Investigation into the Physiological State of Heat Stressed Escherichia coli Used in the Evaluation Testing of an Intrinsic Fluorescence-Based RMM.

Rapid microbiological methods (RMMs) have been used as novel quality control technologies in industry. The ability of RMMs to detect stressed bacteria, in particular, is of continued interest due to the limitations of the conventional method in stressed bacteria detection. Accordingly, there is a need to better characterize an RMM's ability to detect stressed microorganisms. Previously we reported on the detection ability of an intrinsic fluorescence-based RMM using a 50% injured (determined based on colony-forming ability) bacterial cell group after heat treatment at 55°C for 8 min. In this study, we added further information about the physiological state of the heat treated Escherichia coli, besides proliferation ability, by investigating respiratory activity using CTC fluorescent staining and expression of DnaK, a heat shock protein. It was found that 89% of cells (control 96%) retained respiratory activity, but only 20% (control 41%) retained proliferation ability after heat treatment. The difference between the percentage of cells with respiratory activity versus that of cells still capable of proliferation further supports the existence of viable but non-culturable stressed cells in the test sample. Also, we suggest such analysis would be one approach to confirming the use of stressed as opposed to dead cells when evaluating an RMM's ability to detect stressed microorganisms.

Investigation of the detection ability of an intrinsic fluorescence-based bioaerosol detection system for heat-stressed bacteria.

A number of rapid microbiological methods capable of aerosol-based microbial detection are quickly emerging for use in the pharmaceutical and food markets. A subset of these technologies utilizes intrinsic microbial fluorescence as the basis for bioaerosol detection. This fundamental method of detection is relatively new to the pharmaceutical and food industries, which rely on traditional culture-based methods implemented decades ago to gain an understanding of their manufacturing environments. When combined with real time and continuous assessment, intrinsic fluorescence-based detection provides a new level of information and monitoring in these environments. One aspect of this monitoring relates to the detection of stressed micro-organisms. Bacteria found in pharmaceutical and food manufacturing environments can be in a stressed state due to heat, UV, or chemical exposure, desiccation, and so forth. As a result, the ability of an environmental monitoring system to detect stressed microbes is of particular interest. A commercially available, intrinsic fluorescence-based bioaerosol detection RMM was utilized in this study to determine the ability of such systems in the detection of heat-stressed microorganisms. An assessment of culturability and growth delay in control and heat-stressed samples was performed to confirm stress. Furthermore, the performance of the intrinsic fluorescence-based bioaerosol detection systems were compared to the SAS Super 100, MAS-100 NT, and SMA air samplers in the detection of heat-stressed Escherichia coli, Staphylococcus epidermidis, and Bacillus atrophaeus spores. These bacteria were selected because they are industry-relevant organisms, commonly found in various manufacturing environments, that represent a Gram-positive, Gram-negative, and spore-forming bacteria, respectively. It was found that the intrinsic fluorescence-based bioaerosol detection systems can detect heat-stressed microorganisms, including those that are not detected by the traditional culture-based method due to the inability of the stressed microbes to form colony-forming units. LAY ABSTRACT: Rapid microbiological methods capable of aerosol-based microbial detection are emerging for use in pharmaceutical and food markets. A subset of these technologies utilizes intrinsic microbial fluorescence as the basis for bioaerosol detection, a method relative new to the pharmaceutical and food industries, which rely on traditional culture-based methods. Bacteria found in such environments can be in a stressed state due to heat, UV, or chemical exposure, desiccation, and so forth. As a result, the ability of an environmental monitoring system to detect stressed microbes is of particular interest. A commercially available, intrinsic fluorescence-based bioaerosol detection RMM was utilized in this study to determine the ability of such systems in the detection of heat-stressed microorganisms. An assessment of culturability and growth delay in control and heat-stressed samples was performed to confirm stress. Furthermore, the performance of the intrinsic fluorescence-based bioaerosol detection systems were compared to the SAS Super 100, MAS-100 NT and SMA air samplers in the detection of heat-stressed Escherichia coli, Staphylococcus epidermidis, and Bacillus atrophaeus spores. These bacteria were selected because they are industry-relevant organisms, commonly found in various manufacturing environments, that represent a Gram-positive, Gram-negative, and spore forming bacteria, respectively. It was found that the intrinsic fluorescence-based bioaerosol detection systems can detect heat-stressed microorganisms, including those that are not detected by the traditional culture-based method due to the inability of the stressed microbes to form colony-forming units.

A green Paramecium strain with abnormal growth of symbiotic algae.

Some hundred cells of Chlorella-like green algae are naturally enclosed within the cytoplasm of a single cell of green paramecia (Paramecium bursaria). Therefore, P. bursaria serves as an experimental model for studying the nature of endo-symbiosis made up through chemical communication between the symbiotic partners. For studying the mechanism of symbiotic regulations, the materials showing successful symbiosis are widely used. Apart from such successful model materials, some models for symbiotic distortion would be of great interest in order to understand the nature of successful symbiosis. Here, we describe a case of unsuccessful symbiosis causing unregulated growth of algae inside the hosting ciliates. Recently, we have screened some cell lines, from the mass of P. bursaria cells survived after paraquat treatment. The resultant cell lines (designated as KMZ series) show novel and unusual morphological features with heavily darker green colour distinguishable from the original pale green-coloured paramecia. In this type of isolates, endo-symbiotic algae are restricted within one or two dense spherical structures located at the center of the host cells' cytoplasm. Interestingly, this isolate maintains the host cells' circadian mating response which is known as an alga-dependent behaviour in the host cells. In contrast, we discuss that KMZ lacks the host-dependent regulation of algal growth, thus the algal complex often over-grows obviously exceeding the original size of the normal hosting ciliates. Additionally, possible use of this isolate as a novel model for symbiotic cell-to-cell communication is discussed.