1          Material

·          Petri dish (diameter: 9 cm)

·          4 ml glass bottles

·          Pasteur pipette

·          Pipetter

·          1, 2, 5, 10 ml plastic pipettes

·          200 µl and 1000 µl for automatic pipette

·          Medium distributor

·          Screw caps

·          Laminar flow hood type PSM.

2          Reagents

Preparation of specific RL medium: charcoal medium

·          Weigh out 51 g oxoid charcoal medium

·          Dissolve by stirring in 1 litre double-distilled H2O

·          Dispense 15.3 ml of the medium using a fractionating distributor or conventional pipetting into washed glass tubes

·          Seal with single-use screw caps

·          Autoclave for 20 minutes at 120°C

·          Store at +4°C (cold room) for four weeks.

Preparation of cefalexin

·          Reconstitute a bottle of 20 mg with 2 ml of bi-distilled sterile water

·          Dilute these 2 ml of solubilized cefalexin with 48 ml of bi-distilled sterile water

·          Store the cefalexin solution (0.4mg/ml) in 1.5 ml sterile Eppendorf tubes per aliquot of 850 µl

·          Record the preparation data and the validity limit date in the preparation book

·          Store at –20°C for 6 months.

Sheep blood or horse blood

For a plate: 1.7 ml of blood.

Reference strain

Bordetella pertussis  Tohama I (CIP 8132 which can be purchased from the Institut Pasteur) for quality control in growth support and testing incubation conditions for cultures.

3          Preparation Reagan Lowe medium agar dishes

·          Petri dishes of medium are prepared under clean air conditions using a laminar flow hood or burning Bunsen burner.

·          Place the tubes containing Reagan Lowe medium in a water bath at 52°C in order to melt the medium.

·          Petri dishes should be removed under sterile conditions from their plastic wrappings within the laminar flow hood and filled under sterile conditions.

·          Remove five tubes of medium from the waterbath at 52°C, rinse them and then place them in a holder under the laminar flow hood or close to the burning Bunsen burner.

·          Extract 8.5 ml blood (for 5 tubes) using a 10 ml pipette and distribute 1.7 ml blood into each of the five tubes, seal them and mix the contents by gently inverting each tube three times: this will homogenize the preparation.

·          Pour the total mixture of 17 ml (15.3 ml + 1.7 ml) into a Petri dish and spread it uniformly using a slow circular motion.

·          Remove the dishes from the laminar flow hood when the charcoal agar has solidified, identify individual batches (in case one part of the blood is contaminated) and leave them overnight at room temperature.

·          Perform sterility and growth support tests. Plates without bacteria and plates cultured with bacteria are put in the oven for a few days. Those without bacteria must remained sterile, and on those cultured with bacteria, growth must be observed after three days.

Preparation of bottles containing Reagan Lowe medium for transport

·          Prepare the exact number of bottles requested.

·          Unscrew the caps under the laminar flow hood.

·          Pipette in 1.5 ml of the mixture of (blood–charcoal medium into each bottle).

4          Preparation of Reagan Lowe medium agar dishes supplemented with
cephalexin

·          Thaw the volume of cefalexin solution required (0.4 mg/ml).

·          Add 170 µl of cefalexin (0.4 mg/ml) per tube of Reagan Lowe medium agar after adding blood (2.5 ml). The final concentration of cefalexin is 40 µg per tube.

·          Record the batch reference number in the preparation logbook. Identify prepared plates by inscribing a sign on the cover.

·          Seal the bottles, tilt them to allow the mixture to solidify. The cap of the sealed bottle can be laid against the side of a petri dish (height: 1 cm).

Store dishes in plastic bags and keep them at 4°C.

Prepared petri dishes and bottles can be stored at 4°C for 2 weeks and should be discarded afterwards.

