菌密度对青海弧菌生物毒性测试的影响

Effect of the bacterium suspension density on the acute toxicity tests with the fresh luminescent bacterium Vibrio qinghaiensis sp.-Q67

青海弧菌（Vibrioqinghaiensissp．，Q67）已广泛应用于环境样品的急性生态毒性检测，但新鲜培养的Q67菌液的试验结果重现性较差。为了提高采用新鲜培养Q67菌液进行急性生毒性试验的重现性，采用生物毒性测试微孔板法，以紫外分光光度计600rim处的吸光度值（OD600）表征菌密度，分别利用不同OD600值的菌悬液测定1rZnS04·7H20、CdCl2·2．5H20、氟硅唑、十二烷基苯磺酸钠、三氯异氰尿酸5种不同性质化学物质及焦化废水、油田采出水、电镀废水3种不同类型工业废水对Q67的急性生物毒性。结果表明，发光细菌生物毒性试验不适合采用培养时间作为细菌的培养终点。菌液的菌密度对急性毒性测试结果影响很大。当新鲜培养的Q67菌悬液的OD600值介于1．6～1．8时，水样的EC50值远大于其他阶段，灵敏度最低；部分低浓度的工业污水对OD600位于2．5左右的菌液会产生较大的刺激性作用，毒性感应滞后于菌密度低的Q67菌悬液。当Q67菌悬液的OD600值控制在2．o左右时检测的灵敏度最高。固定菌液的OD600值可获得重现性良好的试验结果，并排除接种量、菌种保存时间对急性毒性测试的影响。

This paper is inclined to identify and evaluate the effects of the bacterium suspension density on the acute toxicity tests with the fresh luminescent bacterium Vibrio qinghaiensis sp.-Q67, which has been widely used for detecting the acute toxicity of the environment. However, for the time being, it is far being satisfactory to use the fresh cultured Q67 bacterium suspension as the test bacterium for re- producibility. It is just for this reason that we have been attempting to use the absorbance of UV spectrophotometer at 600 nm （OD600） for the density of bacterium suspension. Cherishing this goal in mind, we have, firstly, studied the growth curve （OD600-time） of Q67 strains with different storing time lengths. Secondly, we have measured the luminous intensity by using the microplate luminometer for the acute toxicity tests with five different chemical substances, including ZnSO4·7H20, CdC12·2.5H20. In the above said tests, we have also conducted the tests with flusilazole, SDBS and TCCA, along with the oil-field sewage, electroplating sewage and coking sewage on the Q67 in the middle and late growth stage with different kinds of OD600. The growth curve indicates that it is ditficult to use the culturing time to control the growth state of Q67 since the storage time has a great in- fluence on the growth speed of Q67, while the density of bacterium suspension is a good indicator to determine the state of bacteria. In addition, the results of the acute toxicity tests are greatly influenced by the bacteria density, the ECs0 values of all the samples are much higher than in the other growth stages during the ODr00 value between 1.6 - 1.8. Some kinds of the industrial sewage in the low concentra- tion have strong stimulating effects on Q67 during the ODr0o value around 2.5, but they may have a certain-degree inhibition effect on Q67 when the OD600 value is between 1.6 - 2.0. The sensitivity of the luminescent bacterium toxicity test is the highest when the OD600 of Q67 bacterium suspension is about 2.0. Furthermore, a good re- producibility will be obtained while the OD600 of bacterium suspension remains constant, then it would be possible to eliminate the influence of the amount of inoculation and the storage time of bacterium.