腐败微生物及腐败产物检测推断死亡时间的研究
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摘要
[背景]
死亡时间(Postmortem interval，PMI)推断一直是法医病理学实践和研究的一项重、难点课题，尤其对晚期、腐败的尸体，推断其死亡时间更缺乏客观、准确的方法。以往研究主要关注于死后机体内源性物质随时间延长而降解的过程，因受到检材保存和内源性物质降解时限的影响，研究时间范围多局限于死后72h内，如何拓展PMI推断研究的时间范围，寻找死后更长时间的PMI推断方法，是法医学家面临的新挑战。
死亡后，尸体在微生物的作用下逐渐发生腐败，腐败产物也逐渐在组织中生成并积聚，腐败微生物和腐败产物的量随着死亡时间的延长，而呈现一定的变化规律。借鉴食品卫生学中检测食品中腐败微生物和腐败产物的含量判断食品质量、预测食品货架期的思路，本实验对尸体腐败过程中微生物和腐败产物含量的检测方法及其随时间变化的规律进行了一系列初步研究，探讨利用腐败过程推断PMI的可行性。
[目的]
1．建立利用生物发光法检测尸体组织中微生物ATP含量的方法。
2．检测死后0～10d大鼠尸体组织中微生物ATP的含量，并分析其变化与PMI的关系，建立推断PMI的回归方程。
3．观察尸体腐败过程中腐败细菌的生长情况，并对优势腐败菌种进行鉴定。
4．建立检测尸体组织中腐败产物三甲胺-氮(TMA-N)的实验方法。
5．检测死后0～10d大鼠尸体组织中TMA-N的含量，并分析其变化与PMI的关系，建立推断PMI的回归方程。
[方法]
1．处死健康SD大鼠，于死后不同时间取待检组织样本100mg，加入4℃无菌生理盐水匀浆后，以3000r／min离心5min。取离心后上层清液50μl于检测管内，加入50μl的Triton X-100和三磷酸腺苷双磷酸酶(Apyrase)混和液(其中Triton X-100浓度0．2％，Apyrase浓度0．1％)，37℃下孵育10min以消除组织细胞内ATP，然后于沸水中水浴1min以灭活Apyrase。平衡至室温后，加入100μl Bac Titer-Glo~(TM)检测液，用TD-20／20 Luminometer检测发光值(设置延时2s，检测时间10s，连续测3次，取最高值为结果)。以无菌生理盐水作空白对照，按标准曲线法计算ATP浓度。进行日内检测偏差和日间检测偏差的重复性实验。
2．于死后即刻、1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等11个时间点，按无菌操作要求，各取6只大鼠尸体的肌肉、肝脏、脾脏和肾脏组织，准确称取各组织样本100mg，用生物发光法检测各样本中微生物ATP含量，并进行统计分析。
3．死后1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等10个时间点各取1只大鼠尸体的肌肉、肝脏、脾脏和肾脏组织少量，将样品加无菌生理盐水匀浆后，每份样品以分区划线法接种于2个血液增菌培养基上，分别置于37℃有氧和厌氧环境下培养24h。蘸取长出的菌落进行涂片，革兰氏染色后显微镜下观察。分离纯化优势菌种后，用VITEK-32型全自动微生物分析仪进行菌种鉴定。
4．大鼠尸体组织样本100mg加入4℃10％三氯乙酸匀浆处理后，以3000r／min离心3min，取上层清液1 ml依次加入0．2ml 10％甲醛溶液、2ml无水甲苯、0．6ml 1：1碳酸钾溶液，上下振摇60次，静置后吸去下面水层，加入少许无水硫酸钠脱水。取1ml上层液体加入已有1ml 0．02％苦味酸甲苯溶液的比色皿中，于410nm波长下读取光密度(OD)值，标准曲线法计算TMA-N含量。进行日内、日间重复检测及加样回收率实验。
5．取健康SD大鼠处死，于死后即刻、1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等11个时间点，分别各取6只大鼠尸体的肌肉、肝脏、肾脏组织，用分光光度法检测TMA-N浓度，对所得数据进行统计学处理。
[结果]
1．在10~(-6)mol／L～10~(-12)mol／L 6个数量级范围内，发光读数(RLU)的对数值与ATP浓度([ATP])对数值呈现良好的线性关系，以ATP浓度的对数值(Log([ATP]))为横坐标，以相应荧光读数的对数值(Log(RLU))为纵坐标，一元线性方程为y=0．815x+9．0148，R~2=0．9977，P＜0．001。重复性实验显示，平均日内检测偏差为1．40％；平均日间检测偏差为1．74％。
