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

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 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up 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 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.

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

“…Bacteriophages were concentrated by centrifugation at 6,000 ϫ g for 10 h at 4°C, and phage pellets were gently resuspended in 10 ml of Dulbecco's phosphate-buffered saline and sterilized again by filtration. The sterilized phage suspensions were subjected to ultrafiltration and 1-octanol extraction to remove lipopolysaccharide (LPS) as previously described (30). In most cases, this final treatment was performed at the Laboratory for Viral Information at San Diego State University.…”

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

“…This reveals some of the mechanism behind the PLE's successful anti-phage activity and lays the groundwork for asking classic questions about evolutionary theory in respect to optimal virulence and selective pressures in complicated parasitic relationships, all the while providing insight into an ongoing evolutionary arms race that impacts human health. Bacteriophages were propagated on PLE (-) V. cholerae hosts and prepped via polyethylene glycerol precipitation or concentration and media exchange on Amicon Ultra -15 (Millipore) centrifugal filters (73,74). Stocks were stored in sodium chloride-tris-ethylenediaminetetraacetic acid buffer (STE), and quantified via the soft agar overlay method (73).…”

Bacteriophages targeting Acinetobacter baumannii capsule induce antimicrobial resensitization

“…1A). We used the Phage-on-Tap protocol (36) to isolate and purify phages into high-titer preparations targeting the nine strains. Eight bacteriophages were isolated and used to establish an A. baumannii -specific phage library.…”