Study Of Detecting Spoilage Microorganism And Spoilage Product For PMI Estimation

[BACKGROUND]Estimation of Postmortem interval (PMI) has always being a crucial and difficult topicfor forensic pathology study and practice.Up to now,no objective and accurate method isoffered for the estimation of PMI,especially for late period after death and decomposedcorpse.The former researches focused on the decomposition of endogenous substances.Dueto the time limit of conservation and existence of these endogenous substances,most of theformer researches were not beyond 72h after death.How to expand the time range of studyand find new method for late PMI estimation is a new challenge in front of forensicpathologists.With the propagation of microorganisms after death,the body shall spoilage graduallyand spoilage products accumulate in tissues.Quantity of microorganisms and spoilageproducts shall change with the time after death.Mirroring the research in food hygiene aboutputrefactive organisms and spoilage products,this research is a pilot study of microorganismsand product in the spoilage of corpse in order to approach its value in PMI estimation.[OBJECTIVES[1.To build a bioluminescent method for the determination of microbial ATP in the tissues ofcorpse.2.Study the relationship between changes of the concentration of microbial ATP in fourtissues of cadaver and PMI during 0～10d since death.The regression equations for PMIestimation were established. 3.To observe the putrefactive bacteria growth during the spoilage of corpse and identify thespecies of the overwhelming strain.4.To build an experimental method for the determination of trimethylamine-nitrogen(TMA-N),a spoilage product,in tissues of corpse.5.Study the relationship between changes of the concentration of TMA-N in three tissues ofcadaver and PMI during 0～10d since death.The regression equations for PMI estimationwere established.[METHODS]1.Healthy SD rats were put to death and 100mg of each the objective tissue was sampled atdifferent time after death.Each sample was given homogenate with 4℃sterilephysiological saline and the homogenate was centrifuged for 5 minutes under 3000r/min.To eliminate the ATP from somatic cells,a 50μl sample from the supernatant wasincubated for 10 min at 37℃with 50μl of a Triton-apyrase solution consisting of 0.1%apyrase and 0.2% Triton X-100.After bath in boiling water for 1min to deactivateApyrase,the samples were cooled to room temperature,and then,100μl ofBacTiter-Glo^TM reagent was added to the sample (100μl),luminescence was measuredfor 10s after 2s delayed by TD-20/20 Luminometer.The highest reading of threecontinuously measures was recorded as the final reading.The repetitive detections werecommitted to calculate the relative standard deviation (RSD) within day and betweendays.2.At 0d,1d,2d,3d,4d,5d,6d,7d,8d,9d and 10d since death,6 rats' corpses were sampled.100mg of muscle,liver,spleen and kidney tissues were removed from each corpse andmicrobial ATP in each sample was detected by bioluminescent method.The data wasstatistical analyzed.3.At 1d,2d,3d,4d,5d,6d,7d,8d,9d and 10d since death,small amounts of muscle,liver,spleen and kidney tissues were removed from rat corpse.Each sample was givenhomogenate with 4℃sterile physiological saline.The homogenate was transferred to twoenrichment medium plats respectively by sectional streak method.After 24h aerobic or anaerobic cultivation under 37℃,the colonies were smeared,Gram stained and observedunder microscope.The overwhelming strain was isolated and identified by VITEK-32Auto Microbic System.4.100mg of tissue sample was given a rapid homogenate with 4℃10 % trichloracetic acidand the homogenate was centrifuged for 5 minutes under 3000r/min.To lml of supernate,0.2ml of 10% formalin,2ml of Anhydro-methylbenzene,0.6ml of 1:1 solution ofcarbonicum kalium was added in order.The solutions were mixed well by shaking up anddown for 60 times.After about 3 minutes standing,the underlayer water was drawn off bypipette and suitable aliquot of natrii sulfas exsiccatus was added for dehydration.1ml ofupper layer liquid of each tube was added to the glass cell,which already has 1ml of0.02% solution of 2,4,6-trinitrophenol in it.The optical densities (OD) of the solutionswere measured at 410nm.The concentration of TMA-N in the sample was calculatedaccording to the calibration graph.5.Healthy SD rats were executed and at 0d,1d,2d,3d,4d,5d,6d,7d,8d,9d and 10d sincedeath,6 rats' corpses were sampled.100mg of muscle,liver,and spleen tissues wereremoved from each corpse.The concentration of TMA-N was detected usingspectrophotometric method and the data was statistical analyzed.