Bordet Gengou medium

1          Equipment and materials

·          Plastic or glass sterile plates (diameter: 9 cm)

·          Wheaton 4 ml glass bottle

·          Pasteur pipette

·          Automatic pipetter (200 µl and 1000 µl) or conventional pipettes

·          Plastic pipettes of 1, 2, 5 or 10 ml

·          200 µl and 1000 µl  for automatic pipetter

·          Wheaton medium distributor

·          Screw stoppers

·          Laminar flow hood.

2          Reagents

Preparation of specific Bordet Gengou medium

Bordet Gengou agar medium (DIFCO) ref. 248200: (Beckton Dickinson Biosciences, 2350 Qume Drive, San Jose, CA 95131-1807, USA; Tel: 408.432.9475; Fax: 408.954.2347). Storage period: 1 month. Storage at 4°C.

·          Weigh 30 g Bordet Gengou medium and dissolve by:

—     boiling in a mixture of glycerol 10 ml

—     adding 5N NaOH until pH = 7.4 and

—     distilled H₂O to make 1 litre

·          Record batch reference number in the preparation logbook.

·          Distribute 14.5 ml of medium per tube.

·          Seal with single-use screw stoppers.

·          Autoclave for 20 minutes at 121°C.

·          Allow to cool and store at 4°C.

·          This medium can be kept for up to 12 weeks at 4°C.

Preparation of cefalexin

·          Reconstitute a bottle of 20 mg with 2 ml of double-distilled sterile water.

·          Dilute these 2 ml of solubilized cefalexin with 48 ml of bi-distilled sterile water.

·          Store the cefalexin solution (0.4 mg/ml) in 1.5 ml sterile Eppendorf tubes per aliquot of 850 µl.

·          Record the preparation data and the validity limit date in the preparation book.

·          Store at –20°C for 6 months.

Reference strain

Bordetella pertussis: Tohama I (this strain can be purchased from the Institut Pasteur as CIP 81.32).

3          Preparation of Bordet Gengou agar dishes

·          Petri dishes are prepared under clean air conditions using a laminar flow hood, burning Bunsen burner or a hood.

·          Place the tubes containing Bordet Gengou medium in a water bath at 52°C in order to melt the medium.

·          Petri dishes should be removed under sterile conditions (clean air) from their plastic wrappings beneath the laminar flow hood and filled under sterile conditions in batches of five.

·          Five plates should be prepared at the same time under the laminar flow hood or close to the burning Bunsen burner.

·          Remove five tubes from the waterbath at 52°C, and put them in a holder under the laminar flow hood or close to the Bunsen burner.

·          Extract a 8.5 ml sample of blood using a 10 ml sterile pipette and distribute 1.7 ml blood into each of the five tubes; seal them three times by inverting each tube, and gently mix the agar medium and blood in order to homogenize the preparation.

·          Pour total mixture of 17 ml (15.3 + 1.7 ml) into a dish and spread it uniformly by using a slow circular motion.

·          Remove the plates from the laminar flow hood when the BGS is cold. Identify batches (in case one part of the blood is contaminated) and leave overnight at room temperature before storage at 4°C.

·          Perform sterility and growth test. Plates without bacteria and plates cultured with bacteria are put in the incubator for a few days. Those without bacteria must remain sterile and on those cultured with bacteria,  growth must be observed after three days.

4          Preparation of Bordet Gengou agar plates supplemented with cefalexin

·          Thaw the volume of cefalexin solution required (0.4 mg/ml).

·          Add 170 µl of cefalexin (0.4 mg/ml) per tube of Bordet Gengou agar medium after adding blood (2.5 ml). The final concentration of cefalexin is 40 µg per tube.

·          Record the batch reference number in the preparation logbook. Identify prepared plates by inscribing a sign on the cover.

Store dishes in plastic bags and keep them at 4°C.

Prepared petri dishes and bottles can be stored at 4°C for two weeks and should be discarded afterwards.

