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Method for freeze-drying of Bordetella isolates
Hans Hallander
Prepare fresh freeze drying freezing medium i.e. Albumin & SPG (solution 1 & 2 below)
Resuspend the 48-72 hour pertussis culture to an optical density of approximately 1.0 at 650 nm.
Aliquot 500 ?l of prepared freeze drying buffer into each ampoule.
Freeze the bacterial suspension at -70 oC overnight or at least 3 hours.
Place samples into freeze drier.
Dry overnight.
Follow the freeze-drying apparatus manual
Seal the ampoules
Solution 1.
Albumin, BSA, 25% in sodiumphosfate buffer, 10Mmol/L, pH 7.3.
Di-sodium hydrophosphate, Na2HPO4x2H2O 0.45 g
Sodium dihydrophosphate, NaH2PO4xH2O 0.078 g
Albumin, BSA, Sigma A-9647 62.5 g
Sterile/ultra-pure water 250 ml.
Weigh out salts and albumin in a beaker and dissolve in approx. 200 ml of water with magnetic stirrer.
Adjust the pH to 7.3.
Pour the solution into a volumetric flask and adjust the final volume to 250 ml with water.
Sterile filter the solution in a sterile bottle in the LAF hood.
Aliquot the solution in sterile bottles, 10 ml/flask.
Freeze at -20 oC, store for one year.
Solution 2.
SPG buffer pH 7.2.
Di-sodium hydrophospahte, Na2HPO4x2H2O 1.38 g
Sodium dihydrophosphate, NaH2PO4xH2O 0.39 g
Sacarose 85.6 g
L-glutamic acid sodium salt 0.94 g
Sterile/ultra-pure water 1000 ml
Weigh out materials in a beaker and dissolve in approx. 900mls water, stirring continuously.
Measure pH and adjust to 7.2.
Pour the solution into a volumetric flask and adjust the final volume to 1000 ml with water.
Sterile filter the solution in a sterile bottle in LAF hood.
store at room temperature/fridge 2-8 oC for 1 year.
To make storage/freezing buffer: Add 1 full bottle of Albumin 25% (10ml) to 1 full bottle of SPG buffer(100ml). enough for 200 tubes.
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