Dispensing Aspirations

Date: October 13, 1997 Test Results Chart 2 Anyone who has manually filled and rinsed all 96 wells of a microplate knows what a chore that is and can surely appreciate the labor-saving benefits of an automated microtiter plate strip washer. Curious about the attributes of the latest crop of strip washers, LabConsumer invited four manufacturers to submit their instruments for a hands-on review. All the strip washers arrived with an 8 channel manifold for washing one 8 well strip at time, although 12 and 16 (2 by 8) channel manifolds were available as accessories. Three use syringe pumps to dispense buffers; the fourth, the Stat Fax 2600 from Awareness Technologies, employs a pressurized liquid dispensing system. Two or 2.5 liters are the standard volumes for waste, wash, and rinse reservoirs. Tri-Continent's MultiWash PLUS and the Stat Fax 2600 feature waste bottle fill-level monitoring systems, but the Stat Fax 2600 was the only instrument tested that also incorporates a standard empty warning mechanism for its wash and rinse reservoirs. The ballet of plate and manifold movements comes in two choreographed modes. Tri-Continent's machine holds its plate stationary while moving the manifold across the wells. The others raise and lower the manifold while the plate travels under it on a moving table. The moving table also confers plate-shaking capabilities to the Bio-Tek ELx50 and the Tecan Columbus Washer. All four have an automatic manifold rinsing program to prevent evaporation and crystallization of buffer salts in the manifold needles. The Bio-Tek and Awareness machines include a standard aerosol shield, while Tri-Continent's is an optional accessory. Tecan's Columbus Washer has an optional manifold with a positive aerosol protection system. With the exception of the Tri-Continent instrument, all offer crosswise aspiration, or the ability to aspirate each well twice, once on each side. Software, of course, drives the strip washers through their cycles of dispensing, soaking, aspirating and shaking, and it's the range of adjustable parameters that sets the four washers apart across a spectrum of increasing complexity and versatility. All washers were examined for ease of assembly, programming capabilities, clarity of the user interface, visibility of the plate during washing, and any unique features. Three tests, as explained below, were also designed to evaluate washer performance. All tests were performed using flat bottom COSTAR 96 Well Cell Culture Clusters. The amount of time required for each washer to wash five plates was determined using a standard protocol. Each washer was set to wash each plate completely using three cycles and dispensing 750µl of distilled water into each well. No soak times were used. For those machines that had a range of values for dispensing and/or aspiration flow rates or times, middle range values were selected. When a plate was completed, it was removed and replaced before the next program began. Each washer was programmed to dispense 200µl of a 1:2000 dilution of Schilling red food coloring in Tris-buffered saline, pH 7.6, with 0.05% Tween 20 (TBST) into all 96 wells of a plate. The optical density of the filled wells was then measured on a Molecular Devices THERMOmax microplate reader at 490 nm and compared to a standard curve consisting of a range of volumes from 150 to 250µl of the dye solution. A new standard curve was prepared for each plate; all were linear and had correlation coefficients between 0.997 and 1.00. The average of the volumes dispensed into all 96 wells was determined. The plates were also examined for gradients and other signs of uneven distribution. Aspiration Test One hundred microliters of a 1:200 dilution of Schilling red food coloring in TBST were pipetted with a Rainin EDP-Plus M8 eight channel pipettor into 11 strips on a plate. The 12th strip contained 100µl per well of TBST for use as a blank. Next, the food coloring solution was aspirated away by each plate washer with no further rinsing performed. The aspirated wells were then filled with 100µl of TBST, mixed, and then measured for absorbance at 490 nm. The average value across the plate was compared to a standard curve of 0.5 to 20µl of dye diluted into 100µl of TBST, and the average residual volume in microliters was calculated. Also, the separate values over the whole plate were examined for evidence of gradients or uneven aspiration. Both single and crosswise aspirations were tested. Here's what LabConsumer found: Tri-Continent Multiwash PLUS Tri-Continent MultiWash PLUS Tri-Continent's MultiWash PLUS is an easy-to-use and uncomplicated little plate washer. Its user friendliness is obvious as soon as the shipping box is opened, revealing the 11 x 16 inch quick setup instructions. Assembly is aided by color-coded bottle labels and tubing connectors, and it does not take long before the MultiWash PLUS is up and running. Programming the Multiwash PLUS is easy and straight forward. All the necessary parameters are accessed on the front of the instrument by scrolling through them with arrow keys, and then set to the required value by pushing "+" and "-" buttons. A single button then starts the program, and a beep signals its completion. Also, a single push of the prime button purges the lines quickly and easily. The simplicity of the Multiwash PLUS comes at the expense of some features, like direct access to aspirate and dispense programs, but these functions are available through a series of key strokes. Although the Multiwash PLUS operates at a slower speed, its ease of use and user friendliness made it a favorite. Awareness Stat Fax 2600 Awareness Technology, Inc. Stat Fax 2600 Microplate Washer After fumbling with the manifold height positioning adjustments on the other instruments in the review, it was a pleasure to discover the Stat Fax 2600's automatic well depth detection feature. This washer automatically determines the depth of the plate's wells at the beginning of a program and retains that information to optimize aspirations. Manual adjustments are also possible. Assembly of