2．大鼠死后肌肉组织中微生物ATP含量在7d内随时间延长而增加，第8～9d下降，第10d再次升高；肝、脾和肾脏组织中微生物ATP含量在死后8d内随时间延长而增加，8～10d下降。因肝、脾和肾脏组织中微生物ATP含量检测结果无统计学差异性，故合并三个器官的数据为内脏组。PMI在0～9d内与肌肉组织中微生物ATP含量拟合最佳，以PMI为自变量的三次多项式回归方程为：y=0．02x~3-0．166x~2-0．666x+13．412(R~2=0．989，P＜0．01)；在0～10d内与内脏组织中微生物ATP拟合最佳，以PMI为自变量的三次多项式回归方程为：y=0．016x~3-0．127x~2-0．809x+13．324(R~2=0．986，P＜0．01)。
3．大鼠死后3天起，肌肉、肝脏、脾脏和肾脏组织中均有细菌生长，菌种并不单一，可见G~-、G~+杆菌、G~+球菌等，但四种组织匀浆液培养的细菌中始终有一种G~-小型杆菌存在，在有氧和无氧条件下均可生长。死后7～9d，四种组织中先后出现一种G~+的大杆菌，至死后10d，四种组织有氧和厌氧培养均可见。对优势菌种进行菌种分析，鉴定结果为奇异变形杆菌。
4．在1 mg／L～10 mg／L范围内，光密度(OD)值与TMA-N浓度呈现良好线性关系，一元线性方程为y=0．053x+0．001 8(y为OD值，x为TMA-N浓度)，R~2=0．9991，P＜0．001。平均日内检测偏差为2．37％，平均日间检测偏差为3．2％。平均加样回收率为98．4％。
5．肌肉、肝脏和肾脏组织中TMA-N含量随死亡时间延长而增加，肌肉组织中TMA-N含量于死后第7d，肝脏和肾脏组织于死后第8d达到浓度高峰，随后开始下降，于死后第10d再次升高；因肝、肾组织内TMA-N含量无统计学差异性，故合并两个器官的数据进行回归分析；PMI在2～7d内与肌肉中TMA-N浓度变化拟合最优，以PMI为自变量的三次多项式回归方程为：y=-0．457x~3+6．51 9x~2—24．574x+27．207(R~2=0．969)；在3～8d内与肝肾中TMA-N浓度变化拟合最优，以PMI为自变量的三次多项式回归方程为：y=0．509x~3-9．153x~2+55．727x-95．819(R~2=0．953)。
[结论]
1．本实验方法可灵敏、准确、快速的检测尸体组织中的微生物ATP含量，填补了利用生物发光法检测尸体组织内微生物ATP含量方法的空白。
2．尸体组织中微生物ATP含量变化可用于PMI推断，高度腐败或不完整的尸体仍可以取材检测，可大大拓宽PMI推断研究的时间范畴。
3．尸体组织腐败的优势菌种为奇异变形杆菌，死后7～10d组织中出现一种新的G~+大杆菌，其出现时间与微生物ATP、TMA-N含量变化趋势发生波动的时间范围一致。
4．分光光度法检测死后尸体组织中的TMA-N灵敏、准确、稳定、重复性好，可用于进一步检测尸体组织内TMA-N含量变化与PMI推断的研究。
5．腐败产物TMA-N含量可以用于推断PMI，为晚期PMI推断提供了新思路和新指标。
[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~(-12)mol/L to 10~(-6)mol/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=0.02x~3-0.166x~2-0.666x+13.412 (R~2=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=0.016x~3-0.127x~2-0.809x+13.324 (R~2=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 tissuse
4.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 R~2 was y=-0.457x~3 +6.519x~2-24.574x+27.207 (R~2=0.969),whichcorresponding 2-7d range of PMI;for group liver-kidney,the equation with highest R~2was y=0.509x3 - 9.153x~2+55.727x - 95.819 (R~2=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.
