Method Establishing And Application Resrarch Of "Microtitre" Fluctuation Test For Detecting Mutagenicity

With the development of industrialization, the environmental problems resulted from chemical and radiation factors have become increasingly prominent. These factors lead to the mutagenesis effects on organisms and alteration of genetic materials of organisms, causing genetic damage to the organisms and their offspring. Therefore, accurate and rapid evaluation of biological hazards caused by these factors is very important for contemporary environmental assessment. Traditional mutagenicity detection is mostly carried out by Ames test, which is complicated, time-consuming and has some limitations in the practical application. Much improvement is needed.Based on the principle of Ames test, this study proposed the best reaction conditions and their specific application of "microtitre" fluctuation test by improving the culture and analysis methods. "Microtitre" fluctuation test was carried out with liquid medium to observe the medium turbidity and the color changes of pH indicator instead of solid medium used for colony count in traditional Ame test, with the aim to improve the experimental simplicity and sensitivity.Salmonella typhimurium strain TA98 and mutation inducing substance diamino fluorene were first used to determine the optimal reaction conditions of "microtitre" fluctuation test. By analyzing the ratio of the bacterial concentration, S9 concentration and the amount of mutation-inducing substance, 10ul/ml of bacterial concentration and 20ul/ml S9 concentration were determined. Based on this, TA98, TA97, TA100, TA102 were further used as test strains, water samples collected from eight rivers inflowing to the Dianchi Lake in Kunming were used for a confirmatory test. GC-MS analysis was used to detect the complex organic components in water samples collected from the eight rivers and preliminarily reveal the mutagenic effects of polluted water from the biological point of view.Using the above established method, "microtitre" fluctuation test was used to detect the mutagenicity of tobacco smoke condensate (TPM) of the standard cigarette 3R4F. The resultant experimental results were further analyzed and verified by the traditional Ames plate test. Results revealed that "microtitre" fluctuation test using the strains TA98 and TA100 showed a good linear relationship in concentration and even a better result, compared to the traditional Ames test, However, "microtitre" fluctuation test was too sensitive and not stable enough for the TPM analysis of smoke.As whole smoke exposure can reflect the mutagenicity of tobaccothe system of whole smoke exposure was analyzed comprehensively in this study after referring to similar research carried out by other labs. Based on the self-developed devices of whole smoke exposure and the above mentioned "microtitre" fluctuation test, analysis of whole smoke mutagenicity was carried out. Some corresponding technical parameters of whole smoke exposure applicable to "microtitre" fluctuation test were obtained.