Flow Cytometry for Probiotic Enumeration

Creative Biolabs provided custom solutions for each live biotherapeutics (LBPs) project. Our expert technical team has a wide range of research experience in Next-Generation Probiotics (NGPs), from strain isolation and screening to animal studies, we can flexibly apply our experience to meet your needs.

Overview

Most promising new strains are strictly anaerobic (e.g. Akkermansia municiphila, Eubacterium hallii, Faecalibacterium prausnitzii, etc.), and in addition to the technical challenges of developing them at an industrial level. The probiotic industry faces difficulties enumerating them. Classical plate counting methodology is arduous to implement on these new species due to their often-unknown growth requirements, lack of enumeration methodology, and necessity to operate in oxygen-free laboratories. In this context, the application of flow cytometry (FC) from the early phase of a product design (e.g. strain characterization) to the latest phase (e.g. cells administration in a clinical trial, finished product stability) would be a key factor for the development of NGPs or LBPs-based products. In this context, FC is bound to become a major opportunity to enumerate and characterize bacterial heterogeneity.

Principle of FC

FC can be described as automated microscopy with the advantages of automation, objectivity, and speed, as many thousands of cells can be analyzed in a second. The basic principle of FC is the measurement of the optical characteristics of a single microbial cell. The sample in liquid form is introduced to a fast-flowing fluid stream that forces the cells to pass in a single file through a laser beam. The results obtained with the cytometry method are expressed in units of fluorescence activity (Active Fluorescent Unit AFU/g). In addition, the value of the inactive fluorescence unit (n-AFU/g) can be quantified. Total Fluorescent Unit (TFU/g) indicates the total cell number, which is the sum of AFU and n-AFU.

FC Techniques at Creative Biolabs

FC can complement plate counting as it detects a ratio of intact versus total bacteria. The FC technique is a conventional method to detect the total number of bacteria in milk. It was used successfully for the assessment of viability and physiological activity of LAB and bifidobacteria subjected to different stress conditions, such as heat, drying or freezing, osmotic stress, and the presence of bile salts, as well as for stability testing. In addition, future studies will allow the use of FC for bacterial enumeration and identification, thanks to species-specific or even strain-specific primers for cell classification.

Generalised relationship between the viability of probiotic bacteria measured by Plate Counts and FC data.Fig.1 Generalised relationship between the viability of probiotic bacteria measured by Plate Counts and FC data. (Wilkinson, 2018)

Advantages

The main advantages of FC for microbiology are rapid assay times and data generation (1-2 min), high numbers of cells that can be analyzed per sample (10,000 and upwards), minimal sample volume potential high throughput, the multiplicity of stains available to examine various aspects of cell viability, structure and/or metabolism, and less labor and space required compared with conventional plating techniques. In addition, it should be emphasized that because of the use of cytometry, viable but nonculturable cells (VBNC) can be determined. In addition, the FC method has higher repeatability and better accuracy.

Creative Biolabs, one of the most experienced live biotherapeutics companies in the world, Our expert scientists and talented technical support team have got you covered all the way. If you would like a quote to discuss your requirements, please do not hesitate to contact us.

Reference

  1. Wilkinson, M.G. Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review. Trends in Food Science & Technology. 2018, 78: 1-10.

For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.

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For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.

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