Simple Detection of Pseudomonas Cells in Milk

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Table of Contents

Introduction
Techniques for bacteria analysis
Microfluidic chip-based systems
Materials and methods
Discussion
References

Introduction

Bacterial spoilage is caused by harmful or food spoiling bacteria, which result in diseases and contamination, leading to the outbreak of food-borne diseases. The result is an impact on the economic side of the food industry. It is possible to detect these harmful bacteria using conventional culture methods. These conventional methods do not expose all the bacteria found in injured cells. As a result, there are a variety of methods used to identify food spoiling bacteria in food (Yamaguchi, Ohba, and Nasu 631).

Techniques for bacteria analysis

“Gene amplification and PCR provide species-specific detection with high sensitivity, though the process is arduous, as it requires the extraction of DNA or RNA from microbial cells” (Yamaguchi, Ohba and Nasu 631). According to Yamaguchi, Ohba, and Nasu, flow cytometry is used in the rapid identification of bacteria in milk through methods such as “total bacterial counts, detection of pseudomonas cells by fluorescence in situ hybridization, and detection of respiring Escherichia coli O157:H7 cells by double staining with Fluorescein Isothiocyanate -labeled antibody and 5-cyano-2, 3-ditolyl tetrazolium chloride”. Though the flow cytometry technique is fast, responsive, and consistent, the equipment is expensive, and its operation is complex for the untrained personnel (Yamaguchi, Ohba, and Nasu 632).

Microfluidic chip-based systems

According to Yamaguchi, Ohba, and Nasu, these systems have been used on criteria such as bacteria cell sorting, on-chip staining as well as the simple identification of protozoa or bacterial cells in river water. The chip-based systems are highly advantageous since they can be automated, and have low reagent consumption. In addition to this, they are small, cost-effective fast, and extremely reproducible. The chips are used once and the system is implemented when closed, which reduces the risk of biohazard. The efficiency of the system has been tested on small amounts of pseudomonas cells, which cause spoilage in milk. The pseudomonas spp. can contaminate milk in various phases of the growth cycle, as it “grows actively during refrigerated storage and produces enzymes such as proteases and lipases that degrade milk compounds, which result in off-flavours and reduced shelf life” (Yamaguchi, Ohba and Nasu 632).

Materials and methods

In the investigation, Pseudomonas cells in their stationary phase were acquired by incubation at 30°C in a liquid medium, while starved cells were obtained by a process involving centrifugation and washing with sterile phosphate-buffered saline, before being stored in the dark for a month, at 4°C. The milk used was inoculated with the pseudomonas cells at various densities, after being heated at 140°C, before the samples were prepared for Fluorescence in situ hybridization (FISH) in the liquid phase (Yamaguchi, Ohba and Nasu 632).

The chip-based method of detecting bacteria in milk requires the milk to be cleared, in order to avoid “false-positive results due to nonspecific binding of fluorescent dyes and probes to milk compounds” (Yamaguchi, Ohba and Nasu 633). Another way in which negative results can be obtained is interference during fluorescent staining and FISH. The system was designed for simple flow cytometry, and requires no training on operation, as it is also used in identification of bacterial cells in both culture and natural river water samples. The results are indicated as dot plots of the fluorescence values and the specificity of the optimized FISH procedure are proven using the total direct counting method (Yamaguchi, Ohba and Nasu 633).

Discussion

The research conducted shows that it is possible to identify small amounts of pseudomonas cells in milk when on-chip flow cytometry is preceded by FISH, in a time frame of 16 hours. The FISH procedure is only necessary when it is required that there be specific detection of targeted cells, based on their rRNA sequence, while any other detection of bacteria in milk can use the on-chip flow cytometry, after nucleic acid staining, within a period of 12 hours (Yamaguchi, Ohba and Nasu 635).

References

Yamaguchi, Nobuyasu, Hojo Ohba and Masao Nasu. “Simple detection of small amounts of Pseudomonas cells in milk by using a microﬂuidic device.” Letters in Applied Microbiology (2006): 43(6), 631-635. Print.

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Simple Detection of Pseudomonas Cells in Milk

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