This study introduces an optimized integration of flow cytometry and fluorescence in situ hybridization (Flow-FISH) as an approach for the specific enumeration of gram-positive bacteria in probiotic products, overcoming the limitations of conventional methods. The enhanced Flow-FISH technique synergizes the rapid and automated capabilities of flow cytometry with the high specificity of FISH, facilitating the differentiation of viable cells at the species level within probiotic blends. By analyzing lyophilized samples of Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium animalis subsp. lactis, and a commercial product, the study highlights the optimized Flow-FISH protocol\'s advantages, including reduced hybridization times to 1.5 h and elimination of centrifugation steps. Comparative evaluations with the widely accepted enumeration methods plate count and Live/Dead (L/D) staining were conducted. The study revealed that Flow-FISH produces higher viable cell counts than plate count, thereby challenging the traditional \"gold standard\" by highlighting its predisposition to underestimate actual viable cell numbers. Against L/D staining, Flow-FISH achieved comparable results, which, despite the different foundational premises of each technique, confirms the accuracy and reliability of our method. In conclusion, the optimized Flow-FISH protocol represents a significant leap forward in probiotic research and quality control. This method provides a rapid, robust, and highly specific alternative for the enumeration of probiotic bacteria, surpassing traditional methodologies. Its ability to enable a more detailed and reliable analysis of probiotic products paves the way for precise quality control and research insights, underscoring its potential to improve the field significantly.

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm2 and 10 CFU/mL present in both surface and water samples, respectively.

Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current \"gold standard\" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell\'s ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

Food processing, e.g., freeze-drying, exerts strong pressure on bacteria in the food matrix, decreasing their viability/activity and even forcing them to become viable but unculturable (VBNC), which are often underestimated by traditional plate count. The strict standards of bacterial viability in probiotic products require accurate cell viability/activity enumeration. We developed a staining (5(6)-carboxyfluorescein diacetate succinimide ester, propidium iodide)-based flow cytometry rapid method for detecting the viability/activity of Lacticaseibacillus (Lb.) casei Zhang, a widely used probiotic in the dairy industry in China. We optimized the procedural and instrumental parameters for generating results comparable to that of standard plate counts. This method was also applied to freeze-dried Lb. casei Zhang, yielding 7.7 × 1011 CFU/g, which was non-significantly higher than the results obtained by plate count (6.4 × 1011 CFU/g), possibly due to the detection of VBNC cells in the freeze-dried powder. We anticipated that this method can be used for detecting lactic acid bacteria in other probiotic food/beverages.

The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

Isothermal microcalorimetry can provide a general analytical tool for the characterization of bacterial growth. Methodologies and equipment have been studied to expand the application and disseminate the use of the technique. The MCDSC is a microcalorimeter capable of measuring in the range of 0.2 μW that can operate at a temperature range of -20 to 140 °C or under isothermal conditions. Here, we present the first investigation of MCDSC for E. coli growth with the Baranyi and Roberts modeling application. This study presented the calorimetric E. coli fingerprint at MCDSC and compares it with the plate count technique, giving the data more biological meaning. The calorimeter was able to accurately detect growth metabolism and discriminate E. coli at different inoculum densities. Additionally, the MCDSC can offer a new point of view for evaluating microbial growth, such as the significant reduction in error due to dispersed data by the viable counting method.

Biofilms are often polymicrobial in nature, which can impact their behavior and overall structure, often resulting in an increase in biomass and enhanced antimicrobial resistance. Using plate counts and locked nucleic acid/2\'-O-methyl-RNA fluorescence in situ hybridization (LNA/2\'OMe-FISH), we studied the interactions of four species commonly associated with catheter-associated urinary tract infections (CAUTI): Enterococcus faecalis, Escherichia coli, Candida albicans, and Proteus mirabilis. Eleven combinations of biofilms were grown on silicone coupons placed in 24-well plates for 24 h, 37°C, in artificial urine medium (AUM). Results showed that P. mirabilis was the dominant species and was able to inhibit both E. coli and C. albicans growth. In the absence of P. mirabilis, an antagonistic relationship between E. coli and C. albicans was observed, with the former being dominant. E. faecalis growth was not affected in any combination, showing a more mutualistic relationship with the other species. Imaging results correlated with the plate count data and provided visual verification of species undetected using the viable plate count. Moreover, the three bacterial species showed overall good repeatability SD (Sr ) values (0.1-0.54) in all combinations tested, whereas C. albicans had higher repeatability Sr values (0.36-1.18). The study showed the complexity of early-stage interactions in polymicrobial biofilms. These interactions could serve as a starting point when considering targets for preventing or treating CAUTI biofilms containing these species.

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability - up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

The measurement of microbial contamination is of primary importance in different fields, from environmental monitoring to food safety and clinical analysis. Today, almost all microbiology laboratories make microbial concentration measurements using the standard Plate Count Technique (PCT), a manual method that must be performed by trained personnel. Since manual PCT analysis can result in eye fatigue and errors, in particular when hundreds of samples are processed every day, automatic colony counters have been built and are commercially available. While quick and reliable, these instruments are generally expensive, thus, portable colony counters based on smartphones have been developed and are of low cost but also not accurate as the commercial benchtop instruments. In this paper, a novel computer vision sensor system is presented that can measure the microbial concentration of a sample under test and also estimate the microbial growth kinetics by monitoring the colonies grown on a Petri dish at regular time intervals. The proposed method has been in-house validated by performing PCT analysis in parallel under the same conditions and using these results as a reference. All the measurements have been carried out in a laboratory using benchtop instruments, however, such a system can also be realized as an embedded sensor system to be deployed for microbial analysis outside a laboratory environment.

BACKGROUND: Safe water supply is vital and can result in significant benefits to health. However, contaminated bottled water poses a great health risk due to the poor microbiological quality of water.
METHODS: A retrospective study was conducted on 222 Bottled water samples collected from various regions of Ethiopia from January 2008 to December 2015, tested and recorded in Ethiopian Public Health Institute to determine heterotrophic plate count and Staphylococcus aureus by pour plate method; for coliforms using multiple tubes fermentation techniques; for mould and yeast count using spread method, and for Salmonellae and Shigella spp. using ES ISO 6579 and ES ISO 21567. The data was analyzed using SPSS 20 statistical package.
RESULTS: Among the total samples examined from 44 brands, detections of heterotrophic plate count, mould, yeast, total and thermotolerant coliforms, Escherichia coli and Staphylococcus aureus were observed in 114 (51.4%), 33 (14.9%), 5 (2.3%), 2 (0.9%), 1 (0.5%), 1 (0.5%) and 1 (0.5%) samples respectively, but there were no detections of Salmonellae nor Shigellae species.
CONCLUSIONS: About 40% of bottled water samples were mycologically and bacteriologically unsafe for human consumption. To prevent public health hazards, regular monitoring of bottled water using quality indicators should be a priority agenda.