*3.5.1. General considerations*

One of the major concerns with any high throughput microplate handler device is its com‐ patibility with plates of various types and sizes. While most high throughput instruments are designed to accommodate labware with dimensions conforming to ANSI/SBS standards, an ideal plate washer is also able to support flat, v-shaped and round-bottom plates.

Cleaning of the other detachable or fixed plate washer components should also be per‐

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Plate washers serve as an excellent alternative to time consuming manual wash procedures for many applications. Since all the wash parameters should be optimized for each specific application during the assay development stage, a tedious troubleshooting process may be inevitable while setting up wash protocols to meet specific assay needs. Table 1 presents a summary of wash parameters/components and their contributions to the wash performance along with various troubleshooting tips. Different assay types may require distinct consider‐ ations. With biochemical assays, minimizing the background signal and well-to-well varia‐ tions are the most important tasks in the optimization process. Low background signal levels can be achieved by reducing the leftover liquid volume in each well. Decreasing the aspiration height and lowering the aspiration rate can greatly affect the residual volume leading to minimal liquid amounts in the wells. In order to prevent high standard devia‐ tions in the assay readouts, equal residual volumes should be attempted by optimizing the aspiration/dispense heights and rates. Depending on the viscosity of the wash buffer, high aspiration rates or low dispense rates may lead to unequal volumes. Inadequate priming volumes, unadjusted dispense or aspiration heights, clogged tubing, and physical misalign‐ ments between the manifolds and plate carrier should also be avoided to prevent high sig‐ nal variations. The effect of the aspiration height on the final residual volume is presented in Fig. 6 for both 96- and 384-well black plates with clear bottom. The volume of the residual liquid (water) per well was measured with the gravimetric technique at several selected as‐ piration heights on a Biotek EL405 microplate washer, while all the other wash parameters were kept constant. A rising trend is observed in the final volume as the aspiration height is

**Figure 6.** Effects of aspiration height on residual volume. Residual volume was measured in a) 96-well and B) 384-well plates at various aspiration heights. Residual volume was increased as the aspiration height from the bottom of the

formed periodically.

*3.5.4. Troubleshooting*

increased.

well was increased.

Both the vacuum assembly and the bottle setup are also important aspects of the plate wash‐ er. Although most washers operate through changes in vacuum pressure, pump-based vac‐ uum-free and pressure-free systems are also offered.

Plate washers functioning by positive displacement principle are also available, enabling non-contact washing with no residual volume (Rudnicki and Johnston 2009). For assays where more than one wash buffer may need to be used, plate washers with multiple dis‐ pense channels and automatic buffer switching capability are preferred to minimize both operation time and contamination. Examples of other optional features for safe instrument operation include waste liquid level sensors and plate detection sensors to avoid unwanted overflows and jams. For BSL2 or higher level experiments, a washer with aerosol cover should be chosen to prevent spread of the contagious material.
