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Quantification of Bacterial Attachment to Tissue Sections

组织切片细菌附着定量

BI Batya Isaacson
TH Tehila Hadad
GB Gilad Bachrach
Ofer Mandelboim Ofer Mandelboim

发布: 2018年03月05日第8卷第5期 DOI: 10.21769/BioProtoc.2741 浏览次数: 8355

评审: Andrea PuharSofiane El-Kirat-ChatelMigla Miskinyte

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Abstract

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) search Bacterial attachment (细菌附着) search Bacterial colonization (细菌定植) search

Background

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)

Equipment

  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)

Software

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

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

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分类

免疫学 > 宿主防御 > 鼠

微生物学 > 微生物-宿主相互作用 > 离体模型

细胞生物学 > 组织分析 > 组织成像

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