Stainer Scholte medium

1          Material

·          Miscellaneous glassware

·          Scales

·          Magnetic stirrer and magnetic bars

·          pH-meter (or litmus paper with pH range 7–8)

·          Autoclave

·          Plastic bottles and tubes

·          150 ml stericup; 0.22 µm Millipore filter.

Note:

·          Use sterile labware.

·          Prepare all media with double-distilled sterile water.

·          Substances should be weighed following the instructions supplied with the scales.

·          The pH-meter is calibrated and the temperature measured with the probe.

2          Reagents

Solution

1% calcium chloride solution freshly prepared thus:

Calcium chloride                       250 mg

H₂O                                        to make            25  ml

Concentrated 10X stainer

·          Reagent

Sodium glutamate (C₅H₈NO₄Na)                      214 g

L-proline (L-C₅H₉NO₂)                                    4.8 g

Sodium chloride (NaCl)                                    50 g

Potassium dihydrogen phosphate (KH₂PO₄)      10 g

Potassium chloride (KCl)                                 4 g

Magnesium chloride (MgCl₂)                           2 g

Tris base (C₄H₁₁NO₃)                                     30.05 g Réf: T1503; Purity 99.9%; Sigma

1% calcium chloride (CaCl₂₎                             40  ml

Distilled H₂O                                                   1000 ml

HCl                                                                 adjust to pH 7.6

H₂O                                                                to make 2000 ml

Aliquot by 200 ml before storing in –20°C.

·          Protocol

—     Put a 1 liter beaker on a magnetic stirrer.

—     Place a clean magnetic bar inside the beaker.

—     Pour 700 ml of pure distilled water into the beaker.

—     Regulate the rotation of the magnetic bar in order to allow effective, homogenous and silent mixture of the solutions.

—     Weigh out the various chemical products required to make the medium.

—     Slowly pour the dry products one after the other into the liquid so that dissolution takes place without clumps being formed.

—     Add 20 ml of 1% calcium chloride.

—     Carefully raise the pH to 7.6 with concentrated hydrochloric acid.

—     Exactly make up the final volume to 1 litre after ensuring the magnetic bar has been removed.

—     Homogenize the medium by inverting it several times after first covering it with parafilm.

—     Distribute it into plastic bottles either in 200 ml or 20 ml fractions depending on workload needs.

—     Label the bottles with the preparation date, expiry date and product name.

—     Freeze at –20°C.

—     Complete the medium preparation logbook.

—     Store at –20°C for 6 months.

10X supplement

Dissolve in a beaker:

·          L-cystine                                                   8 g

·          concentrated HCl                                       20 ml

Add 120 ml of a solution containing:

·          FeSO₄, 7 H₂O                                            2 g

·          Ascorbic acid                                            4 g

·          Nicotinic acid                                             0.8 g

Make up to 200 ml

Distribute into 5 ml tubes

Freeze at –20°C.

1X Stainer Scholte medium

·          Verify that the bottles have not been damaged during freezing.

·          Use a waterbath at 37°C to thaw the 10X concentrated medium.

·          Rinse all the labware to be used with double-distilled water.

·          Use a 2 litre flask.

·          Rinse before measuring out 1800 ml of sterile double-distilled water.

·          Add 200 ml of  thawed Stainer 10X medium.

·          Cover with parafilm and homogenize the medium by inverting it several times.

·          Distribute 200 ml of 1X medium into pre-rinsed 2 litre Erlenmeyer flasks.

·          Sterilize in the autoclave (120°C for 20 minutes).

"Enriched" 1X Stainer: to be prepared freshly

·          Enrich sterile 1X Stainer medium by adding 1X culture supplement.

1X supplement:

100 mg             Glutathione

1 ml                 10X supplement

9 ml                 H₂O à filter through a 0.22 µm stericup

·          “Enriched” sterile 1X Stainer

Add 2 ml 1X supplement to 200 ml 1X Stainer and use immediately.