the Stat Fax 2600 is aided by color coded tubing and connectors and a clear diagram on the instrument's back panel. The keypad provides immediate access to all the separate direct operations, such as dispense, aspirate, rinse, and prime, with the press of the appropriate button. This simplifies operation and was a well-liked feature, as were the dynamic graphics of the display. Compared to the other instruments, this display shows the most amount of information during a run, including the cycle number and the status of the strip being washed. Programming involves selecting the direct operations in the desired order and then simply storing them as a numbered program. Other than allowing the choice of the dispense volume or single or double aspirations, however, the separate operations are largely preset. After compensating for the Stat Fax 2600's maximum dispense volume of 350µl, the lowest in the review, it still had the fastest performance in the time test. Tecan, U.S. Inc. Columbus Washer Tecan, U.S., Inc. Columbus Washer The Columbus Washer moves versatility and complexity of operation up a few notches with many choices offered in its operating parameters. For example, three washing modes, normal, overflow, and bottom, are presented, along with three plate shaking modes and nine shaking speeds. Dispensing rates, from drops to a heavy stream, aspiration rates and speed (the speed at which the needles move into the wells), and specific strip selection are also available. The range of choices to select from complicates programming, but once one becomes accustomed to the programming paths, the Columbus Washer is not difficult to operate. The most striking aspect of the Columbus Washer is the number of available optional accessories. It's fill verification option is particularly noteworthy because none of the other instruments reviewed has any system to verify that wells are being filled. After running one machine with an empty wash reservoir, the value of this feature became self-evident. Other options include a bar code pen, a liquid sensor system, and a manifold detection device. Altogether, the options give the Columbus Washer plenty of adaptability. Bio-Tek Instruments, Inc. ELx50 Automted Strip Washer Bio-Tek Instruments, Inc. ELx50 Automated Strip Washer Bio-Tek has designed the most versatile, and hence the most complicated, strip washer examined in this review. It took some time to grasp the programming paths and to understand the nature of all the available parameter choices, but once that was done, the value of the ELx50's versatility became apparent. This instrument allows the user to optimize programs for all types of applications, from gently washing cells to vigorously washing sticky hepatitis ELISAs. When a program is optimized, Bio-Tek's Download software enables Bio-Tek to then program it directly into the ELx50 for ease of repeated use, such as in clinical settings. After they were understood and optimized, the ELx50's aspiration parameters produced the best aspiration test results in the review. The most difficulty was encountered with the aspiration height adjustments because of reduced visibility of the plate under the manifold. Also, entering numbers into the ELx50 required some persistence. With all things considered, if versatility and lots of parameters to adjust for optimization are of interest, then the ELx50 is the machine of choice. Ease of use comes at the expense of versatility. It's up to the readers to decide what best suits their particular needs and then to find an instrument to satisfy those. We hope our experiences will help. Due to the proverbial circumstances beyond our control, testing was not completed on several plate washers. We regret this and include table information on these instruments: Bio-Rad Model 1575, Immunowash Microplate Washer, Dynex Multi-Reagent Washer, EG &G Wallac Columbus Washer and Lab Systems Aquarius. Ain't got no money? Prefer something simple? Then check out the Elcawash Plate Washer from Elcatech, Inc. Designed to wash inverted plates like a 96 well cuvette washer, the Elcawash relies on gravity to feed buffers from elevated carboys or attaches directly to tap or distilled water lines. A two-way, stainless steel valve is the only moving part, so there's no need to worry about programming, manifold positions, or even where to plug in the Elcawash. Hand-crafted by Dr. George Doellgast, Elcatech's president, the Elcawash sells for $1495, and has a 30-day money back guarantee. Call Elcatech at (800) 544-8613 for more information. Unlike the other instruments, the Stat Fax 2600 dispensed only 350µl per well, instead of 750µl The Columbus washer dispensed more along outer rows than inner rows. The Columbus washer aspirated inner rows better than outer rows. Crosswise aspiration test performed without Bio-Tek's crosswise manifold. The exact volume dispensed by the strip washers is not critical because the wells are filled and emptied several times during a washing program. Aspiration is more important. Our tests showed that 2 to 3 washes were sufficient to remove all traces of the dye from the plates (data not shown). Researchers interested in performing their own tests are urged to contact the manufacturers for specific instructions. Please note that these tests were designed to provide insight into the operation of the strip washers and to generate data for comparison. The data presented are not meant to be taken as evidence for the best or worst possible performance of the instruments, nor are they meant to contradict the manufacturers' performance claims. The author thanks the Hitachi Chemical Research Center in Irvine, California for providing the laboratory space and supplies necessary to complete this review. The author also thanks Bio-Tek, Awareness Technology, Tecan, and Tri-Continent for sending reps and offering advise on the protocols. Michael Brush can be contacted at mdbrush@compuserve.com. * Testing and write-ups were provided to LabConsumer by an independent scientist and author.

Dispensing Aspirations

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