In response to our recent postings related to our July focus on Real-time Microbial Viability Assessment and specifically regarding the research developing at Ghent University, we received comment from Dr. Volker R. Stoldt, Member of the BioStruct Research Group at Heinrich Heine University Düsseldorf, where research is also being conducted in this critical area of optimization and worker safety. Dr. Stoldt provides us with additional insight into flow cytometry research and his opinion of the future of real-time assessments:

Beyond the slogan "Rapid Assessment Of The Biostability Of Metalworking Systems Is The Future" the future for consumer and provider of MWF is different. Only a small and decreasing number of admitted biocides (see Biocidal Products Directive 98/8/EC, Product-type 13: Metalworking-fluid preservatives) is available here in Europe. It is obvious that microbial growth inhibition in used MWF will be a problem for the consumer in the future. What we really need are tools to characterize killing of microorganism in used MWF.

The use of flow cytometry with the fluorescent dyes propidium jodid (PI) and Syto 9 (USA Patent No. 5,436,134, 12. July 1993) is not new but a promising tool. Using laboratory strains with planktonic growing cells this method has also some limitations and it has to be considered that:
- PI and Syto 9 bind to the DNA
- fluorescence signals depend on the amount of intracellular DNA
- the intracellular DNA can be influenced by growth conditions and biocides
- we have a long lasting debate about the intermediate cell
- acquisition of hyha-like structures is not possible

We measured the killing of bacteria in MWF by flow cytometry. The binding of biofilm-derived extracellular DNA to the surface of bacteria, the species specific amounts of DNA in the mixed bacterial populations and the growth conditions etc. can cause problems when analyzing MWF.

The future of this method lies in the characterization of intermediate cells, because those cells can be sorted by flow cytometry sorter. Intermediate cell can be a reservoir for persister cells or resistant cells and those cells should be our target in the future to establish new antimicrobial and adjuvant strategies for MWF. For this purpose we need classical and cell biological tools to be successful in the fight against microorganism in our MWF.

Volker R. Stoldt Ph.D. Biological Safety