I am currently working on a spot assay for a bacteriophage that infects Ralstonia solanacearum. There are 3 types of stock bacteriophages that I use:
1. Original stock isolated from 2016, I think without the addition of glycerol
2. Stock of bacteriophage - ralstonia solanacearum which has been filtered using a syringe filter 0.2 and stored without glycerol (2021)
3. Stock of bacteriophage from plaque stored in a mixture of ralstonia solanacearum : glycerol 40% (1 : 1). This plaque is from stock restorations 1 and 2 (also 2021)
all three stocks were stored at -80. The results of the spot test, stocks with glycerol (number 3) showed a clear zone/plaque, other stocks did not. I doubt whether the clear zone that appears is plaque or because the growth of bacteria is inhibited by glycerol. Please help with the information if anyone has experience with this. Thanks.
The best answer you can get will be to perform a +glycerol, no phage control.
I agree with Pierre Béguin that it seems you can't really be certain without running the controls. But I doubt you are seeing any phage lysis in these spots and it probably is just a glycerol artifact.
Dear Anggi Anwar Hendra Nurdika
A universal and effective method for long-term bacteriophage storage has yet to be described well and It differs from phage to phage. Phage are known to be obligate intracellular parasites that require a suitable bacterial host cell to multiply and proliferate. Phage could be stored inside an infected host cell with Glycerol at -80°C without significant loss of phage and host viability, which is a standard method for phage preservation.
I agree with your third method of phage storage, and I hope you will accept Pierre Beguin's guidance.
good luck.
No glycerol has not much inhibitory effects,but in this pictures some phage activity seems to be there
Thanks for the advice, I've done a spot test with only 40% glycerol as suggested and the result is a clear zone.
It's a bit odd that glycerol produces a clear zone like plaque, maybe the concentration is too high. Then I decided to store the bacteriophages only in SM Buffer without glycerol and host bacteria. Maybe any other suggestions for storage ?
Glycerol can cause osmotic stress on the bacteria leading to clearings on your bacterial lawn if too concentrated.
You can try to produce stocks with varying concentrations of glycerol (as long as phages remain stable) and dilute with SM buffer to reduce glycerol concentration before use. Phage titre will also be reduced.
Note that phage tolerance to freeze varies and you may need to try a different approach.
I'll leave this article for other options: Article A reliable method for storage of tailed phages
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