[RESULTS]1.When the concentration of ATP was in the range of 10^-12mol/L to 10^-6mol/L,a calibrationgraph was obtained with a positive slope and the equation being y= 0.815x+9.0148 wherey is the logarithm of relative light unit (Log (RLU)) and x is the logarithm of ATPconcentration (Log ([ATP])).The correlation coefficient was 0.9977 (P＜0.001).Theaverage RSD within day was 1.40%,that between days was 1.74 %.2.The concentration of microbial ATP in muscle increased while PMI extended.The peakappeared at the 7th day since death,and at the 10th day,microbial ATP in muscle tissueincreased again.In internal organs,the peaks of microbial ATP were observed atthe 8thday since death and decrease were seen in 8～10d.There was no statistic differencebetween microbial ATP concentration in liver,spleen and kidney.During 0d to 7d sincedeath,there was the best correlation between PMI and microbial ATP in muscle;having PMI as the independent variable,the cubic polynomial regression equation is y=0.02x³-0.166x²-0.666x+13.412 (R²=0.989,P＜0.01) .In internal organs,there was the bestcorrelation between PMI and microbial ATP during 0d to 10d;having PMI as theindependent variable,the cubic polynomial regression equation is y=0.016x³-0.127x²-0.809x+13.324 (R²=0.986,P＜0.01)3.After the third day since death,there were floras developed in muscle,liver,spleen andkidney of rats' corpses,including G^- bacillus,G⁺ bacillus and G⁺ coccus.There was asmall G^- bacillus which was observed in all smears of aerobic or anaerobic cultivations.After 7～9d since death,there was a large G⁺ bacillus observed on the smears ofcultivations of four tissues successively.At the 10th day after death,the large G⁺ bacilluswere observed in aerobic and anaerobic cultivations four tissuse4.When the concentration of TMA-N was in the range of lmg/L to 10 mg/L,a calibrationgraph was obtained with a positive slope and the equation being y = 0.053x + 0.0018where y is the OD and x (mg/L) is the concentration of TMA-N.The correlationcoefficient was 0.9991 (P＜0.001).The average RSD within day was 2.37%,that betweendays was 3.2%,and the average recovery rate was 98.4%.5.The concentration of TMA-N in muscle,liver and kidney increased while PMI extended.The TMA-N peak in muscle appeared at the 7th day since death and that in liver andkidney appeared at the 8th day since death.At the 10th day,TMA-N in all above tissuesincreased once again.Changes of TMA-N in liver and kidney had homoplastic pattern andthe ANOVA showed there was no significant difference between data of liver and kidney.Having PMI as the independent variable,TMA-N concentration in muscle andliver-kidney as dependent variable respectively,the regression analysis was subjected andthe cubic polynomial regression equation was optimal.For group muscle,the equationwith highest R² was y=-0.457x³ +6.519x²-24.574x+27.207 (R²=0.969),whichcorresponding 2-7d range of PMI;for group liver-kidney,the equation with highest R²was y=0.509x3 - 9.153x²+55.727x - 95.819 (R²=0.953),which corresponding 3～8drange of PMI. [CONCLUSIONS]1.The bioluminescent method presented in this study was an initial way to detect themicrobial ATP concentration in postmortem tissues,which proved to be sensitive,accurateand rapid.2.There were high correlations between PMI and microbial ATP concentration inpostmortem tissues of rat.Since slight tissue was needed for the detection and the samplewas not influenced by self-decomposition,the method may broaden the time range of PMIestimation.3.Proteus mirabilis was an overwhelming flora developed in corpse,after 7～9d since death,there was a kind of large G⁺ bacillus observed on the smears of cultivations of postmortemtissues.At the same time,an undulant tendency was seen in both microbial ATP andTMA-N concentration in corpse.4.The spectrophotometric method presented in this study was an initial way to detect theTMA-N concentration in postmortem tissues,which proved to be sensitive,accurate,reliable and repetitive.It could be use in the further research of PMI estimation byTMA-N concentration.5.The concentration of TMA-N,a spoilage product,could be use to estimate PMI.It offereda new parameter and a new way to the study of PMI estimation.