QUALITY CONTROL CHECK OF HOST/PLASMID COMBINATIONS

LABORATORY PROCEDURES FOR PHAGES

REFERENCE NO: PH/2003/03/01

TITLE: QUALITY CONTROL CHECK

INTRODUCTION

This document describes the basic procedure to be followed when checking the quality of a phage for the public collection. Methods may vary, due to individual requirements of a phage or phage host system. All the information gathered from the following steps has to be documented in detail in the lab books

PROCEDURE

1. Arrival of phage

2. Recommended host, sterile fresh media, buffer, agar plates and soft agar (in tubes, contains half of the agar concentration) are prepared for use

3. Take aliquot of original phage sample (Master Stock) for dilution series (see pt. 4.) for initial lysis procedure on plates in order to classify plaque morphology. Leave the rest of the original phage suspension for long-term storage, preferably in liquid nitrogen

4. For initial lysis on plates, use a fresh growing culture of the host and make a dilution series of the original phage suspension in 1:10 or 1:100 steps and vortex gently

5. Melt sterile soft agar in tubes and keep the tubes at 45°C in water bath or heated block until use. Mix into each soft agar tube 0.1 mL of respective phage dilution plus 0.1 mL of the undiluted host strain suspension or any other recommended combination, mix gently (avoid vortexing) and pour on the plates. The procedure asks for rotating the wrist at the same time as the tube contents are poured onto the plate.

6. Incubate as appropriate

7. If lysis is visible, count the plaques and calculate the phage titre of the original phage suspension and confirm that the plaques are homogenous. Describe plaque morphology or make photographs.

8. Store the rest of the original phage suspension in liquid nitrogen using appropriate cryoprotectant like DMSO or glycerol or as recommended by the depositor.

9. Phage propagation can be performed in liquid culture using the appropriate host strain and growth conditions as described for the host or phage/host system. This can result in high titre suspensions, the purity (plaque uniformity) of which has to be tested on plates (see above and below). In case of plate lysis, use those plates showing confluent or semi confluent lysis of the bacterial lawn for recovering phages in order to get a high titre stock by flooding/overlaying the plates:

10. Gently flood/overlay the plates at room temperature with 5 mL of phage buffer, as appropriate; after flooding, either scrape the top agar layer immediately into a centrifuge tube (avoid incubation as it may result in phage inactivation by readsorption) or simply decant the liquid containing the free phage particles into a centrifuge tube. Centrifuge in order to pellet bacterial cell debris, then filter the supernatant through a 0.45 µm sterile filter. Some workers may choose to add a few drops of chloroform either before or after this process to ensure maximum recovery of phage and as an insurance that bacterial contamination is reduced, but this step is not mandatory. This WORKING AND DISTRIBUTION STOCK is to be tested again for purity and titre estimation, therefore, start again at step 4.

11. If the titre is acceptable (phage-dependent) and purity assessed, this stock can be divided into aliquots for storage in liquid nitrogen (using glycerol) and a suspension for storage at either a different location or at a different temperature or using a different cryoprotectant. Titre and plaque morphology should be controlled regularly. High titre suspensions have 109 pfu/mL and more

12. ADDITIONAL QUALITY CONTROL CHECKS:

As mentioned in the General Procedure Guidelines, electron microscopic examination of phage suspensions (negative staining procedure) and testing host specificity is desirable. Testing the host specificity is mandatory in case of male-specific phages or in cases where the host specificity is published or described by the depositor

13. If a contamination of the phage suspension occurs, single plaque isolation can be performed:

Recover a single plaque with a sterile Pasteur pipette and resuspend this single plaque in 100 µL of recommended phage buffer, use this for preparing a dilution series and a new phage stock (see steps above). This procedure can also be used to recuperate a phage the titre of which is too low to attempt confluent lysis by serial dilutions from the stock.

14. ADDITIONAL STORAGE METHODS

Phages may be stored freeze-dried on filter paper or as freeze-dried suspension, which can make shipping easier. In these cases, use methods as described by the depositor. See book

reference: T. Kieser, M.J. Bibb, M.J. Buttner, K.F. Chater, D.A. Hopwood: Practical Streptomyces Genetics, published by the John Innes Foundation, ISBN 0-7084-0623-8, Chapter 12

15. The database/catalogue entry is produced and the phage material is made available to the public according to the CABRI Guidelines for Catalogue production

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