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.

Bacteriophage uptake by Eukaryotic cell layers represents a major sink for phages during therapy

“…Cells were first grown on glass bottom slides for two days to generate an ~ 80% confluent cell layer, followed by fluorescence staining of the nucleus and plasma membrane. T4 phages were prepared using the Phage-on-Tap method (45), labelled using SYBR-Gold, subsequently washed to remove residual stain, and then directly applied to cell layers observed using live cell imaging.…”

“…The bacterial strains used in this study, Escherichia coli B strain HER and E. coli B strain W3350, were cultured in lysogeny broth (LB) media (10 g tryptone, 5 g yeast extract, 10 g NaCl, in 1 litre of distilled water [dH2O]) at 37 ºC shaking overnight and used to propagate and titre phages T4, T3 and lambda supplemented with 10 mM CaCl2 and MgSO4. Phages T4, T3 and lambda were cleaned and purified using the Phage on Tap protocol (PoT)(45) and titred up to a concentration of approximately 10 10 phages per ml. After purification, phages were stored in a final solution of SM Buffer (2.0 g MgSO4•7H2O, 5.8 g NaCl, 50 ml of 1M Tris-HCl pH 7.4, dissolve in 1 litre of dH2O) at 4 ºC.…”

Characteristics of lytic phage vB_EcoM-ECP26 and reduction of shiga-toxin producing Escherichia coli on produce romaine

“…Resuspended phage was filtered through a 0.22 μm filter to remove bacterial debris. Subsequently, isolated phage was stored at -80 °C in LBC broth containing 20% glycerol until further use [34,11].…”

Temporal stability and genetic diversity of 48-year old T-series phages

“…In instances of no plaque formation, amplification was attempted using the original lysate and its host in broth culture, then replated. Lysates with viable phages were propagated following the Phage-on-Tap protocol (Bonilla et al 2016).…”