Application Of Mtt Assay And Atp Bioluminescence Assay For Detecting The Total Bacteria On Eggshell Surface

Eggs as an important raw material of dietary,which may be contaminated with varying degrees of micro-organisms in the process of transportation,storage,processing and consumption after output.Eggshell surface microbial will cause some hazards:firstly,the eggshell surface microbial may direct threat to human health;secondly,in the process of food processing,eggshell surface microbial is easy to produce cross-contamination with other foods,which is one of the reason of outbreak food borne illness;finally,the eggshell surface microbial will invade into the egg,causing bacterial spoilage of egg.Therefore,establishing a rapid and accurate method to detect the eggshell surface microbial has a very important significance to control the potentially hazard.This study was aimed at establishing two rapid detection methods to detect the total number of bacteria:MTT assay and ATP bio luminescence assay,and then detect the total number of bacteria on the surface of the egg shell,which providing practical guidance to achieve real-time monitoring of microorganisms in the field of food safety.The main research contents and results are as follows:1.Establish MTT assay for detecting the total number of bacterialSince the formazan crystals generated from MTT reduction reaction do not dissolve in water,it is generally divided MTT colorimetric assay into the MTT reduction reaction stage and the formazan crystals dissolving stage,and the MTT reduction reaction stage is also called the formazan crystals forming stage.In the formazan crystals dissolving stage,optimized the species of dissolving agent and the dissolving time respectively;in the MTT reduction reaction stage,optimized the amount of MTT,pH value and the reaction time respectively,establishing the optimal MTT assay to detect the total number of bacteria.Experimental results showed that:in the formazan crystals dissolving stage,the mixture of alkaline solution and DMSO showed a better dissolve effect,and determined the mixture of 32 mM NaOH and DMSO as the formazan crystals dissolving agent,which was mixed with the volume ratio of 1/9,and determined 20 min as the formazan crystals dissolving time;in the MTT reduction reaction stage,the optimum volume ratio of the amount of MTT(5 g/L)and the sample was 4/100,the optimum pH value of the reaction system was 8,reaction time was 4 h,and proved the dead cells would not affect the results.2.Establish ATP bioluminescence assay for detecting the total number of bacterialIn this study,we studied the effect factors of ATP bioluminescence reaction system,the stability of firefly luciferase and the method of extract ATP from bacterial cell,respectively.The results are as follws:firstly,in the optimization of the ATP bioluminescence reaction system,the best combination of enzyme and substrate concentration was 60 mg/L luciferin and 70 mg/L firefly luciferase,the optimum Mg2+concentration was 18.75 mM,pH value was 7.50,the reaction temperature was 25-30?;secondly,in the study of the stability of the enzyme,determined the firefly luciferase preservation solution was firefly luciferase diluent,which containing 7 g/L BSA,0.75 mM GSH and 160 mM DTT;finally,in the study of the method of extract ATP from bacterial cell,the best extraction agent was 0.2 g/L BAB and found that DEAE-Dx could clear the inhibition of ATP extraction agent for ATP bioluminescence reaction,the optimum concentration was 0.4%.3.The application of MTT assay and ATP bioluminescence assay for detecting the total number of bacterial on eggshell surfaceThe preliminary study of laboratory has shown that the composition of the dominant genus on the eggshell surface was 59.57%staphylococcus,25.62%bacillus and other genera.Based on this,we mixed the Salmonella enterica,Staphylococcus aureus and Bacillus amyloliquefaciens with the concentration ratio of 1:3:6,and regard this as the microbial species model on eggshell surface,establishing the standard curve of MTT assay and ATP bioluminescence assay.The results showed:the absorbance detected by MTT assay and the total number of bacteria of analog broth was positive correlation,and the limit of detection was 5.2×105cfu/ml;the logarithm of luminous intensity detected by ATP bioluminescence assay and the total number of bacteria of analog broth was positive correlation,and the limit of detection was 9.5×104 cfu/ml;there is no significant differences between the results of total number of bacterial on the surface of eggshell detected by MTT assay,ATP bioluminescence assay and plate count method.Therefore,MTT assay and ATP bioluminescence assay can be used to detected the total number of bacterial on the surface of eggshell.