Bacteriophages (phages) are all around us, they are soil dwellers, water dwellers and possibly even air dwellers. The time has arrived for the 2019 Phage Hunt as six classmates set off to attempt successfully isolate phages. The first step of extracting a phage from their environment is to isolate them. The sample is processed through filtration and then progressing to plating collected solution onto a bacterial lawn of base agar dosed with the target host bacterial strain.
This year, there seemed to be an inconsistency regarding the typical frequency of successfully finding a phage using host strain Mycobacterium smegmatis mc2155. Hence a protocol was incorporated into the direct isolation stage (before protocol 5.3) in attempt to increase the chances of phage isolation- phage precipitation by zinc chloride (referred to here as ZnCl2 precipitation).
ZnCl2 precipitation, as proposed by Santos, was more commonly used for rapid extraction and purification of phage genomic DNA (Santos, 1991). However, the scope of ZnCl2 precipitation is being redefined, as it is now also being adapted for use in the isolation phage particles from environmental samples (Czajkowski, Ozymko, & Lojkowska, 2016).
As phage particles are mostly comprised of a protein coat that protects genetic material( i.e. found in head and tail proteins), ZnCl2 precipitation works alongside the process of “salting out” proteins in assays for protein precipitation(Arakawa & Timasheff, 1984). Likewise, when phage particles are in a solution containing high salt content, precipitation occurs due to electrolyte-nonelectrolyte interaction (Czajkowski et al., 2016). This causes the nonelectrolyte phage particles to be less soluble at high ZnCl2 concentrations (Czajkowski et al., 2016). Experiments done by Czajkowski et al. suggest the sensitivity of phage detection, which were seen as plaque-like formations on the top agar, using this precipitation method was 10-100 times higher (Czajkowski et al., 2016).
The effects of ZnCl2 precipitation method was experienced first-hand as analysis of plates resulting from samples processed using this method had significantly more plaque counts compared to plates of which the samples were not processed in this way. However, although the sensitivity of phage detection increased, the actual chances of finding phage had not risen. Many plaque-like formations were picked and spot tested however none had given a positive result of a presence of a phage.
Using this method had given many of our classmates false hope, but we had learnt that research is not all about positive findings. A deeper understanding of phage structure and how components of their structure are able to interact with the environment in vivo.
Arakawa, T., & Timasheff, S. N. (1984). Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry, 23(25), 5912-5923. doi:10.1021/bi00320a004
Czajkowski, R., Ozymko, Z., & Lojkowska, E. (2016). Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts. Folia microbiologica, 61(1), 29-33. doi:10.1007/s12223-015-0411-1
Santos, M. A. (1991). An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Res, 19(19), 5442. doi:10.1093/nar/19.19.5442