Detection method for acute toxicity of water quality using luminescent bacteria method

The luminescent bacteria method is suitable for acute toxicity testing of water quality, industrial wastewater, contaminated water bodies, and soluble chemical substances under laboratory conditions. Luminescent bacteria assay is a simple and fast biological toxicity detection method. It can not only test the single factor indicators that can be measured by physical and chemical methods, but also quickly and accurately test the comprehensive toxicity indicators of the environment. It has unparalleled characteristics compared to physical and chemical methods and traditional biological monitoring methods. 1. The method summary is based on the significant negative correlation between the relative luminosity of luminescent bacteria and the total concentration of toxic components in water samples; 0.05), thus the relative luminosity of water samples can be measured using a bioluminescent photometer to represent their acute toxicity level. The acute toxicity level of water quality is characterized by selecting an equivalent reference toxic substance mercury chloride concentration (in mg/L) according to the conditions described in 8, or by selecting an ECSO value (half effective concentration - in percentage concentration of the sample solution). 2. Introduction to Luminescent Bacteria Method 2.1 Principle of Luminescent Bacteria Method Luminescent bacteria are a type of non pathogenic Gram negative facultative anaerobic bacteria, with an optimal temperature of 20-30 ℃ and a pH of 6-9. N aCl concentration of 3%. Under normal physiological conditions, they can emit visible fluorescence. The visible fluorescence wavelength is between 450-490nm, which is visible to the naked eye in the dark The luminescent bacteria method utilizes a sensitive photoelectric measurement system to determine the effect of toxins on the luminescent intensity of luminescent bacteria. The toxicity of a toxin can be expressed as EC50, which is the concentration of the toxin when the luminous intensity of luminescent bacteria decreases by 50%. Luminescent bacteria contain luminescent elements such as fluorescein, fluorescent enzyme, and ATP, which produce weak fluorescence through intracellular biochemical reactions under aerobic conditions. When the activity of cells increases and they are in an active state of division, their ATP content is high and their luminescence intensity is enhanced. Under the action of toxins, the cell activity and ATP content of luminescent bacteria decrease, leading to a decrease in their luminous intensity. Experiments have shown that there is a linear negative correlation between the concentration of toxins and the luminous intensity of bacteria. Therefore, the toxicity of toxins can be determined based on the luminous intensity of luminescent bacteria, and the acute toxicity of the environment in which the toxins are located can be characterized by luminosity. This method is a new microbiological monitoring method proposed in the late 1970s, which is unique due to its sensitivity, speed, simplicity, and low cost. 2.2  Method for the determination of luminescent bacteria (1); Fresh luminescent bacteria culture determination method: Inoculate luminescent bacteria in liquid luminescent culture medium, and incubate them under appropriate conditions (about 20 ℃) for 12 hours (logarithmic growth period). Dilute with buffer to appropriate bacterial concentration, then add to the test tube and mix with the test solution for 10-20 minutes. Read the light intensity of the sample tube and control tube (2)   The mixed determination method of luminescent bacteria and algae. Some toxic substances have no direct toxic effect on luminescent bacteria, but have a toxic effect on algae. By utilizing this characteristic, the cultured luminescent bacteria suspension and algae suspension are mixed and added to the test tube to mix with the test solution. After a period of illumination, the changes in the light intensity of the luminescent bacteria are measured. Due to the toxicity of the toxin to algae, it interferes with the photosynthesis of algae, resulting in a decrease in their ability to release oxygen, Due to hypoxia, the luminous ability of luminescent bacteria also decreases randomly. Therefore, the toxicity of toxins can be calculated based on the changes in luminous intensity caused by the amount of oxygen released during photosynthesis (3)   The luminescent bacteria freeze-drying preparation determination method uses a special method to make a dry powder of luminescent bacteria that have been cultivated to the logarithmic growth stage and store it in a refrigerator. When used, the luminescent bacteria are taken out and added to a buffer solution for insulation and equilibrium for 5-10 minutes to restore their original physiological state and luminescent level, and then used for testing. Its characteristic is that the luminescent bacteria used each time have the same physiological state, consistent sensitivity to toxins, easy quantitative determination, good repeatability and comparability of results, Easy to operate and can be stored for a long time, which will be the direction for the development of this technology