Annex 6.2
Preparation of reagents for specimen processing for culture
DEFINITIONS AND ABBREVIATIONS
MW molecular weight
NALC N-acetyl-L-cysteine
CPC cetylpyridinium chloride
EQUIPMENT AND MATERIALS
•Balance (sensitivity 0.01g), spoons or spatulas, weighing paper.
•Autoclave.
•pH meter.
•Dispenser device.
•Appropriate sterile glassware and sufficient sterile screw-capped tubes for reagent dispensing.
•Dark bottles.
•Refrigerator.
•General equipment of a media kitchen
REAGENTS FOR THE SODIUM HYDROXIDE (MODIFIED PETROFF) METHOD
Sodium hydroxide (NaOH) solution, 4%
Sodium hydroxide pellets (analytical grade)4 g
Distilled water100 ml
Dissolve NaOH in the distilled water. Aliquot in 2-ml amounts. Sterilize by autoclaving at 121 °C for 20 minutes.
Phosphate buffer, 0.067 mol/litre, pH 6.8
Mix 50 ml of solution A (9.47 g of anhydrous Na2HPO4 in 1 litre of distilled water) with 50 ml of solution B (9.07 g of KH2PO4 in 1 litre of distilled water). Aliquot in 10-ml amounts. Sterilize by autoclaving at 121 °C for 20 minutes.
REAGENTS FOR THE NALC‒NaOH METHOD
- Sodium hydroxide (NaOH) solution, 4%, 50 ml
Sodium hydroxide pellets (analytical grade)4 g
Distilled water100 ml
Dissolve NaOH in the distilled water. Sterilize by autoclaving at 121 °C for 20 minutes.
- Trisodium citrate·3H2O, 2.94%, 50 ml
- NALC‒NaOH solution, freshly prepared
Mix (1) and (2). Add 0.5 g N-acetyl-L-cysteine (NALC) just before use. Aliquot in 4-ml amounts.
- Phosphate buffer, 0.067 mol/litre, pH 6.8
Mix 50 ml of solution A (9.47 g of anhydrous Na2HPO4 in 1 litre of distilled water) with 50 ml of solution B (9.07 g of KH2PO4 in 1 litre of distilled water). Aliquot in 10-ml amounts. Sterilize by autoclaving at 121 °C for 20 minutes.
REAGENT FOR THE TRISODIUM PHOSPHATE METHOD
Trisodium phosphate, 10%
Trisodium phosphate Na3PO4·12H2O23 g
Distilled water100 ml
Aliquot in 4-ml amounts.
REAGENT FOR THE 5% OXALIC METHOD
5% oxalic acid
Dissolve 5 g of oxalic acid dihydrate (MW 126.01) in 100 ml water. Aliquot in 4-ml amounts. Sterilize by autoclaving at 121 °C for 20 minutes.
REAGENT FOR THE CPC/NaCL METHOD
1% CPC–2% NaCl solution
Dissolve 10 g of cetylpyridinium chloride and 20 g of sodium chloride in 1000 ml of sterile distilled water. Distribute in 4-ml aliquots in sterile bottles.
CPC solution is self-sterilizing and remains stable if protected against light, extreme heat and evaporation. The stock solution should be stored in dark bottles at room temperature.
REAGENT FOR DECONTAMINATION USING MODIFIED KUDOH METHOD
Sodium hydroxide (NaOH) solution, 4%
Sodium hydroxide pellets (analytical grade)4 g
Distilled water100 ml
Dissolve NaOH in the distilled water. Aliquot in 4-ml amounts. Sterilize by autoclaving at 121 °C for 20 minutes.
STORAGE
All solutions can be stored for 3 months at room temperature, protected from direct light.