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Phages - Handling of freeze-dried ampoulesPhages - Handling of freeze-dried ampoules

Revitalisation of Vacuum-Dried Bacteriophages

Several bacteriophages of our DSMZ collection are delivered as lyophilised phage suspensions on filter paper: a tested high-titre suspension of the respective phage had been vacuum-dried on filter paper and checked for viability after this drying process. We only deliver lyophilised phages with high viability and tested survival rates.

For revitalisation of the vacuum-dried phages, please observe the following steps:

  1. Prepare an agar plate with the recommended agar medium (containing 5 mM MgSO4) and plate the host using the “TOP AGAR LAYER METHOD”: 3-4 mL of soft agar (medium with 0.75% agar in a screw-cap vial is heated and cooled down to 48°C) are mixed with 0.1mL of a freshly grown host culture. This top layer mixture is immediately poured onto the bottom agar. Alternatively, 0.1mL of freshly grown host culture is directly poured on the agar and plated. Use ONLY the recommended media (liquid and solid) and buffers. Phages need either 5 mM MgSO4 or 5 mM CaCl2 for adsorption. The host plate is now ready for incubation with the phage. Pre-incubation of the host plate without the phage is not necessary: host and phage will be incubated together (see below).
  2. Open the ampoule of the phage acc. to the procedure as described for our bacterial cultures. With sterile forceps, take out the filter paper (contains the dried phage suspension), place it in the middle of the host plate.
  3. Add 0.1 mL of appropriate medium on the surface of the filter paper, incubate this plate with phage and host under conditions as described for the host, usually overnight. Alternatively, start incubation of the host/phage system in the morning and observe the plate until lysis occurs.
  4. Lysis leads to a clear zone around the filter paper. Several phages cause turbid plaques and therefore also a turbid zone around the filter paper. For preparing a PHAGE STOCK SUSPENSION, phages are resolved after lysis occurred: add 2-5 mL of recommended buffer or medium to get a phage stock suspension: leave the plate at room temperature while slowly rotating on a shaker for at least 4 hrs. Then, harvest the phage suspension and pellet the bacteria by centrifugation at 5000 x g. Filtrate the supernatant (0.45 µm porous size filter) to remove remaining bacteria.
  5. Alternatively to lysis on agar (see above), LYSIS IN LIQUID CULTURE may be performed: add the filter paper directly to a small exponentially growing fresh host culture, followed by further incubating phage and host together: in a first step, leave this culture for 1-2 hours without shaking so that phages can adsorb to bacterial cells, then further incubate the culture by shaking, as required. Observe this culture until lysis occurs. Disadvantage of lysis in liquid culture: phage-resistant bacterial mutants may arise and overgrow the culture so that the lysis effect is not visible anymore. Therefore, carefully observe the lysis process. Don’t incubate overnight without observing the lysis process.
  6. The phage suspension harvested after step 4 is the starting suspension for further purification or phage propagation steps.
  7. For saving and propagating the phages on agar plates and in liquid culture, you might cut the filter paper in two pieces and proceed with the above steps 2 and 5 in parallel.
  8. Suspensions of most phages store well at 4°C for one or two weeks or longer. DSMZ does not guarantee long-term survival of phages while stored in the customer’s laboratory. All biological material delivered by DSMZ is for immediate use. Please, see DSMZ’s Terms and Conditions.

Phage-specific information is given in the respective catalogue information. Please, carefully read all specific information before working with our phages.

DSMZ, December 2011