Phage on Tap – A quick and efficient protocol for the preparation of bacteriophage laboratory stocks

Abstract

14 A major limitation with traditional phage preparations is the variability in titer, salts, and 15 bacterial contaminants between successive propagations. Here we introduce the Phage On Tap 16 (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) 17 stocks. This method produces homogenous, laboratory-scale, high titer (up to 10 10-11 PFU•ml -1 ), 18 endotoxin reduced phage banks that can be used to eliminate the variability between phage 19 propagations and improve the molecular characterizations of phage. The method consists of five 20 major parts, including phage propagation, phage clean up by 0.22 µm filtering and chloroform 21 treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and 22 storage of phage banks for continuous laboratory use. From a starting liquid lysate of >100 mL, 23 the PoT protocol generated a cleaned, homogenous, laboratory phage bank with a phage 24 recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards 25 of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of 26 only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing 27 endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol 28 focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated 29 to sufficient titer, producing homogenous, high titer phage banks that are applicable for 30 molecular and cellular assays. 31 32

Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

“…After incubation at 37 °C for 20 min, the phage-host suspension was mixed with 5 ml of soft agar (0.7% Luria Bertani Agar) and overlaid onto Nutrient agar plates, after incubation at 37 °C for 24 h, PFU/ml was determined. Plates showing 1–10 plaques was used to obtain homogenous plaque morphology by triple purification as mentioned earlier (Bonilla et al 2016 ). Briefly, a single plaque was picked, resuspended in SM buffer, vortexed and centrifuged.…”

Engineering laminated paper for SARS-CoV-2 medical gowns

“…Phi-X174 was propagated using host Escherichia coli C ATCC 13706. The lysate was purified following the Phage-on-Tap protocol [ 35 ]. Chloroform extraction was not performed during phage purification of the lysate due to incompatibility with the test materials.…”

Clinical Experience of Personalized Phage Therapy Against Carbapenem-Resistant Acinetobacter baumannii Lung Infection in a Patient With Chronic Obstructive Pulmonary Disease

“…The phage preparations were processed in two different ways. The first batch phage preparation was purified with Amicon Ultra-15 centrifugal filter units (MWCO 100 K Da, Merck Milipore, Massachusetts, USA) (Bonilla et al, 2016). The second phage preparation was purified in a cesium chloride density gradient as previously described (Bachrach and Friedmann, 1971) and then dialyzed using a Spectra/Por RC membrane (MWCO 20 K Da, Spectrum, New Jersey, USA) in phosphate-buffered saline to remove cesium chloride.…”