Culture-Based MEMS Device to Track Gordonia in Activated Sludge

Previously, we reported on the use of microelectromechanical systems (MEMS) fabrication technologies to develop paraffin surfaces for miniaturization of culture-based detection and rapid quantification of Mycolata in environmental samples. In the current study, the novel culture-based biochip was calibrated with a broad range of pure cultures of Mycolata including Gordonia spp. isolated from activated sludge foam. The biochip successfully recovered Gordonia amarae spiked into a sample of mixed liquor collected from a municipal activated sludge system. Comparisons of these results with molecular biology-based assays including 16S rRNA-targeted fluorescence in situ hybridization (FISH) and antibody staining demonstrated that the biochip provides a more rapid and user-friendly platform for reliable identification and quantification of Mycolata in full-scale municipal activated sludge sewage treatment plants. The results of this work successfully demonstrate an alternative platform technology for inexpensive monitoring of environmental microorganisms using existing expertise by potential users in the area of bacterial cultivation

Lysogeny in Bradyrhizobium japonicum and Its Effect on Soybean Nodulation

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobium japonicum 123B (USDA 123) but only mildly lytic on strain L4-4, a chemically induced small-colony mutant of 123. Numerous bacteriophage-resistant variants were isolated from L4-4 infected with phage V; two were studied in detail and shown to be lysogenic. The two, L4-4 (V5) and L4-4 (V12), are the first reported examples of temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closely related on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunity tests, and apparently identical morphology when examined by electron microscopy. However, the three phages differed in host range and in virulence. Lysogens L4-4 (V5) and L4-4 (V12) were immune to infection by phages V and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phage V into the genomes of strains L4-4(V5) and L4-4(V12) and also demonstrated the incorporation of phage V into the genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens, L4-4(V5), L4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridization profile data indicated that phage V had not incorporated into any of the known B. japonicum nodulation genes

An Innovative Approach to Detecting Mycobacterium in Drinking Water Systems

By including Mycobacterium avium Complex (MAC) on the Contaminant Candidate List, the U.S. EPA has initiated research to evaluate if MAC represent a significant threat to human health through occurrence in drinking water supplies. Currently, culture-based approaches to monitor for Mycobacterium require weeks before results are available. To reduce the time needed to screen for Mycobacterium, an interdisciplinary team including environmental and electrical engineers, microbiologists, and physicists worked together to create miniaturized culture-based devices to detect Mycobacterium rapidly. The first generation device relies upon the lipophilic properties of Mycobacterium to isolate target microorganisms in an abundance of non-target microorganisms. Water samples are re-circulated through a twenty-liter water distribution system simulator where paraffin chip devices are exposed to Mycobacterium present in the bulk water. After one day\u27s exposure, the devices, are removed, prepared, and analyzed. An occurrence study of environmental MAC in southwestern Ohio hospitals is in progress. The two main questions being addressed by this study are: (1) is MAC in water distribution systems in health care settings, and (2) does the device provide a faster and more accurate way to detect MAC in environmental samples when compared with traditional methods. There is ecological, epidemiological, and pathological significance in rapidly screening for MAC in drinking water systems. It is possible, using the culture-based device described, to detect and identify MAC more quickly, and future developments aim to use this device for real-time MAC screening. This paper was presented at the 8th Annual Water Distribution Systems Analysis Symposium which was held with the generous support of Awwa Research Foundation (AwwaRF)

Developing Culture-Based Biochips for Detecting Microorganisms in Wastewater Treatment

In this paper, we describe a culture-based biochip for detecting microorganisms in environmental samples in an improved, timesaving, and cost-efficient manner. The biochips use the paraffinophilic nature of microorganisms to rapidly detect and differentiate them in sewage samples collected from wastewater treatment plants. A new method of depositing paraffin was developed in order to fabricate such biochips. The developed technique uses butanol solvent to keep paraffin liquid during deposition by spin-coating and thick photoresist for patterning. Using this approach, 18 mm glass biochips with paraffin layers of 5-70 µm have been successfully fabricated. These biochips were used to demonstrate rapid, 5-min detection of microorganisms responsible for reactor foaming in wastewater treatment