Storage of Bacteriophages at 4°C Leads to no Loss in Their Titer after One Year

Lauria Bertani (LB) broth was employed as the storage medium of the bacteriophages. For preservation, at -20°C and -80°C, 30% v/v of glycerol (50%), was utilized with a final 15% glycerol concentration. Each sample (1mL) was stored in 1.7 ml Eppendorf tubes in triplicates.

Bacteriophages were stored in triplicate at 37°C, 25°C, 4°C, -20°C and -80°C for 12 months. The titers of bacteriophages JHP, RLP, RSP, SaPL and IttPL before storage was 1.6x1013, 4.6x1011, 4.0x1017, 9.3x1015 and 2.0x108, respectively, for storage at 37 oC, 25 oC and 4 oC, while, the titer for storage at -20 oC and -80 oC for bacteriophage JHP, RLP, RSP, SaPL and IttPL was 1.1x1013, 3.2x1011, 2.8x1017, 6.5x1015 and 1.4x 108, respectively.

The titer of the all the bacteriophages before and after storage was counted utilizing double layer agar overlay method (Khawaja et al., 2016). In double layer agar overlay method, dilution of bacteriophage, incubated with host bacterium for 10-15 min, was overlaid on the agar plate followed by addition of 3-5 mL of semi solid agar and incubated overnight. The plaques were counted after incubation.

The titer of tested bacteriophages diminished from 8 to 17 logs when the bacteriophages were stored at 37oC and this loss in titer was from 84 to 100 percent. Similarly, a 69-100 percent titer decrease (8-15 logs) was noted when put at 25oC. Interestingly, we detected no decrease in bacteriophage titer of all investigated viruses when the bacteriophages were stored at 4oC. Surprisingly, the bacteriophage titer also decreased (5-12 log; 25 to 70 percent) and (5-12 logs; 45 to 100 %) when stored at -20oC and -80oC, respectively (Table I).

In our investigation, the most reasonable condition observed for bacteriophage storage with minimal titer drop was 4 oC, which is in agreement with others working with tailed bacteriophages (Ackermann et al., 2004). At 4oC temperature, no substantial drop in the titer occurred and zero log decrease across. In contrast, freezing conditions of -20 oC and -80 oC appeared to substantially reduce bacteriophage titer. Storage at 25oC or 37oC, however, had the greatest impact on phage titer as few infectious virions were recovered from these phage suspensions after 12 months. Except for SaPL and JHP, zero percent survival was observed in other investigated bacteriophages (RLP, RSP, and ITTPL) at 37 oC, while at 25 oC, zero percent survival was observed in case of SaPL. The reductions in phages titer stored at 37, 25, 4, -20 and -80 oC is presented in Figures 1 and 2.

The comparative detail in terms of percent log decrease at 37, 25, 4, -20 and -80 oC for the 5 bacteriophages under investigation is presented in Figure 2.

Table I.- Titers of bacteriophage lysates before and after (one year) storage at 37, 25, 4, -20 and -80oC for bacteriophage JHP, RLP, RSP, SaPL and IttPL.

* name of bacteriophage.

Bacteriophages have been studied for over 100 years, which served us to understand many modern biological aspects, however, no common method for their preservation is available and in use. Despite this, there are very few published studies, focusing on this issue. It is perhaps owing to the disparate nature of bacteriophages that fewer efforts on this aspect of the bacteriophage biology are available. In this investigation, the activity of five tailed bacteriophages JHP, RLP, RSP, SaPL and IttPL, isolated from different sewage water sources of Lahore, Pakistan, was examined after their long-term storage. The novelty of this research is that no special preservatives were used in our phage solutions. The medium chosen for long-term storage was standard LB broth, which is regularly employed for the propagation of bacteriophages.

In earlier studies, different conditions for bacteriophage preservation were documented, including storage at 4, -20, -80oC and even Nitrogen freezing at -196oC, however, the suitable storage temperature vary with phage type (Clark, 1962; Clark et al., 1962; Warren and Hatch, 1969). In this study, 4oC was found to be the suitable temperature for storage of these bacteriophages, similar to results described by others working with tailed bacteriophages (Ackermann et al., 2004). They found that the tailed and filamentous phages without lipids can be stored for over 5-10 years, but their results varied with different bacteriophages. The results of our study are in accordance with another study which revealed no change in titer of bacteriophages Staphylococcus aureus and P. aeruginosa after one year of storage at 4oC (Merabishvili et al., 2009).

There are reports of bacteriophages sensitivity to low temperatures, including seven log reductions in titer observed after only t three months of storage. Interestingly, the Bacillus phage CP-54Ber showed the most stability at room temperature (Ackermann et al., 2004). None of the bacteriophages under study demonstrated significant viability after one year of storage at room temperature and 37oC, except JHP where a 30 % viability was observed. Sometimes, bacteriophages are also stored at chilling conditions with the addition of glycerol. It has also been documented that freezing with glycerol was not as effective as preservation at 4oC, which is evident in the current study (Clark and Geary, 1973). We observed a 25 to 71% and 45 to 100% decrease in bacteriophage titer, when phages were stored at -20oC and -80oC, respectively for 1year.

Based on this study, it was concluded that 4oC is the best storage temperature for bacteriophages, followed by -20oC and -80oC, in terms of minimally impacting viability suitability. Whereas, the temperature between 25 and 37oC were not suitable for bacteriophage storage. To further enhance our knowledge regarding storage stability of bacteriophages, storage stability studies of more than 1-year are required.

The authors declare no conflict of interest.