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Storage of Bordetella spp. bacteria
When the bacteria have been identified, they must be stored for further analysis. The bacteria are stored in the medium described below at -80°C.
1. Material
Cryotubes volume (2 ml)
Petri dish
Spectrophotometer OD650nm
Automatic pipetter, 200 µl and 1000 µl
1, 2, 5, 10 ml plastic pipettes
Tips for 200 µl and 1000 µl automatic pipetters
1.6 ml polystyrene spectrophotometer cuvettes
Laminar flow hood
Plastic or glass rake
0.2 µm filter.
2 Reagents
Products
90° ethyl alcohol
Sodium glutamate - C5H8NaNO4 -H2O
Di-sodium hydrogen phosphate, dihydrous - NaH2PO4 -2H2O
Di-sodium hydrogen phosphate, monohydrous - NaH2PO4 -H2O
Sodium and potassium phosphate pH 7.2 = PBS 10X: KCl 26.8 mM; KH2PO4 14.7 mM; NaCl 1.36 M; NaH2PO4-7H2O 80.57 mM
Saccharose - C12H22011
Bovine serum albumin (BSA).
Solutions
25 % BSA solution - storage period: 6 months at + 4°C:
Bovine serum albumin 25 g
PBS 1X pH 7.2 to make 100 ml
Sterilize by filtering through a 0.2 µm membrane.
Saccharose-phosphate-glutamate (SPG) solution - storage period: 6 months at + 4°C:
Saccharose 85.6 g
Sodium glutamate 0.94 g
NaH2PO4 -2H2O 1.38 g
NaH2PO4 -H2O 0.39 g
Adjust to pH 7.2
H2O: to make 1000 ml
Sterilize by filtering through a 0.2 µm membrane and store at +4°C.
Solution for use: BSA/SPG - freshly prepare the volume required:
Dilute the BSA solution to 2.5 % with SPG buffer (same as SPG solution above).
Use this solution under very strict conditions of sterility.
3. Protocol
Freezing
Day 0
Take bacteria from isolated colonies (72 h growth for B. pertussis and B. parapertussis) in order to prepare a bacterial suspension.
Measure OD650
nm
With the appropriate medium, and using the same batch of agar medium dishes, perform two layered cultures and an isolation by spreading 100 µl of a bacterial suspension with OD650 nm= 1 using a sterile loop spreader.
Incubate dishes at 37°C for 24 to 48 h.
Day 1 or day 2:
Prepare a freezing bath filled with a mixture of dry ice and alcohol.
Take the first dish of agar medium in its entirety and put it in 5 ml of sterile BSA/SPG buffer.
Allocate 1 ml to each of 5 cryotubes identified with the number of the isolate, the species code (see below), the date of freezing, the freezing medium used: BSA/SPG.
Bordetella pertussis: Bp
Bordetella parapertussis: Bpp
Freeze the tubes rapidly by placing them in the freezing bath.
Inscribe the type and storage site of isolates in the "strains in" log book and in the file for the strains concerned (isolates of B. pertussis, B. parapertussis, B. bronchiseptica and other Bordetellae).
Note: The following procedure is strongly recommended:
In parallel, remove all of the second layer and put it into 5 ml of sterile saline solution, measure and record the OD 650 nm, then adjust to OD = 1.0 in order to obtain X ml which can be used for various analyses such as adenyl cyclase haemolysin assay and immune blot tests.
Storage
Distribute the tubes to different storage sites.
Place 2 cryotubes in the freezer at -80°C for everyday use and producing secondary batches.
Place 2 cryotubes in the freezer at -80°C for producing the primary batch, not intended for distribution.
Place 1 cryotube in liquid nitrogen at -196°C for producing the primary batch, not intended for distribution.
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