ISO 19344 Starter cultures, probiotics, and fermented products – Quantification of lactic acid bacteria by flow cytometry.

For different applications Biosisto measures alive and dead cells according to ISO 19344 protocol B using SYTO® 24 (green fluorescent cell-permeant nucleic acid stain) and PI (Propidium iodide is a red-fluorescent nuclear and chromosome counterstain.

According to ISO 19344

“Flow cytometry is a technique for rapid quantification of cells combining light source, optics, flow chamber, liquid sample delivery system, light detectors, electronics, and software. The flow cytometer provides a constant flow of sheath fluid through a cuvette, singularizing and preparing the cells for analysis. The cuvette is traversed by a beam of light to illuminate flowing cells. Around the cuvette, detectors collect scattered light in two angles [forward scatter channel (FSC) and side scatter channel (SSC)] and fluorescence in different colors (e.g. green, yellow, red). When a cell reaches the flow cuvette, a proportion of the light beam is scattered and cellular fluorescence markers are excited to emit fluorescence. The detection of the scattered light and the concomitant emission of fluorescence is referred to as an event, i.e. each cell passing through gives rise to one event, each of which shows the state of the cell. Whereas the detected fluorescence is linked to the state of the cell, i.e. enzyme activity, membrane integrity, and/or membrane potential, the FSC provides information on cell size and optical density. The SSC provides information on cell morphology, reflectivity, and granularity. The recorded events per microlitre sample indicate how many cells are counted and at the same time differentiate, based on fluorescence parameters, the cells into two categories: active fluorescent and non-active fluorescent.”

The staining principle is based on a dual nucleic acid staining with cell-permeant dye SYTO® 24 (fluorescence emission maximum at 515 nm) and cell impermeant dye PI (fluorescence emission maximum at 620 nm). SYTO® 24 permeates the membrane of total cells and stains the nucleic acids with green fluorescence. PI is not permeant to live cells and is used to detect dead cells in a population. PI penetrates only bacteria with damaged membranes, causing a reduction in SYTO® 24 green fluorescence when both dyes are present. Thus, live bacteria with intact cell membranes fluoresce bright green (defined as active fluorescent cells AFU/ml). Bacteria with slightly damaged membranes exhibit green and red fluorescence (defined as damaged cells), and bacteria with broken membranes fluoresce red (defined as non-active fluorescent cells n-AFU/ml). The total fluorescent units are expressed as TFU/ml, this is the sum of AFU and n-AFU.

 

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