All's Well that Ends Well: A Profile of Specialty Microwell Plates

Date: September 27, 1999Table of Specialty Microplates The story of the microplate is one of those tales of history that either has been forgotten or was never clearly understood. According to Barry Lazar of Dynex Technologies, formerly Dynatech Laboratories, the origin of what is now commonly referred to by Dynex's registered trademark of Microtiter plates began with Gyola Takatsy, a Hungarian-born scientist who was trying to scale down serology tests. His first prototype became available in 1955 and was a 100-well plate cast in acrylic. Corning's Costar spectrum of microplates The first commercially available microplates were designed by National Institutes of Health researcher John Sever. He published on an improved form for the Microtiter system in 1962. The plates were designed simultaneously by John Liner (Linbro) and Nelson Cooke (Cooke Engineering). These acrylic plates already had the familiar 8x12 format that remains the most commonly used configuration. The 96-well format was dictated by microdiluters that served as the multichannel pipettors of their day. Typically, eight of these tulip-shaped microdiluters could be manipulated at one time, resulting in the characteristic eight rows that have persisted. Nalge Nunc debuted its 8x15 well plate about the same time. This plate, as described by Lazar, was considered ideal for antibiotic-susceptibility assays, which, along with hemoagglutination assays, became the most popular application of this product. Yet in spite of Nalge's early success, the 8x12 format eventually triumphed over the 120-well configuration. The dodecimal system has stuck ever since. Even though the 96-well format remained a constant, John West of In Vitro Scientific explains that the individual wells themselves were undergoing perceptible changes. "U" bottoms began to emerge, making it easier to retrieve material and facilitating mixing within the well. "V" bottoms were designed to concentrate material at the bottom of the plate for hemoagglutination, producing a small pellet at the base of the V. The "C" bottom resulted as a compromise between the U bottom and the flat bottom. Its rounded corners encouraged mixing, while the flatter bottom improved its optical characteristics. Each of these developments, one falling on the heels of the next, served to expand the possible applications of microplates. However, it was the revolution in plastics that proved to be the primary force behind the rapid evolution and specialization of the microplate. Plastics were cheap and disposable, they resisted high temperatures, and visualization of the button form was improved. Polystyrene offered a clarity that had not been afforded by previous plates such as Cooke Engineering's original 96-well plate, which was solid white. In addition, the binding characteristics of polycarbonate had the advantage of keeping dilutions in suspension, which was ideal for serology and antibiotic-susceptibility assays. In the age of HIV, plastics have almost entirely replaced reusable glass products. However, for tissue culture applications, plastics require special surfaces that encourage cell adhesion. One of the biggest concerns while running an experiment is knowing whether your tools are interfering in a natural process, producing artifacts. Does cell growth in a microplate resemble that seen in a more natural environment? A solution was found by the application of radiation or plasma treatment and the use of special coatings such as collagen, poly-D-lysine, streptavidin, and fibronectin. However, as one might have guessed, the use of pretreated plates gave rise to growth properties not seen under native conditions--differences measurable in quantity and quality. Today, a number of companies--such as Becton Dickinson, Corning, Nalge Nunc, NEN™ Life Science Products, Pierce Chemical, Sarstedt, and Xenopore--offer custom plate-coating services for tissue culture and immunology microplates that are designed to meet the specific needs of researchers across a number of fields and disciplines. Becton Dickinson's BIOCOAT line includes a wide selection of tissue culture ware, from streptavidin coatings for protein detection to collagen for neuronal cells. Each well format can be purchased in a variety of options, which include hydrophobic plates; enhanced hydrophilic plates for absorption of proteins; and vacuum-gas plasma-treated Primaria® plates that improve the attachment, spreading, and growth of tumor cells. Becton Dickinson's proprietary formulation and manufacturing technique also makes collagen and poly-lysine products stable at room temperature. Corning Science Products has a new 96-well UV-Plate made for determining protein and nucleic acid concentrations. The special UV-transparent bottom is coated with a proprietary acrylic material that permits short wavelength transmission. In fact, the plate can be read down to 220 nm and is certified to have background O.D.s of less than 0.05 at the two key wavelengths of 260 and 280 nm. As much as 80 percent of light is held back in other plates used for the same type of application. The UV-Plate also eliminates the need for quartz cuvettes and is compatible with readers from more than 50 manufacturers. One of the newest products offered by NEN Life Science Products is the FlashPlate HTS Plus. The intended purpose behind the FlashPlate HTS 384-well microplate is to provide a practical assay platform for miniaturized high-throughput screening. The design of the microplate reduces capillary action that causes contamination in many 384-well plates. The interior of each well is coated with a permanent layer of polystyrene-based scintillant, which has a variety of benefits