Bacteria in Food

Quantification of Total Bacterial Cells in Water and Drink

A safe and secure supply of drinking water is an essential requirement for human health. Due to the potential occurrence of various microbiological contaminants in drinking water and beverages, the total bacterial count represents a key parameter for quality assessment. Currently, water dispensers are commonly used, and the fluctuation of water quality, especially the bacterial count of unsealed barreled water, has attracted much attention. The cultivation-based heterotrophic plate count (HPC) has long been a firmly established tool for the assessment of water quality. However, it is labor-intensive, time-consuming, and of limited use in certain circumstances. On the other hand, bacteria in a state of very low metabolic activity, such as viable but non-cultivable (VBNC) bacteria, can be overlooked by HPC.

By utilizing the nucleic acid dye PicoGreen to label particles in water, particles that show burst traces in both side scatter and fluorescent channels simultaneously are recognized as bacteria. Compared with HPC, the Flow NanoAnalyzer-based approach not only shortens the analysis time but also reveals the presence of dead and VBNC bacterial cells.

Combined with PicoGreen nucleic acid fluorescence staining, rapid and accurate quantification of total bacteria in drinking water and tea beverages is achieved, and the results correlates well with conventional HPC method.

Anal. Methods, 2015, 7(7), 3072-3079.

Rapid Quantification of Live/Dead Lactic Acid Bacteria in Probiotic Products

Positive probiotic effects have been demonstrated in specific population groups, especially in patients with diseases such as necrotizing enterocolitis, antibiotic-associated diarrhea, and acute infectious diarrhea. Lactic acid bacteria (LAB), the most common microbes employed as probiotics, are widely used in probiotic products like yogurts and fermented milk beverages to endow them with beneficial effects on intestinal health. To exert beneficial health effects, probiotic bacteria need to be viable and available at a high cell count (at least 10^6 cfu/ml). Probiotic products sold with claimed health benefits should meet this minimum viable count of bacteria. Thus, rapid and accurate quantification of viable LAB is of great importance for the quality assurance of probiotic products. The most frequently used method in the dairy industry to assess bacterial viability is plate counting, which is labor-intensive, time-consuming, and fails to detect bacteria that do not form colonies but are nevertheless metabolically active.

The Flow NanoAnalyzer was employed for the rapid and accurate detection of LAB and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, Propidium Iodide (PI), which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the Flow NanoAnalyzer. The method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands.

Figure 1. The Flow NanoAnalyzer analysis of UHT milk spiked with live LAB with SYTO 9 staining.

Figure 2. The Flow NanoAnalyzer analysis of UHT milk spiked with live/dead LAB with SYTO 9 and PI staining.

The Flow NanoAnalyzer has many potential applications in monitoring the fermentation process, assessing the quality, and studying the effect of prolonged storage of probiotic products.

Methods Appl. Fluoresc., 2017, 5(2), 024002.

Label-Free Detection of Bacteria in Fruit Juice

Rapid quantification of microbial contamination in fruit juice is highly desired for food safety control. Yet, the complex sample matrix and the diversity of bacterial contaminants present a great challenge. Employing the Flow NanoAnalyzer, a label-free approach for the detection of the bacterial population in fruit juice was reported. The weak autofluorescence of bacterial cells was used as a hallmark for the identification of bacteria. The sample pretreatment protocol was optimized to reduce fluorescence background, lyse residual plant cells and debris, and attain a good recovery of bacteria from juice samples. The as-developed method was successfully applied to the bacterial measurement of freshly made orange juice and apple juice purchased from grocery stores. This approach shows great potential in the microbial control analysis of other liquid drinks.

The bacterial concentrations measured by Flow NanoAnalyzer complimented those of the plate counter, with a correlation coefficient of 0.97. Morever, the experimental procedure, including sample pretreatment and result analysis, can be accomplished within one hour, which is far more efficient than the plate counting method.

Anal. Chem., 2020, 92(3), 2393-2400.