Prepare BactoBox® diluent

The conductivity is our contrast agent to distinguish the conductive, salty cytoplasm inside the intact cells from the conductivity in the liquid that surrounds the exterior of the bacteria (the diluent). The conductivity of the sample to be measured must be within a specific range (1,600–2,100 µS/cm) for the electrical detection principle in BactoBox® to work.

It is essential that the diluent

1

Has the right conductivity

2

Must be rid of bacteria and other particles as these will otherwise give false-positive background when doing the actual measurements. We recommend using a diluent consisting of a diluted phosphate buffered saline (PBS) and deionized water such as MilliQ, water for injection (WFI) or similar

NB! We recommend buying the WFI and PBS ready-made. See “Our go–to reagents and consumables” in the last page of this booklet for suggestions on these. PBS and WFI are not expensive to buy ready-made and the risk of errors are reduced substantially.

If you need to remove bacteria and/or particles from your diluent, then it must be sterile-filtered and not autoclaved. Autoclavation will evaporate water and change the conductivity of the prepared diluent, and the process won’t remove particles.

If you’re using freshly opened containers of PBS and sterile water (such as WFI) you don’t have to do any filtration on the day of use. If you use a LAF bench to prepare 10 mL aliquots in sterile 15 mL centrifuge tubes, you can use these as long as there is no growth. It’s always a good idea to do a test run of the presumed sterile diluent on the BactoBox® to confirm the absence of intact bacterial cells. Store solutions at room temperature to avoid fluctuations in conductivity. In most cases, 1:9 PBS is the best choice for a diluent, but there have been other cases, where 1:10 PBS was more appropriate. This will depend on the temperature in your lab as temperature is a big influencing factor on conductivity. A guide for making both diluent solutions is provided in the next section.