Project duration: 2020–2021
Funding: Deutsche Bundesstiftung Umwelt (DBU)
Project webpage: https://www.dbu.de/
The Ames test developed by Ames and Maron in 1971 is one of the most important bacterial tests with regard to mutagenicity. The Ames test is based on the back mutation of histidinauxotrophic bacteria (Salmonella typhimurium) towards histidine prototrophy. The more bacteria that reverse mutate to histidine prototrophy, the more bacteria will grow in a histidine deficient medium. Growth is detected by the detection of visible bacterial colonies on agar plates (Ames agar plate test) or using color indicators in liquid culture in microtiter plates (Ames fluctuation test). Both test methods are standardized by the OECD and ISO, respectively. Within the framework of a DBU project, a new Ames test system was developed in microtiter plates using the µRAMOS technology developed at the Chair of Biochemical Engineering (Ames-RAMOS project, AZ 32654). The RAMOS technology allows defined, well-mixed, non-O2-limited culture conditions and a very precise measurement of the oxygen consumption of the test microorganisms (oxygen transfer rate, OTR) of submerged microbial cultures. Based on the OTR, significant conclusions can be drawn about the metabolic activity and growth of a culture. This has already resulted in an optimized, more resource-efficient and, above all, more informative Ames test in 48-well microtiter plate format (Ames-µRAMOS test). Mutagenicity is determined by the time frame of OTR increase due to growth of back-mutated bacteria. However, the previous project also revealed glaring deficiencies in the original Ames fluctuation assay that were equally applicable to the newly developed Ames µRAMOS assay. In part, these deficiencies have already been corrected in the newly developed Ames µRAMOS test. However, further investigations and optimizations are also required here, just as the already standardized Ames fluctuation test should be optimized with regard to these deficiencies. In order to exploit all the advantages of the Ames µRAMOS test, it must first be transferred from the 48-well prototype format to the 96-well µRAMOS system to be commercialized by Kuhner Shaker GmbH. Only in this way can the Ames µRAMOS test be applied on a broader basis in other laboratories. This will allow increased throughput and resource savings. In addition, the increased reproducibility already achieved in the Ames µRAMOS assay (Ames-RAMOS project) should also be transferred to the Ames fluctuation assay through rational preculture management without histidine carryover into the main culture. By investigating and eliminating the batch differences of the bacterial test strains (Salmonella typhimurium TA 98 and TA 100) already uncovered in the Ames-RAMOS project, both the Ames-µRAMOS test and the already standardized Ames-fluctuation test are to be made even more reproducible. The batches of test strains are to be more closely examined and standardized on the one hand by RAMOS technology (Chair of Bioprocess Engineering, RWTH) and on the other hand by molecular biological identification by means of sequencing (Teaching and Research Area Ecosystem Analysis RWTH or in future by the appointment of Prof. Hollert to the Goethe University Department of Evolutionary Ecology and Environmental Toxicology in Frankfurt via a service at a sequencing facility). New quality control systems for the test strains will be developed.
Both the optimized Ames µRAMOS test and the optimized Ames fluctuation test will subsequently be evaluated with regard to improved reproducibility (GU and SME Hydrotox). The results of the internal validation of the optimized Ames-µRAMOS test at the Chair of Biochemical Engineering will be incorporated into the further commercialization of this test system. Both the optimized Ames µRAMOS test and the process optimization of the Ames fluctuation test are to be implemented in the existing guidelines (ISO and OECD) in the future.
Prof. Dr. Dr. h.c. Henner Hollert
Goethe University Frankfurt
Biologicum, Campus Riedberg
60438 Frankfurt am Main, Germany
Phone: +49 (0)69 798 42171
Fax: +49 (0)69 798 42161