*3.2.1. Uneven dispensing*

plate, which is weighed immediately after dispensing to prevent evaporation. %bias can be calculated based on the total weight of the dispensed solution (Wtotal) and the number of dis‐

Environmental conditions (e.g. temperature and humidity) have major effects on the relia‐ bility of gravimetric methods, which facilitates evaporation and water uptake for hygro‐ scopic solvents such as dimethyl sulfoxide (DMSO). These factors of variation can be minimized by placing gasketed lids on the microtiter plates immediately following dispense

Absorbance and fluorescence are the most common photometric methods utilized to test the accuracy and precision of the transferred volumes of a liquid handling device. In a study comparing the performance of the two methods on determining the precision in 96-/384-/ 1536-well plates, no significant difference was observed between the 96- and 384-well plates (Petersen and Nguyen 2005). However, to achieve similar results for both fluorescence and absorbance measurements in the 1536-well plate, a centrifugation step was required because of the irregular meniscus shape enhanced by the small well geometry. In another study per‐ formed on liquid handlers with two different mechanisms, absorbance was found to be a more reliable method as long as the pH stability of the dye-buffer solution is maintained

Fluorescence signal is also known to be susceptible to photobleaching, which can be pre‐ vented by shorter excitation times, suitable buffer solutions and adequate concentration of fluorophore (Diaspro et al. 2006; Harris and Mutz 2006). To overcome the problems encoun‐ tered due to signal quenching in DMSO, sulforhodamine 101 was presented as an alterna‐ tive fluorescence dye (Walling 2011). Fluorescein was found to be a suitable probe to use in liquid handling performance quantification as long as the DMSO concentration in the buffer solution does not exceed 1% and the stock solutions are stored in 70-100% DMSO in a dark environment (Harris and Mutz 2006). While photobleaching is not an issue in absorbance, the method is limited by high background levels and lower sensitivity compared to fluores‐ cence (Bradshaw et al. 2007). Based on the physical characteristics of a transferred sample and the material of the consumables, unforeseen interactions may be observed influencing the assay results. Especially, DMSO-containing samples are highly affected by the hydro‐

scopic properties of the solvent, which inflates sample volume (Berg et al. 2001).

**3.2. Considerations for using bulk reagent dispensers: Peristaltic-based devices**

A single screening experiment can be costly, requiring valuable compounds and biological reagents. Routine evaluation of liquid handlers, in particularly prior to each run, is a neces‐ sary mean for preventing disastrous outcomes. Simple procedures can be integrated to iden‐ tify problems in a relatively short period of time, which in many instances, can be easily corrected. Routine analysis should be performed with the actual reagents, because there are

( Wtotal n×ρ )-VT VT

(3)

%bias per well (gravimetric)=100×

pensed wells (n):

182 Drug Discovery

(Taylor et al. 2002).

(Rhode et al. 2004).

Tubings tend to stretch after certain period of use, affecting the intended volume to be deliv‐ ered. When not in use, the cartridges should be placed in the "rest" position. In addition, autoclaving the cassettes should be minimized. Dispensing speed and the height of the tips in relation to the plate have to be optimized for the intended reagent, as viscous solutions could miss the targeted well at low dispensing speed and large spacing between the tips and microtiter plate. When working with cells, uneven dispensing can be reduced by increasing the prime volume, constant mixing/stirring the cell suspension source and minimizing cell clumps. Solutions should be dispensed in the center of the well, and plates have to be centri‐ fuged when dispensing low volumes to force droplets at the walls to the bottom of the well. Cassettes should be calibrated regularly as recommended by the supplier and checked for tip clogging.
