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Spotlight on Flow Cytometry

Probiotical Leads the Field with State-of-the-Art Probiotic Enumeration Methods 

In the rapidly evolving field of probiotics, precise methods for counting bacteria are essential for maintaining both product quality and effectiveness. The very definition of probiotics, as outlined by FAO/WHO in 2001, hinges on the quantity and viability: "live microorganisms that, when consumed in sufficient amounts, offer health benefits to the host." While the classical Plate Count (PC) method has been the gold standard for bacterial enumeration for more than a century, it comes with significant limitations and inaccuracies. To meet the demands of high-quality probiotics, more advanced methods offering better speed, accuracy, and consistency are needed. 

Probiotical is at the forefront of this shift, being one of the first manufacturers to widely adopt Flow Cytometry as a cutting-edge technique for bacterial enumeration. This adoption positions Probiotical as a leader in the industry, setting new standards for the production of effective, high-quality probiotic products.

Limitations of the Plate Count Method

The Plate Count (PC) method, introduced in the late 19th century and refined by pioneers like Robert Koch and Julius Richard Petri, has been a foundational technique for bacterial enumeration. The process involves taking a diluted sample of bacteria and spreading it across the surface of a nutrient-rich agar plate. The plate is then incubated at a specific temperature to allow bacterial colonies to grow. After a set period, often ranging from 24 to 72 hours, the visible colonies are counted. Each colony is assumed to have originated from a single bacterial cell (a colony forming unit, of CFU), and the number of colonies is used to estimate the concentration of bacteria in the original sample. By the early 20th century, PC had become an integral part of microbiological research and a staple in both academic and industrial settings due to its simplicity and effectiveness.

However, despite its long-standing use, the PC method has limitations that are becoming more apparent in the nuanced field of probiotics:

  1. The method lacks universal applicability across different probiotic organisms due to variations in species and strains' responses to plating procedures. This has led to a plethora of internal methods developed by manufacturers, making cross-comparison of strain quantities challenging.

  2. PC is labor-intensive and time-consuming, often requiring up to 72 hours for results due to long incubation periods. This impacts both laboratory workload and sample throughput.

  3. Establishing the appropriate growth conditions for each bacterial strain can be technically challenging. This is particularly true for strains that are sensitive to oxygen and are adapted to specific environments like the gastrointestinal tract.

  4. The method suffers from low precision. The variability in results is so significant that different guidelines and quality seals have been proposed to account for this, such as the Italian Ministry of Health's recommendation of listing an "uncertainty of 0.5 log" on product labels.

  5. PC may significantly underestimate the actual count of viable cells. This is because Viable But Not Culturable (VBNC) cells do not form colonies, and aggregates or chains of cells may form only a single colony, skewing the results.

As the probiotics and live biotherapeutic product sectors continue to mature, the limitations of traditional methods like the PC technique underscore the urgent need for innovative, accurate, and efficient enumeration methods to usher the industry into a new era of quality and efficacy.

Flow Cytometry, a Modern Method for Viability Measurement

Flow cytometry is emerging as a groundbreaking technology that addresses the shortcomings of traditional PC methods, offering a more comprehensive and efficient approach to bacterial enumeration. The technique is based on the principle of passing individual cells through a focused laser beam. This laser analyzes various cellular attributes, including size, which is determined by forward light scatter, and granularity and morphology, assessed through side scatter. 

Additionally, flow cytometry employs a range of staining protocols, each designed to evaluate different aspects of bacterial viability. For instance, the membrane integrity protocol uses dual colorants to distinguish between bacteria with intact membranes (viable) and those with damaged membranes (non-viable) (Fig. 1). Cells with undamaged membranes are denoted as Active Fluorescent Units (AFU), signifying the portion of the bacterial population that is viable but may not necessarily proliferate when subjected to PC methods.

Figure 1: A readout of Flow Cytometry membrane integrity protocol

Figure 1: A readout of Flow Cytometry membrane integrity protocol 

Flow cytometry has manifold advantages over the PC method:

  • Universal Applicability: Unlike PC methods, which often require species-specific protocols, flow cytometry can be applied universally. It is capable of enumerating a wide range of microbial species, from common probiotics like Bifidobacteria and Lactobacilli to yeasts and even contaminants.

  • Speed: One of the most striking benefits is its speed. A complete analysis, including triplicates, can be accomplished in as little as 30-45 minutes. This is a significant improvement over Plate Count methods, which can take up to 72 hours for results.

  • Independence from Growth Conditions: Flow cytometry does not rely on bacterial growth for enumeration. This eliminates the need for laborious preparatory work to determine optimal growth conditions, although it does necessitate careful sample preparation to minimize background noise.

  • High Accuracy: The technique offers a level of precision far superior to traditional methods. International standards based on extensive analyses have demonstrated high repeatability and reproducibility, which can be further improved with proper training.

  • Insight into Functional Responses: Beyond mere counting, flow cytometry can provide valuable insights into the functional behavior of bacterial strains under various conditions.

  • Detection of Dormant and Non-Culturable Cells: Crucially, flow cytometry can identify cells that are metabolically active but not culturable, known as VBNC cells. These cells can be biologically active and may regain their replicative abilities under favorable conditions.

By offering these advantages, flow cytometry is not just an alternative but a significant upgrade to traditional bacterial enumeration methods, positioning it as the method of choice for the future of probiotics and live biotherapeutic products.

The Significance of Measuring Viable but Non-Culturable Cells

Metabolically active viable but non-culturable (VBNC) cells present a significant consideration in bacterial enumeration, especially in the context of probiotics and pathogen detection. These cells can enter a VBNC state due to various stressors encountered during industrial production processes like fermentation and lyophilization. Importantly, VBNC cells are not just dormant; they can recover and become viable under favorable conditions, such as when they enter the gastrointestinal system. Traditional PC methods may significantly underestimate these cells, capturing only about 1% of the total bacterial population in some cases. This has implications not only for understanding microbial diversity but also for assessing the efficacy of probiotics, as these cells can resuscitate and become active under specific conditions. Moreover, even dead bacterial cells can have biological roles, such as gene transfer to other microbial community members, challenging the conventional wisdom that only culturable cells are biologically significant. Flow cytometry offers a more comprehensive approach, capable of detecting VBNC cells and providing greater insights into bacterial viability and heterogeneity, thereby broadening our understanding of their role in health.

Probiotical Pioneers Next-Generation Quality Control with Flow Cytometry

As a pioneer in the probiotics industry, Probiotical is among the first manufacturers to adopt flow cytometry, setting a new standard for quality and efficacy in both raw materials and finished products. This cutting-edge technology is particularly crucial as the definition of probiotics expands to include non-culturable and heat-killed strains, as well as "probiotic-derived factors" that also confer health benefits. The implementation of flow cytometry is even more vital for emerging "novel" probiotics, which are often strictly anaerobic and present unique challenges for traditional enumeration methods like Plate Count. These next-generation strains frequently have complex growth requirements and may even require the presence of other strains for cultivation, making them difficult to isolate and enumerate using conventional methods.

By integrating flow cytometry into its quality control processes, Probiotical not only enhances the reliability and reproducibility of its products but also positions itself at the forefront of innovation in the probiotics industry. This technology allows for the nuanced assessment of bacterial viability, thereby ensuring that Probiotical and their customers are able to create products of the highest quality and efficacy.