Compounds are typically diluted in complete K-medium or 1% DMSO, depending on aqueous solubility. A number of other vehicles have also been evaluated and may be useful for solubilizing certain chemicals including ethanol (≤ 0.1%), cyclodextrins, and PEG-60.
For chemicals that alter the pH of the exposure solution, an alternative buffer should be used in place of complete K-medium. For chemicals that increase the pH above 8.5, M9 buffer is substituted for complete K-medium. In cases for which the pH decreases below 4.5, complete K-medium should be buffered with 1N KOH up to a pH of 5.5.
One limitation of the COPAS Biosort REFLX tool is carryover, the cross-contamination of a sample well with nematodes from previously sampled sample well(s) or treatment group(s). As nematodes are aspirated from the wells, they are trapped against a filter within the Biosort and then washed into the flow cell. Carryover nematodes may be stuck to the aspirating tool, bubbling filter, or tubing, especially if compounds affect the viscosity of the exposure solution. To minimize carryover, rinse wells containing 1% DMSO are placed between samples of different treatment groups.
A number of conditions may affect COPAS Biosort sampling efficiency or data quality including clogging of the flow cell, sample tubing, or aspirating tool; leaking of the waste tubing; disrupted flow due to pressure changes; and excessive sampling due to air bubbles, waste, or chemical precipitates (see Note 4.5). If the Biosort is observed to be clogging or leaking, the run should be aborted and the machine cleaned and primed. If clogging or noise is not observed until after sampling is complete, in some cases, the data may be ‘cleaned’ using mathematical modeling.
A certain level of noise is normal in COPAS Biosort data. This routine noise may include dead bacteria, shed cuticles from molting (especially if development is delayed due to chemical exposure), dead nematodes, and air bubbles in the system. Routine noise may be modeled and removed as described in Section 3.3.