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Optimization of FISH Protocol

Bacteria and Archaea in Paddies Detected by FISH Jiangtao Qin, T. Eickhorst, Bin Zhang, R. Tippkötter. Optimization of FISH Protocol. Purpose: 1)Better understanding of the number and distribution of bacteria and archaea of paddies; 2) A proper format to applied FISH analysis to soil samples.

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Optimization of FISH Protocol

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  1. Bacteria and Archaea in Paddies Detected by FISHJiangtao Qin, T. Eickhorst, Bin Zhang, R. Tippkötter • Optimization of FISH Protocol Purpose: 1)Better understanding of the number and distribution of bacteria and archaea of paddies; 2) A proper format to applied FISH analysis to soil samples. • Decreased probe concentrations (5 to 1 ng µl-1) • Increased tyramide concentration (1:50 to 1:10). • Increased temperature during hybridization and washing (35℃ / 37℃ to 46℃ / 48℃) • SDS, formamide, Triton X-100, acetone, proteinase K, and lysozyme, were examined. a)Normal FISH without ultrasound treated b)Normal FISH without agrose added c)Normal FISH without lysozyme added d)CARD-FISH without ultrasound treated e)Normal FISH treated with all procedures f)CARD-FISH treated with all procedures

  2. Quantification of Cells 0-7cm: A) Bacteria in NF-ZM-f; B) Bacteria in FC-f; C) Bacteria in FC-s; 7-14cm:D) Archaea in NF-SM-f; E) Archaea in FC-f; F) Bacteria aggregates in FC-s. • By proper pretreatment and improved CARD-FISH protocol, microbes can be quantified in paddies. • More specific rRNA-targeted probes to determine specific taxa or functional groups. • Development of thin section ---applying FISH in undisturbed soil samples.

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