for nonseparation assays. Pierce Chemical provides specialty coated 96-well plates utilizing standard and custom coating methods. Pierce Reacti-Bind™ standard coatings include NeutrAvidin and Streptavidin coated plates for binding biotinylated peptides, nucleic acids, and proteins; metal chelate, glutathione, and dextran coated plates for binding fusion proteins; protein A, protein G, and protein L coated plates for effective antibody immobilization. Pierce ChoiceCoat™ Microwell Plate Coating Service provides customized coated and derivatized 96- and 384-well plates to meet specific needs of the customer. ChoiceCoat services also include statistical quality analysis of coated plates, bar coding, and bulk packaging. Not to be outdone, Sarstedt, the perennial manufacturer of plastics and lab consumables for all occasions, has Cell+ 96-well plates for cell cultures. The Cell+ tissue culture products enhance growth by providing three growth surface options that imitate proteinlike structures. The advantage of Cell+ is that pretreatment with poly-lysines, collagens, and gelatins may not be necessary. Furthermore, the Cell+ line has shown certain benefits in culturing primary cells and epithelial cell lines. Xenopore has one of the most extensive offerings of specialty coatings in the industry. What began as a consulting firm in 1988 has developed into a major sources for custom coatings. In addition to its streptavidin plates and some of the more common covalent plates, Xenopore provides such exotic creations as its glutathione plates for binding GST-tagged proteins and maleimide plates for binding sulfhydryl groups. The company also makes thin-wall polycarbonate plates for simultaneous PCR and capture or immuno-PCR. Although special coatings have resulted in faster and more accurate results, product design remains one of the critical factors in microplate effectiveness. In this respect, microplates still present engineering challenges to their designers. Though advanced microplate readers and chemical treatment may enhance the performance of plates, innovative designs continue to emerge that extract minor improvements from what is essentially an established technology. Citing the significant advances in PCR techniques for industrial and academic research, Vince Prezioso of Eppendorf Scientific explained how his company was required to rethink the fundamental design of its microplates. "DNA fragments can be amplified exponentially via the rapid cyclic repetition of three temperature steps. However, to enable this technique to be practiced efficiently, our Eppendorf tubes had to be reengineered," said Prezioso. Modern Peltier elements can produce temperature differences of more than 50°C in a matter of seconds. The design of Eppendorf's micro tubes has followed these developments by incorporating extremely thin walls enabling the transmission of temperature in a fraction of a second. Eppendorf's most advanced PCR microplates include such features as thin walls, bar code identification, and grooves for thermal cycler positioning and robotic handling. The low profile of these plates also improves contact with the lid, decreasing evaporation. The new CyclePlate-384DW Thin-Wall PCR Plate is one of a number of high-throughput PCR plates offered by Robbins Scientific. Intended for use with Robbins' Hydra® line of microdispensers as well as its CycleSeal® plate sealer, the CyclePlate-384DW maintains well-to-well uniformity before and after thermal cycling. According to company claims, this is an improvement over other thin-wall plates that may warp after use. Robbins is also the only manufucturer that certifies its plastics for use in PCR by performing functional tests on each lot of plates. Samples are inspected for the presence of PCR inhibitors, human DNA, and nucleases. MJ Research is another major producer of microplates that has taken advantage of the proven benefits of thin-wall plates. MJ's Microseal™ 384-well thin-wall microplates are specially designed for high-throughput thermal cycling applications. The wells in these plates are spaced at half the common 96-well spacing so that they are compatible with standard multichannel pipettors. The plates also feature raised rims around the wells, which provide a greater surface area for pressure sealing whether using MJ's Microseal™ "A" film or one of the many brands available from its competitors. MJ's newest plate is its version 2.0 384-well plate. These thin-wall plates are designed to conform to the Society for Biomolecular Screening's proposed standards for automated equipment. These plates are available with metal positioners, which hold the plate in a precise X-Y axis. The more predictable shape makes pipetting more reliable at smaller volumes. One of the most useful options for the Microseal 384-well is an off-the-shelf bar coding system that helps to identify, monitor, and control each plate. MJ also makes low-profile 96-well plates that are nonskirted and have a rigid deck for easier handling. The low profile also cuts down on air space above the well, which causes water vapor to recondense at a faster rate. As simplistic as it may seem, sometimes the easiest solutions reap the greatest rewards in terms of performance and economy. This is certainly true when considering the geometry of microplates. What many companies are discovering these days is that the binding characteristics achieved through the use of special plastics and chemical coatings can be designed into the plate simply by altering its shape. Labsystems has taken a serious look at design issues in improving its line of microplates. The new Cliniplate™ takes advantage of this concept. By observing the physics of liquid adhesion that take place within a