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LABORATORY PROCEDURES FOR PHAGES
REFERENCE NO: PH/2003/02/01
TITLE: REQUEST AND RECEIPT OF PHAGES
This document describes the sequence of the principal steps when a new phage
is to be accessioned with a culture collection
- New phages for admission in the public collection are identified as a follow-up of one
of the events specified hereunder:
- an application by a depositor (1.1.)
- a customer request (1.2.)
- screening scientific literature databases (1.3.)
In case the collection cannot accept
the offered phages (due to national regulations, outside the interest of
the collection, …), the potential depositor is informed.
Only phages conform to the accession criteria of the collection can
- In cases (1.2.) and (1.3.) potential depositors are invited by letter to deposit the
plasmid with the collection.
- In order to receive the information about the plasmid in a standard format, the
depositor is requested to complete and return the collections Accession Form. The
data given in the Accession Form should enable the collection to decide whether the
plasmid can be accepted or should be refused according to the criteria established. The
form should also provide the information necessary for handling and preservation of the
plasmid, as well as information of interest to the scientific community. Examples of such
Accession Forms are annexed: PH/2003/02/01 Appendix 1,
PH/2003/02/01 Appendix 2 and PH/2003/02/01
- The depositor sends the completed Accession Form to the collection, preferably in
advance to the despatch of the plasmid.
- Upon receiving the phage, it is stored as appropriate until
culturing/propagation can be started. It has to be found first if the phage
bears any health hazard for the lab personnel or if it is genetically
manipulated. The potentially high or unknown phage infectivity for bacteria
should be considered. Generally, work with phages should be performed on a
bench that is separated from other parts of a bacteriological laboratory, as
some phages including coliphages are highly infective and transmissible even
by air drifts. They may cause devastating effects on bacterial cultures.
Sterilisation should be perfectly done after working with phages.
- Only freshly prepared buffers and sterile equipment should be used for all
phage experiments. When experiments with phages are finished and results
documented, all phage-containing material should be stored as appropriate or
sterilised before disposal.
- A suitable host strain has to be selected and prepared for phage
propagation. Medium and buffer have to be prepared as required. It has to be
made sure that the host is under good condition, uncontaminated and growing
in the recommended medium or agar whether it is a host strain, which is
already a collection strain or a host sent along with the new phage.
- Minimal criteria are to be checked before a new phage is being
accessioned: phage viability and homogenous plaque morphology. The latter is
to be documented photographically or descriptively in words; it is
phage-specific and important for testing the phage purity, see PH/2003/03/01.
- Additionally, electron microscopic examination of the phage morphology and
purity is desirable, but not mandatory. The host specificity of a phage
should be tested, if applicable.
- The host specificity of a male (F+ cell) specific phage has to be verified
by using the recommended host and at least one negative control (F minus
- Mutant strains (e.g. point mutants, temperature sensitive mutants, amber
mutants) will require being tested using permissive and non – permissive
growth conditions or hosts and/or complementary phage tests where
- If any of these criteria does not fulfil the descriptions given by the
depositor or literature, the collection is responsible for discussing and
solving the problem and contacts the depositor requesting new material if
- If all tests fulfil the criteria, an aliquot of the original phage
suspension should be stored for long-term purposes, preferably in liquid
nitrogen, and the remaining part of the original suspension should be used
for preparing a high-titre working and distribution stock.
- In case the phage host is a new strain for accession with the culture
collection, the CABRI guidelines for bacteria fully apply as this host will
be the recommended host of the phage and should be available along with the
prepared for CABRI by DSMZ in cooperation with NCCB and NCIMB
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