Phage on tap: A quick and efficient protocol for the preparation of bacteriophage laboratory stocks

Natasha Bonilla, Jeremy J. Barr

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

Abstract

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 1010–12 PFU/mL), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations, improve the molecular characterizations of phage, and may be applicable for therapeutic applications. The method consists of five major parts, including phage propagation, phage cleanup by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of >100 mL, the PoT protocol generated a cleaned, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just 2 days. In contrast, the traditional method took upward of 5 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 3000-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.

Original languageEnglish
Title of host publicationThe Human Virome
Subtitle of host publicationMethods and Protocols
EditorsAndrés Moya, Vicente Pérez Brocal
Place of PublicationNew York NY USA
PublisherHumana Press
Chapter4
Pages37-46
Number of pages10
Volume1838
ISBN (Electronic)9781493986828
ISBN (Print)9781493986811
DOIs
Publication statusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1838
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Bacteriophage
  • Cesium chloride
  • Dialysis
  • Endotoxin
  • Phage bank
  • Speed vacuum
  • Top agar
  • Ultrafiltration

Cite this

Bonilla, N., & Barr, J. J. (2018). Phage on tap: A quick and efficient protocol for the preparation of bacteriophage laboratory stocks. In A. Moya, & V. Pérez Brocal (Eds.), The Human Virome: Methods and Protocols (Vol. 1838, pp. 37-46). (Methods in Molecular Biology; Vol. 1838). New York NY USA: Humana Press. https://doi.org/10.1007/978-1-4939-8682-8_4