Effective control of tuberculosis transmission in vulnerable population groups is dependent on rapid identification of the infectious agent and its drug susceptibility. However, the slow growth rate of mycobacteria has undermined the ability to quickly identify antimicrobial resistance. These studies describe a mycobacterial growth assay based on microencapsulation technology used in conjunction with flow cytometric analysis. Mycobacteria were encapsulated in agarose gel microdrops approximately 25 m in diameter, and colony growth was monitored by using flow cytometry to evaluate the intensity of auramine staining after culture for various times at 37؇C. By this method, colony growth of Mycobacterium bovis and M. smegmatis could be quantified within 1 to 3 days after encapsulation. Inhibition of growth by rifampin and isoniazid was also evaluated in this time period, and the presence of an isoniazid-resistant subpopulation representing 3% of the total microorganisms could be detected. This use of encapsulation and flow cytometry has the potential to facilitate rapid and automated evaluation of inhibition of growth by antimicrobial agents and shorten the time frame for analysis of clinical specimens.
Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization (MISH) method which facilitates hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosomes using hybridization rather than nucleic acid binding dyes. Key terms: GMD, flow cytometry, MISHGel microdrop (GMD) technology (33) evolved from an interest in encapsulating biological materials such as mammalian cells or microorganisms in agarose microspheres. Growth (25,34), secretion (25,26,34), metabolism (34,35,36), cytotoxicity (2,15,35), and electroporation (14) can be measured rapidly in the defined microdrop environment. GMDs are usually composed of agarose. They are prepared by dispersing molten agarose into an excess of a hydrophobic fluid, such as inert silicone oil, to form an emulsion. After the emulsion is transiently cooled, GMDs are separated from the silicone oil by centrifugation and remain physically distinct and robust.Various methods are available to analyze metaphase chromosomes, including flow cytometry (7,17,2 1 ), in situ hybridization (16,19,29,30,31), and staining (3,10). In order to provide stability, chromosomes are typically adsorbed onto slides for analysis. Consequently, analysis requires microscopic evaluation of individual slides which limits automation. Attempts to perform in situ hybridization on chromosomes in solution have been hindered by clumping, breakage, and aggregation ( 1 ). Current methods ( 18) could be improved by increased stabilization. Integration of GMD technology with hybridization techniques provides a system for analyzing chromosomes in suspension, facilitating sample handling, flow cytometry analysis, and hybridization-based isolation.GMDs provide assay microenvironments which are compatible with most in vitro cell manipulations. Our studies have indicated that GMDs can be pipetted, centrifuged, filtered, and analyzed by flow cytometry. The gel matrix provides physical protection, while the small size and porosity result in high permeability and rapid diffusion (35). These properties make the GMDs compatible with enzyme manipulation, fluorescence staining, and repeated washing.The results presented here demonstrate successful stabil ization and flow cytometric analysis of hybridized chromosomes. Development of this technology involved optimization of buffers, emulsion speeds, surfactants and agarose type. Successful encapsulation of chromosomes into GMDs was first confirmed using fluorescence microscopy and image analysis. Protocols were then developed for in situ ...
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