well, engineers at Labsystems discovered a subtle effect that may have implications for enhanced plate reading. They noted that square wells produce a "wicking effect" in which fluid rises up toward the corners of the well. Round wells, by comparison, do not exhibit this same tendency and have been incorporated into all of Labsystems' new Cliniplates. Labsystems also uses polypropylene, which prevents DNA from sticking to the sides of the wells. In combination with round wells, the polypropylene material is thought to encourage reactions taking place in the center of the well, where they can be more accurately measured. Perhaps the most revolutionary new design in microplates is EmbiTec's Morph Expandable Plate. This plate expands and contracts like an accordion, permitting users to switch from thermal cycler to sequencer in less time. This plate can be used for ethanol precipitation or chromatography without sample transfers or pipette adjustments. Before purchasing another expensive multichannel pipettor you might want to give this plate a try. LJL Biosystems has been building detection systems for years, but only since 1998 has it been producing microplates. LJL offers both black and white plates for a variety of modes. Black plates are ideal for fluorescence assays and prevent crosstalk between wells by quenching fluorescence. This background subtraction effect results in more homogeneous assays. White plates, on the other hand, disperse light in such a way that the total summation of light intensity is enhanced. These plates are very bright and nontransparent. They are well suited for intensity, TOF, fluorescence, and luminescence applications. One of the important contributions LJL has made to microplate design is in the conical shape of its wells. The conical design is intended to accommodate confocal optics in which a beam shaped like an hourglass converges on a single spot. It is possible to achieve greater optical collection and emission using this technique. Whatman Polyfiltronics has developed the world's first glass-bottomed plate with superior optical clarity for high resolution and confocal microscopy. The Whatman glass-bottom plate was specifically designed for fluorescence correlation spectroscopy (FCS), which permits the study of interactions at the level of single molecules. The plate is available in either opaque black or opaque white and is configured for 96 wells with a 300 µl capacity. The glass-bottom plate has been used successfully in the PARISS Imaging Spectrophotometer by LightForm. 96- and 384-well plates with bar coding from MJ Research Other specialty plates from Whatman include a 384-well DeepWell Collection Plate. One of the unique features of this plate is that its wells are square for maximum volume capacity but have round bottoms for greater ease of aspiration. The manufacturer has avoided the wicking effect by providing a large internal radius. The DeepWell plate is for high-density storage up to 400 µl and is resistant to DMSO and biological buffers. Packard Biosciences has four different varieties of specialty plates for fluorescence and luminescence. The CulturPlate is a black, culture-treated microplate that comes with a transparent lid. This plate is recommended for low to moderate signal luminescence assays as well as fluorescence and counting. Similarly, the OptiPlate is a black 96- or 384-well microplate designed for fluorescence and high signal luminescence applications under nonsterile conditions. These plates complement Packard's proprietary Homogenous Time-Resolved Fluorescence technology and allow high counting efficiency with reduced cocktail consumption. Packard's LumaPlate is available as a shallow- or deep-well plate and is coated with a solid yttrium silicate scintillant. The LumaPlate's solid scintillant simplifies single and dual label counting by eliminating variable chemical quench and is suited for samples produced by 51Cr release, enzyme inhibition, organic extractions, and chromatography fractions. The PicoPlate is a solvent-resistant, white microplate available in 24- and 96-well configurations that is suggested for use with samples of a highly polar organic nature. This plate is adapted for robotic systems and is well suited for aqueous samples as well. Packard's newest release is its ScreenReady™ FlashPlates® prepared by BioSignal®. These precoated plates for high-throughput screening are treated with a purified G protein-coupled receptor. Radiolabeled ligand is also provided by NEN Life Sciences and shipped directly to laboratories. Amersham Pharmacia Biotech extended its bead-based Scintillation Proximity Assay (SPA) technology to 96-well microtiter plates in 1996. The result is Cytostar-T™ scintillating microtiter plates. These clear bottom plates are tissue culture treated and sterile for use with cell-based assays. Proprietary scintillants incorporated into the base plate allow real time measurement of the uptake or efflux of a radioligand by adherent cells. The transparent base allows researchers to monitor cellular morphology throughout the experiment. Cytostar-T microtiter plates have been successfully used for cell proliferation and apoptosis, mRNA quantification, receptor-ligand interaction, drug uptake and efflux, and toxicology-ADME studies. The manufacture of Cytostar-T microtiter plates has been recently improved to reduce the level of extractables in the plate. The newest release from Hitachi Genetic Systems is a 96-well low-fluorescence assay plate. Designed for use in Hitachi's FMBIO® II Fluorescence Imaging System, the plate provides minimal background fluorescence and light scattering. These black polystyrene plates allow transmission of laser light and yet the opaque walls prevent crosstalk