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LABORATORY PROCEDURES FOR PHAGES

REFERENCE NO: PH/2003/02/01


TITLE: REQUEST AND RECEIPT OF PHAGES


INTRODUCTION

This document describes the sequence of the principal steps when a new phage is to be accessioned with a culture collection

PROCEDURE

  1. 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.) 

Only phages conform to the accession criteria of the collection can be accepted. 
In case the collection cannot accept the offered phages (due to national regulations, outside the interest of the collection, ), the potential depositor is informed.

  1. In cases (1.2.) and (1.3.) potential depositors are invited by letter to deposit the plasmid with the collection. 
  2. In order to receive the information about the plasmid in a standard format, the depositor is requested to complete and return the collection’s 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 Appendix 3
  3. The depositor sends the completed Accession Form to the collection, preferably in advance to the despatch of the plasmid. 
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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 strain).
  10. 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 appropriate.
  11. 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 necessary.
  12. 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.
  13. 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 phage.

     


Guidelines prepared for CABRI by DSMZ in cooperation with NCCB and NCIMB
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