| Coliform,as an important microorganism index for evaluating various foods,water quality and medicines,can be used not only for judging whether the samples are polluted by feces,but also for indicating bacteria contaminated by intestinal pathogens.Detection of coliform has important microbiological significance.At present,the traditional multiple-tube fermentation method is still used to detect the coliform,and there are some shortcomings,such as tedious operation steps,long time consuming and so on.In recent years,the rapid detection technology of coliform on specific enzyme technology has many advantages,such as high specificity,high sensitivity and fast detection.It has been widely recognized and applied.Although the rapid detection technology of coliform on the specific enzyme technology has been greatly improved than the traditional method,so far,the detection results are basically qualitatively judged by visual inspection,and then the number of coliform has been obtained according to the MPN method.The result of this method is easy to misjudge and influence the result of the experiment.In addition,the accuracy of the results obtained by the MPN method is not high,and its multi tube determination also increases the workload of the detection and the use of the reagents.Based on this,a single tube quantitative and rapid detection technology for coliform based on specific enzyme technology is studied in this paper.Combined with the self-made color medium and fast detection instrument,the single reaction tube can be used for quantitative detection of coliform.Tryptone,lactose and sodium chloride were selected as the nitrogen source,carbon source and inorganic salt of the basic culture medium of coliform.The optimal dosage and optimal p H value of tryptone,lactose and sodium chloride were verified by single factor experiment,and the optimal dosage and optimal p H value were further optimized by orthogonal experiment.The appropriate dosage of the chromogenic substrate ONPG was studied,and the suitable concentration of the chromogenic agent was determined.To study the bacteriostasis effect of different kinds of different concentration of bacteriostat on the heterozygous bacteria,select the suitable bacteriostat and add amount.Determination of coliform selective chromogenic medium formula as follows:tryptone 15.0g/L,lactose 4.5g/L,4.5g/L sodium chloride,2.1g/L dipotassium hydrogen phosphate,1.0g/L potassium dihydrogen phosphate,0.2 g/L sodium deoxycholate,0.5g/L ONPG,p H7.0.Through studying the influence of the best detection wavelength,temperature and p H value on the growth of coliform and the absorbance value of culture medium,we determined the most suitable physical and chemical conditions for coliform growth and hydrolysis substrate detection.By studying the quantitative relationship between the initial inoculation quantity of coliform and the time needed to reach the 0.2 absorbance,the quantitative rapid detection technique of single tube of coliform was established.According to the established quantitative detection technique of single tube of coliform group and the selection of appropriate light source and related components,a rapid detection instrument for coliform group was developed.The results showed that at the wavelength of 410nm detection,when the initial inoculation concentration of coliform was within the range of7-10~5cfu/m L(g),the initial inoculation number of coliform had a good linear relationship with the time needed to reach 0.2 absorbance(R~2=0.995).When the bacteria concentration is 7-10~5cfu/m L(g),the detection time is only558-275min.The formula y=-0.014x+8.909 is solidified into the software,and the computer can automatically calculate the concentration of coliform according to the time needed for the sample to reach 0.2 absorbance,so as to achieve single tube quantification.The precision between the eight channels of the single tube quantitative detection instrument for coliform is good,and the detection limit is less than 7 for each gram(m L)sample.The detection of food and environmental water samples by using a single tube quantitative and rapid detection instrument of coliform has no significant difference compared with the national standard method(P>0.05),and the relative standard deviation is far lower than the national standard method.Coliform single tube quantitative rapid detection method has the advantages of simple operation,high accuracy,high data accuracy,detection time is far less than the multi tube fermentation method,single reaction tube can realize the quantitative advantage of saving reagent,coliform can replace the traditional multi tube fermentation method,it has a good application prospect... |
