BACTERIOPHAGES
Biology and Applications
EDITED BY
Elizabeth Kutter
Alexander Sulakvelidze
Written by Karin Carlson
Department of Cell and Molecular Biology,
University of Uppsala, Uppsala, Sweden
Phage Stocks
General Considerations
Phages are fairly fragile and should be handled gently and not to expose them to osmotic shock, or to strong daylight or fluorescent light.
Vigorous mixing of phage solutions with a pipette and aggressive vortexing should also be avoided.
The time during which phages maintain full viability varies considerably.
The titers of phage stocks should be determined at the time they are prepared, and also shortly before their use.
A phage stock that has been stored cold for some time can be warmed to 37° for 5–10 min and gently mixed in order to disaggregate phage clumps.
Phage stocks (like stocks of microorganisms) should not be routinely propagated by serial passage, because variants that grow faster than the original strain will increase in frequency with every round of growth.
An archival or “master stock” of each phage strain should be maintained, preferably in aliquots kept in different places as a precaution against laboratory accidents.
New working stocks should be grown from this stock as required.
Phage stocks can be prepared in any medium that permits growth of the bacterial host strain.
During propagation, mutations will arise.
It is prudent to start new phage stocks from single plaques but there is a risk that the selected plaque was formed by a nondesired variant phage. An appropriate balance must be determined on a case-by-case basis between using single plaques to start cultures and picking 2–3 plaques to avoid the risk of accidentally selecting a variant.
It is advisable that the properties of new phage stocks be compared with the original stock’s known properties.
Lytic phage progeny are harvested after they are released from their host bacteria by cell lysis.
If the phages do not contain lipids, chloroform can be added to the infected cells to release the progeny phages and reduce their adsorption to bacteria and bacterial debris.
A lysed culture is commonly centrifuged or filtered to remove bacterial debris, and the resulting suspension is commonly referred to as a “cleared lysate.”
