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Gram Stain for Intestinal Bacteria
肠道细菌的革兰氏染色   

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Abstract

With this protocol you can perform a gram stain in paraffin embedded tissue sections.

Materials and Reagents

  1. Tissue sections (paraffin embedded, gained from mice)
  2. Paraffin (Engelbrecht, catalog number: 17932 )
  3. Xylene (VWR International, catalog number: 28975 )
  4. Ethanol (Sigma-Aldrich, catalog number: 32205 )
  5. Iodine (Merck KGaA, catalog number: 109261 )
  6. Safranin (Merck KGaA, catalog number: 109217 )
  7. Crystal violet (Merck KGaA, catalog number: 109218 )
  8. Decolorizing agent (Merck KGaA, catalog number: 110218 )
  9. 100% (v/v) ethanol (see Recipes)
  10. 96% (v/v) ethanol (see Recipes)
  11. 80% (v/v) ethanol (see Recipes)

Equipment

  1. Slide

Procedure

  1. Paraffin embedded 5 µm tissue sections were deparaffinized: 1 h on 58 °C, 5 x 6 min in xylene, 4 x 3 min in 100% (v/v) ethanol, 2 min in 96% (v/v) ethanol, 2 min in 80% (v/v) ethanol and 2 min in aqua dest.
  2. Tissue sections were incubated for 1.5 min with crystal violet staining reagent and washed for 30 sec under running tap water.
  3. The slide was flooded with Gram's iodine for 3 min followed by a second washing step for 20 sec with tap water.
  4. The slide was flooded with decolorizing agent for 20 sec and once again washed for 20 sec under tap water.
  5. The counterstain was safranin (1 min) which was followed by a last washing step under tap water.

Representative data



Figure 1. The intestine was stained by gram stain. Gram+ bacteria appear blue and are indicated by a red arrow.

Notes

The staining can also be performed additionally to a previous immune histological staining.

Recipes

  1. 100% (v/v) ethanol
    Pure ethanol
  2. 96% (v/v) ethanol
    96 ml ethanol and 4 ml aqua dest
  3. 80% (v/v) ethanol
    80 ml ethanol and 20 ml aqua dest
    Note: The staining solutions are ready to use.

Acknowledgments

This work was supported by the DFG (ZE872/1-2 individual grant to RZ)

References

  1. Schwab, L., Goroncy, L., Palaniyandi, S., Gautam, S., Triantafyllopoulou, A., Mocsai, A., Reichardt, W., Karlsson, F. J., Radhakrishnan, S. V., Hanke, K., Schmitt-Graeff, A., Freudenberg, M., von Loewenich, F. D., Wolf, P., Leonhardt, F., Baxan, N., Pfeifer, D., Schmah, O., Schonle, A., Martin, S. F., Mertelsmann, R., Duyster, J., Finke, J., Prinz, M., Henneke, P., Hacker, H., Hildebrandt, G. C., Hacker, G. and Zeiser, R. (2014). Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage. Nat Med 20(6): 648-654.

简介

使用此协议,您可以在石蜡包埋组织切片中进行革兰染色。

材料和试剂

  1. 组织切片(石蜡包埋,从小鼠获得)
  2. 石蜡(Engelbrecht,目录号:17932)
  3. 二甲苯(VWR International,目录号:28975)
  4. 乙醇(Sigma-Aldrich,目录号:32205)
  5. 碘(Merck KGaA,目录号:109261)
  6. Safranin(Merck KGaA,目录号:109217)
  7. 结晶紫(Merck KGaA,目录号:109218)
  8. 脱色剂(Merck KGaA,目录号:110218)
  9. 100%(v/v)乙醇(见配方)
  10. 96%(v/v)乙醇(见配方)
  11. 80%(v/v)乙醇(见配方)

设备

  1. 滑动

程序

  1. 石蜡包埋的5μm组织切片脱蜡:在58℃下1小时,在二甲苯中5×6分钟,在100%(v/v)乙醇中4×3分钟,在96%(v/v)乙醇中2分钟,2 min在80%(v/v)乙醇中,2分钟在水中。
  2. 组织切片用结晶紫染色试剂孵育1.5分钟,并在流动的自来水下洗涤30秒。
  3. 载玻片用克氏碘淹没3分钟,然后用自来水进行第二次洗涤步骤20秒。
  4. 将载玻片用脱色剂淹没20秒,并在自来水下再次洗涤20秒。
  5. 复染色剂是safranin(1分钟),随后是在自来水下的最后一个洗涤步骤

代表数据



图1.肠通过革兰氏染色染色。革兰氏+细菌显示为蓝色,并由红色箭头指示。

笔记

还可以对先前的免疫组织学染色进行染色。

食谱

  1. 100%(v/v)乙醇 纯乙醇
  2. 96%(v/v)乙醇 96 ml乙醇和4 ml aqua dest
  3. 80%(v/v)乙醇 80毫升乙醇和20毫升水渍
    注意:染色溶液即可使用。

致谢

这项工作是由DFG(ZE872/1-2个人授权RZ)

参考文献

  1. Schwab,L.,Goroncy,L.,Palaniyandi,S.,Gautam,S.,Triantafyllopoulou,A.,Mocsai,A.,Reichardt,W.,Karlsson,FJ,Radhakrishnan,SV,Hanke, Graeff,A.,Freudenberg,M.,von Loewenich,FD,Wolf,P.,Leonhardt,F.,Baxan,N.,Pfeifer,D.,Schmah,O.,Schonle,A.,Martin,SF,Mertelsmann R.,Duyster,J.,Finke,J.,Prinz,M.,Henneke,P.,Hacker,H.,Hildebrandt,GC,Hacker,G.and Zeiser,R。(2014)。 在肠细菌移位时募集的中性粒细胞通过组织损伤增强移植物抗宿主病。 a> Nat Med 20(6):648-654。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Goroncy, L. and Zeiser, R. (2015). Gram Stain for Intestinal Bacteria. Bio-protocol 5(6): e1419. DOI: 10.21769/BioProtoc.1419.
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