baker,目录号:4109-1)
  25. 氯化钠(Avantor Performance Materials,J.T.Baker,目录号:3624-19)
  26. 聚氧乙烯20失水山梨糖醇单月桂酸酯(吐温20)(Sigma-Aldrich,目录号:93774)
  27. 牛血清白蛋白(BSA)(VWR,Ameresco,目录号:97061-420)
  28. 胎牛血清(FBS)(Biological Industries,目录号:04-0071A)
  29. Triton X-100(Avantor Performance Materials,J.T.Baker,目录号:X198-07)
  30. LB培养基(见食谱)
  31. LB琼脂平板与抗生素(见食谱)
  32. TBSS解决方案(10倍)(请参阅食谱)
  33. 阻止解决方案(请参阅食谱)


  1. 移液器
  2. 高压灭菌器
  3. 分光光度计(600 nm波长)
  4. 摇床
  5. 微型离心机
  6. 孵化器
  7. Thermoblock
  8. 化学罩
  9. 荧光显微镜(TL-Nikon)


  1. ImagePro Analyzer 7.0软件
  2. 用于统计分析的软件(例如GraphPad Prism软件版本6.0或更高版本)




  1. 主管细菌的制备
    1. 成长E。大肠杆菌菌株在5ml无菌LB培养基中(参见食谱)在50ml试管中过夜(12-18小时),在37℃下以220rpm振荡。
    2. 将500μl的过夜发酵剂培养物接种到50ml预热的LB(37℃)中并在37℃下以220rpm振荡生长2小时直至OD 600在0.3-0.4之间。 />
    3. 在4℃下离心3,220×g 10分钟。
    4. 丢弃上清液,将颗粒放在冰上10分钟。
    5. 将沉淀悬浮在20ml 0.1M冷CaCl 2中。

    6. 在冰上放置25分钟
    7. 如步骤A3所示再次离心,弃上清并悬浮沉淀在2ml 0.1M CaCl 2 + 15%甘油中。
    8. 在冰上孵育90分钟。

  2. 细菌转化
    1. 取100μl感受态细菌放入1.5ml试管中,加入30ng编码GFP(Hansen等,2001)或mCherry(Sason等,2001)的质粒。 ,2009)。

    2. 在冰上孵育20分钟
    3. 将管子转移到加热到42°C的热块上90秒。
    4. 将试管移至冰上5分钟。
    5. 加入1毫升无菌LB并在37℃,220转/分振荡1小时。

    6. 在4,830克xg离心5分钟。
    7. 根据选择的质粒(这里为氨苄青霉素和红霉素,参见食谱)编码的抗性,将沉淀重悬于150μl新鲜LB中,并将种子在补充有适当抗生素的LB琼脂平板上进行选择。

    8. 在37°C孵育过夜。
    9. 第二天,挑选单个菌落,在37℃下以220rpm摇动过夜(12-18小时),在5ml LB(补充适当的抗生素,参见食谱部分)中生长。
    10. 使细菌培养物达到OD 600的1(在无菌1x PBS中稀释)。

  3. 组织结合分析
    1. 用这三种溶液填充三个染色瓶并按照描述进行脱蜡(图2A):
      1. 二甲苯-5分钟,5分钟,2分钟
      2. 乙醇100%-5分钟,5分钟,2分钟。
      3. 乙醇96%-3分钟,2分钟,2分钟。

    2. 使用阻断溶液覆盖切片(见食谱)并在室温下孵育6小时。
    3. 在弃去封闭溶液后,在950μl阻断溶液中,在OD 600 = 1中悬浮50μl细菌,将细菌悬液轻轻放在载玻片上。

    4. 在4°C的湿室(带湿纸巾的线室)中孵育过夜,避光。
    1. 准备2个装满1x PBS的染色瓶和另一个含有0.05%吐温20的PBS(PBST,图2B)的染色罐。
    2. 用PBS清洗两次,每次5分钟。将玻片转移至PBST并清洗10分钟(图2B)。


    3. 在1x PBS中稀释Hoechst 33258 1:5,000,每张玻片约200μl,在室温下孵育20分钟。避光。
    4. 使用安装介质滑动并用盖玻片盖住。
    5. 想象可以在60倍放大的荧光显微镜下完成。


  1. 从荧光显微镜获得的图像通过荧光显微镜图像分析软件转换成8位图像(例如参见图3)。
  2. 应由两个不同的实验人员对荧光细菌进行量化(对于每个场),总组织面积为1,600μm2 。
  3. 将字段转换为mm 2 。计数的每个荧光细菌代表菌落形成单位(CFU),数据表示为Log 10 CFU / mm 2 2。

                           &NBSP ;            (CFU in100μm 2 )=(CFU in 0.1mm 2 2 / sup> )
                           &NBSP ;           10x(CFU在100μm中)= CFU在1mm中 2 2 2 / sup>

    如果计数的CFU在100μm 2 是1,000,则CFU以mm为单位 2 2 sub> 10 CFU / mm 2 是4。 < br />
  4. 单个细菌的每个点称为CFU。

    图3. GFP表达的尿路致病性大肠杆菌(UPEC)粘附石蜡包埋的膀胱组织切片的荧光显微镜图像比例尺=50μm。


  1. LB媒介
  2. 含有抗生素的LB琼脂平板
    高压釜加热过程中,琼脂会溶解 冷却至LB琼脂可以处理,然后固化
  3. TBSS解决方案(10x)
    500毫升0.5 M Tris(pH 7.4)
    800毫升2M NaCl

    添加DDW至2升 充分搅拌
  4. 阻止解决方案
    100毫升1x TBSS
    5.75毫升5%Triton X-100


欧洲研究理事会(ERC)根据欧盟第七框架计划(FP / 2007-2013)ERC拨款320473-BacNK支持该研究。计划和预算委员会的I-CORE计划和以色列科学基金会以及基因调控中的染色质和RNA I-Core,GIF基金会,刘易斯家庭基金会,ICRF教授职位资助,Helmholtz以色列赠款,Kamin赠款和Rosetrees信托(全部给OM)。 O.M是分子免疫学的皇冠教授。作者声明不存在利益冲突。


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引用:Isaacson, B., Hadad, T., Bachrach, G. and Mandelboim, O. (2018). Quantification of Bacterial Attachment to Tissue Sections. Bio-protocol 8(5): e2741. DOI: 10.21769/BioProtoc.2741.