BS ISO 19344 pdf free download
BS ISO 19344-2015 pdf free download.Milk and milk products一 Starter cultures, probiotics and fermented products – Quantification of lactic acid bacteria by flow cytometry.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707 I IDF 501Z1.
A representative sample is collected for analysis. Unless frozen, test samples shall be cooled after sampling to between 3 °C ± 2 °C and kept at that temperature until testing or freezing. The age of the samples at testing and freezing and the storage conditions may influence the counting result.
The sample shall not be damaged or changed during transportation or storage. The sample shall be homogenous and representative of the batch of product to be tested.
Commercial starter cultures and commercial dairy products shall be stored prior to testing as recommended by the manufacturer.
8 Preparation of test sample
8.1 General
General requirements are in accordance with ISO 6887-1:1999 and ISO 6887-5:2010.
The sample shall be handled for testing in accordance with good laboratory practices.
Initial suspensions and, if needed, further dilutions of the samples to be tested, i.e. freeze-dried cultures, frozen cultures and fermented milk products containing cultures, are prepared as specified in &Z to 4. The appropriate dilution required to be analysed by flow cytometry depends on the initial concentration of cells in the sample, the staining protocol and the flow cytometry equipment.
8.2 Freeze-dried cultures
Prior to preparation of the initial suspension, allow the freeze-dried sample to acclimatize to room temperature before opening the pouch or container. Alternatively, mix the content of the original sample thoroughly, remove the needed test portion with a sterile spatula, and transfer it to a sterile container or pour the needed test portion into a sterile container. Allow the sample to reach ambient temperat tire of 20 °C to 25 °C.
Thoroughly mix the contents of the closed container/bag by repeatedly shaking and inverting it.
Prepare an initial suspension (between 10 and 100 fold) by weighing the test sample into a suitable sterile vessel and then adding the required amount of diluent. Alternatively, weigh the test sample directly into the bottle with the diluent. The diluent shall be peptone-salt solution (2). The temperature of the diluent shall be the same as that of the test sample in order to avoid damaging the microorganisms by sudden changes in temperature.
For rehydration, the initial suspension is left at ambient temperature of 20 °C to 25 °C for approximately 10 mm, and no longer than 45 mm, shaking occasionally or continuously (e.g. using a peristaltic homogeniser), before further dilution.
Make sure that the sample is dissolved before proceeding immediately with further testing steps. If needed, further dilutions are prepared by thoroughly mixing a specific amount of the initial suspension with the appropriate volume of diluent.
As an alternative to manual sample preparation, an automated sample preparation module dedicated to sample preparation, dilution and/or injection can be Lised.
8.3 Frozen cultures
Prior to initiating preparation of the initial suspension, the test sample shall be thawed. This can be done by leaving the sample at an ambient temperature of 20 °C to 25 °C until the sample has just thawed. Alternatively, place the sample in a water bath at 21 °C ± 1 °C and keep it in the water bath until the test portion has just thawed, see ISO 26323 I IDF 213Il. Samples shall be tested as soon as they have been thawed. Mix the test sample carefully after thawing.
Prepare the initial suspension, and appropriate dilution(s) if needed, as stated in 8.2. The test sample is measured by volume or weight. The unit of the reported final result shall reflect the choice of unit for the preparation of the test sample.
8.4 Fermented milk products
Samples from fermented milk products, e.g. yogurts, are prepared as follows.
a) Add 90 ml ± 0,1 ml of peptone-salt solution () at an ambient temperature of 20 °C to 25 °C to a sterile bottle.
b) Mix the sample (e.g. yogurt) either by inverting and shaking or by mixing it with a sterile spatula.
c) Weigh 10 g ± 0,1 g of the sample and add it into the diluent in order to prepare dilution 10-’. The temperature of the diluent shall be the same as that of the test sample in order to avoid damaging the microorganisms by sudden changes in temperature.
d) Shake the bottle slowly 10 times by inverting and shaking.
e) If needed, immediately prepare the serial dilution to obtain the adequate dilution for testing.
NOTE Fora final dilution of 10-i to 10-s, there will be so little background interference in the flow cytometer from yogurt matrices that no specific pre-treatment is needed. For yogurt samples containing particles, e.g. fruit pieces or vanilla, the debris might be removed by filtering the final dilution through a 25 pm filter before staining.
9 Procedure
9.1 General
Following preparation of the test sample, testing includes the following steps:
— staining(2);
— flow cytometry analysis (9,);
— gating (9A);
— calculation of concentrations (Clause 10).
9.2 Staining
Initial suspensions, and/or dilutions from the test sample if needed, are processed and stained depending on the chosen protocol as specified in 9.2.L 9I22 and 2JZL prior to quantification by flow cytometry. Diagrams for the three staining protocols are found in Annex A.
The individual staining protocols include dilution steps to achieve an appropriate cell concentration. For further details on appropriate dilution see Clause 11 and for a calculation example see Annex 8.BS ISO 19344 pdf download.
