BS ISO 19344:2015 pdf download.Milk and milk products — Starter cultures, probiotics and fermented products — Quantification of lactic acid bacteria by flow cytometry
5.3.1 Protocol A
5.3.1.1 Phosphate-buffered saline (PBS)
5.3.1.1.1 Composition
— 9 g sodium chloride (NaCl)
— 795 mg sodium hydrogenphosphate heptahydrate (Na 2 HPO 4 ·7H 2 O)
— 144 mg potassium dihydrogen phosphate (KH 2 PO 4 )
5.3.1.1.2 Preparation
Dissolve the components (see 5.3.1.1.1) in water. Add water to a final volume of 1 000 ml. Adjust the pH with HCl to 7,4 ± 0,05, if necessary. Distribute the solution into aliquots and sterilize in an autoclave set at 121 °C ± 1 °C (liquid cycle) for 15 min. The diluent can be stored at cooling temperature (3 °C ± 2 °C) for up to 6 months.
5.3.1.2 5(6)-Carboxyfluorescein diacetate (cFDA) mixed isomers solution
5.3.1.2.1 Composition
— 230 mg 5(6)-cFDA mixed isomers
— 100 ml dimethyl sulfoxide (DMSO)
5.3.1.2.2 Preparation
A 5 mmol/l solution is prepared by dissolving cFDA in DMSO at the amounts specified in 5.3.1.2.1. The
solution can be stored at −18 °C ± 2 °C, protected from light, for up to 6 months.
5.3.1.3 Propidium iodide (PI)
5.3.1.3.1 Composition
— 100 mg PI
— 100 ml ultrapure water
5.3.1.3.2 Preparation
Dissolve the PI in ultrapure water to a final concentration of 1,0 mg/ml, corresponding to approximately 1,5 mmol/l. This can be stored at 3 °C ± 2 °C, protected from light, for up to 6 months.
NOTE The concentration of the PI solution used is 0,1 % and the final concentration is 0,002 %. This is below the potential toxicity level.
5.3.2 Protocol B
5.3.2.1 Phosphate-buffered saline (PBS)
See 5.3.1.1.
5.3.2.2 Propidium iodide (PI)
See 5.3.1.3 for the preparation of the PI solution. The PI solution shall be further diluted to 0,2 mmol/l with water prior to use.
NOTE The concentration of the PI solution used is 0,01 % and the final concentration is 0,000 1 %. This is below the potential toxicity level.
5.3.2.3 SYTO® 24 green fluorescent cell-permeant nucleic acid stain
The stain is a 5 mmol/l solution in DMSO. Store at −20 °C, protected from light, for up to 12 months. The solution shall be diluted to 0,1 mmol/l with water before use.
5.3.3 Protocol C
5.3.3.1 MRS broth
The composition and the preparation are specified in ISO 15214 except for no addition of agar.
5.3.3.2 M17 broth
The composition and the preparation are specified in ISO 7889 except for no addition of agar.
5.3.3.3 Stain mixture
The stain mixture consists of 210 µl 50 % glucose solution (5.3.3.3.1), 210 µl 1,5 mmol/l DiOC 2 (5.3.3.3.2) and 50 ml buffer solution (5.3.3.3.3). The stain mixture is prepared the same day as it is used.
5.3.3.3.1 Glucose solution
5.3.3.3.1.1 Composition
— 50 g D(+)-glucose monohydrate
— 50 g water
5.3.3.3.1.2 Preparation
A 50 % glucose solution is prepared by dissolving the glucose in the water. This is aided by warming the solution to below the boiling point. Avoid evaporation. The solution is autoclaved at 121 °C ± 1 °C for 15 min and can be stored unopened at 3 °C ± 2 °C for up to 3 months.
5.3.3.3.2 3,3’-diethyloxacarbocyanine iodide (DiOC 2 )
5.3.3.3.2.1 Composition
— 69 mg 3,3’-DiOC 2 , ≥ 98 %
— 100 ml dimethyl sulfoxide (DMSO)
5.3.3.3.2.2 Preparation
The DiOC 2 staining is prepared as a 1,5 mmol/l solution by weighing DiOC 2 into DMSO at the amounts specified in 5.3.3.3.2.1. Dispense in, e.g., 1 ml tubes. Keep dark, as DiOC 2 is unstable in light, at 5 °C ± 3 °C for up to 12 months.
5.3.3.3.3 Buffer solution
5.3.3.3.3.1 Composition
— 7,6 g sodium chloride (NaCl; 130 mmol/l)
— 0,5 g sodium dihydrogenphosphate dihydrate (NaH 2 PO 4 ·H 2 O; 3 mmol/l)
— 1,24 g sodium hydrogenphosphate dihydrate (Na 2 HPO 4 ·2H 2 O; 7 mmol/l)
— 1 000 ml water
5.3.3.3.3.2 Preparation
Weigh and dissolve the three salts in the water at the amounts specified in 5.3.3.3.3.1. Stirring is applied until the salts are dissolved. Adjust pH to 6,5 ± 0,05 with 2,5 mol/l HCl. The solution shall then be filtered through a 0,22 μm filter. The mixture can be kept at 3 °C ± 2 °C for up to one week. For longer periods, up to 6 months, storage at −20 °C is recommended.
6 Apparatus
Usual laboratory equipment and, in particular, the equipment required for the preparation of test samples and dilutions specified in ISO 6887-5, as well as the following, shall be used.
6.1 Water bath, capable of operating at 21 °C ± 1 °C.
6.2 Analytical balance, capable of weighing to the nearest 1 mg, with readability to 0,1 mg.
6.3 pH-meter, with temperature compensation, accurate to ± 0,1 pH unit.
6.4 Incubator, heating block or equivalent, capable of operating at the temperatures specified in Table 2.BS ISO 19344 pdf download.BS ISO 19344 pdf download
