Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

PeerJ

article

Natasha Bonilla¹  Maria Isabel Rojas¹  Giuliano Netto Flores Cruz¹  Shr-Hau Hung¹  Forest Rohwer¹  Jeremy J. Barr¹ 

[1] Department of Biology, San Diego State University

关键词: Bacteriophage;    Endotoxin;    Cesium chloride;    Top agar;    Ultrafiltration;    Dialysis;    Speed vacuum;    Phage bank;   

DOI  :  10.7717/peerj.2261

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

【 授权许可】

CC BY