between wells. The flat plate bottoms are 60 percent thinner than conventional polystyrene plates, which serves to further dampen background fluorescence. Each plate has a total well volume of 360 µl and is sterile and nontreated. The FlashPlate HTS 384-well microplate from NEN Life Science Products Certain applications require the use of solutions that are not compatible with plastic labware. The SMP series of quartz microplates from Spectrocell has an inherent resistance to solvents, acids, and hydroxides and is guaranteed to be free of defects and contaminants. They can also be sterilized in an autoclave. The adaptability of these plates under harsh conditions makes them a worthwhile, albeit expensive, alternative for researchers who work with finicky reagents. The quartz plates are available in clear quartz for UV-vis applications and in black self-masking models for fluorescence. Nalge Nunc has an extensive program for treating glass or plastic microplates with specialized formulations of styrene for use in immunology, molecular biology, and cell culture applications. Nunc's MaxiSorp surface is recommended for ELISA assays and is available as a certified surface based on adsorption of IgG, while its PolySorp surface is suggested for use with hydrophobic molecules such as viral antigens. Either of these surface coatings can be combined with Nunc's Nunclon™ Delta Polystyrene, which provides a hydrophobic surface for improved flow attachment. When incorporated into Nunc's 384 Optical plate, the result is a clear thin-bottomed plate that can be read from the top or the bottom. Evergreen Scientific also has ELISA/EIA-treated plates for immunology that are specially treated and gamma-irradiated for protein binding studies. These plates have 96 wells and have the option of a flat or U bottom. In addition, Evergreen has a full line of tissue culture treated plates for animal cell, plant cell, and tissue culture studies. The 96-well treated plates are available with flat bottoms, V bottoms, and U bottoms for easy sample retrieval. Dynex Technologies, billed as The Microtiter Company, was one of the first companies to recognize a need for specialty microplates. For more than 30 years, Dynex has been producing high quality plastics for biological research and has continued to expand its product line in accordance with the rapidly changing requirements of cell and molecular biologists. It has developed four primary product lines that address the demand for absorbance protein binding plates, fluorescence/tissue culture plates, luminescence plates, and styrene and vinyl microtiter plates. Its most recognized and widely distributed plate is the MICROLITE plate, used for applications such as PCR quantitation, reporter gene detection, phagocytosis and luminescent immunoassays, and tissue culture. Greiner America has specialty microplates that fit almost every category of advanced development in microplate technology. Whether you're looking for colored, treated, ultraviolet, deep-well, clear-bottom plates, or even bottomless plates, Greiner has a specific product for each application. Greiner presently has three new products in release. Its 38SV small-volume, 384-well plate, in black or white nontreated polystyrene, allows researchers to miniaturize assays by affording a very small working well volume of 5 to 20 µl. Greiner's UVStar™ plates for reading in the ultraviolet range are competitive with Corning's UV-Plate and offer the added benefit of a 384-well configuration. The 384-well Masterblock™ is a deep-well plate that handles volumes as large as 240 µl/well. This plate has conical bottoms for increased sample recovery and is the latest addition to Greiner's ClearChoice™ polypropylene plate series. Molecular BioProducts' latest release is a 96 deep-well plate with a very large 2 ml working volume per well. This plate is ideally constructed for combinatorial chemistry library storage and is precision molded with round well bottoms to eliminate binding or reactivity. Bel-Art Products also makes a plate with 2 ml wells for serial dilutions with a specially designed lid that separates each well via a sealing ring. The lid not only provides protection against cross-contamination, but allows the plate to be stacked out of the way when not in use. The Bel-Art deep-well plate is recommended for use with robotic workstations from Zymark, Tecan, Hamilton, Rasys, and Beckman. If variety is the spice of life, then some microplate manufacturers should be well seasoned. The ever-expanding market for specialty microplates has driven manufacturers to the extremes of specialization, creating problems of compatibility. Consumers buying microplates are not just getting plates, they are committing to a system that may determine their choice of pipettors, plate readers, and every other accessory or convenience related to microplates. The Society for Biomolecular Screening is attempting to standardize microplate specifications, but getting everyone to agree on an arbitrary set of conventions is a difficult process when brand loyalty and habit are concerned. It is likely that a number of the larger manufacturers will attempt to enforce standards that most closely approximate their existing configurations in order to avoid significant retooling. The result of this standardization will be the selling off of large stocks of microplates by smaller competitors in order to make way for the new formats. Consumers may see some excellent deals in the near future on some of their favorite products. Table of Specialty Microplates Brent Johnson can be contacted at bjohnson@the-scientist.com.

All's Well that Ends Well: A Profile of Specialty Microwell Plates

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