死亡时间(Postmortem interval，PMI)推断一直是法医病理学实践和研究的一项重、难点课题，尤其对晚期、腐败的尸体，推断其死亡时间更缺乏客观、准确的方法。以往研究主要关注于死后机体内源性物质随时间延长而降解的过程，因受到检材保存和内源性物质降解时限的影响，研究时间范围多局限于死后72h内，如何拓展PMI推断研究的时间范围，寻找死后更长时间的PMI推断方法，是法医学家面临的新挑战。
死亡后，尸体在微生物的作用下逐渐发生腐败，腐败产物也逐渐在组织中生成并积聚，腐败微生物和腐败产物的量随着死亡时间的延长，而呈现一定的变化规律。借鉴食品卫生学中检测食品中腐败微生物和腐败产物的含量判断食品质量、预测食品货架期的思路，本实验对尸体腐败过程中微生物和腐败产物含量的检测方法及其随时间变化的规律进行了一系列初步研究，探讨利用腐败过程推断PMI的可行性。
[目的]
1．建立利用生物发光法检测尸体组织中微生物ATP含量的方法。
2．检测死后0～10d大鼠尸体组织中微生物ATP的含量，并分析其变化与PMI的关系，建立推断PMI的回归方程。
3．观察尸体腐败过程中腐败细菌的生长情况，并对优势腐败菌种进行鉴定。
4．建立检测尸体组织中腐败产物三甲胺-氮(TMA-N)的实验方法。
5．检测死后0～10d大鼠尸体组织中TMA-N的含量，并分析其变化与PMI的关系，建立推断PMI的回归方程。
[方法]
1．处死健康SD大鼠，于死后不同时间取待检组织样本100mg，加入4℃无菌生理盐水匀浆后，以3000r／min离心5min。取离心后上层清液50μl于检测管内，加入50μl的Triton X-100和三磷酸腺苷双磷酸酶(Apyrase)混和液(其中Triton X-100浓度0．2％，Apyrase浓度0．1％)，37℃下孵育10min以消除组织细胞内ATP，然后于沸水中水浴1min以灭活Apyrase。平衡至室温后，加入100μl Bac Titer-Glo~(TM)检测液，用TD-20／20 Luminometer检测发光值(设置延时2s，检测时间10s，连续测3次，取最高值为结果)。以无菌生理盐水作空白对照，按标准曲线法计算ATP浓度。进行日内检测偏差和日间检测偏差的重复性实验。
2．于死后即刻、1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等11个时间点，按无菌操作要求，各取6只大鼠尸体的肌肉、肝脏、脾脏和肾脏组织，准确称取各组织样本100mg，用生物发光法检测各样本中微生物ATP含量，并进行统计分析。
3．死后1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等10个时间点各取1只大鼠尸体的肌肉、肝脏、脾脏和肾脏组织少量，将样品加无菌生理盐水匀浆后，每份样品以分区划线法接种于2个血液增菌培养基上，分别置于37℃有氧和厌氧环境下培养24h。蘸取长出的菌落进行涂片，革兰氏染色后显微镜下观察。分离纯化优势菌种后，用VITEK-32型全自动微生物分析仪进行菌种鉴定。
4．大鼠尸体组织样本100mg加入4℃10％三氯乙酸匀浆处理后，以3000r／min离心3min，取上层清液1 ml依次加入0．2ml 10％甲醛溶液、2ml无水甲苯、0．6ml 1：1碳酸钾溶液，上下振摇60次，静置后吸去下面水层，加入少许无水硫酸钠脱水。取1ml上层液体加入已有1ml 0．02％苦味酸甲苯溶液的比色皿中，于410nm波长下读取光密度(OD)值，标准曲线法计算TMA-N含量。进行日内、日间重复检测及加样回收率实验。
5．取健康SD大鼠处死，于死后即刻、1d、2d、3d、4d、5d、6d、7d、8d、9d、10d等11个时间点，分别各取6只大鼠尸体的肌肉、肝脏、肾脏组织，用分光光度法检测TMA-N浓度，对所得数据进行统计学处理。
[结果]
1．在10~(-6)mol／L～10~(-12)mol／L 6个数量级范围内，发光读数(RLU)的对数值与ATP浓度([ATP])对数值呈现良好的线性关系，以ATP浓度的对数值(Log([ATP]))为横坐标，以相应荧光读数的对数值(Log(RLU))为纵坐标，一元线性方程为y=0．815x+9．0148，R~2=0．9977，P＜0．001。重复性实验显示，平均日内检测偏差为1．40％；平均日间检测偏差为1．74％。
2．大鼠死后肌肉组织中微生物ATP含量在7d内随时间延长而增加，第8～9d下降，第10d再次升高；肝、脾和肾脏组织中微生物ATP含量在死后8d内随时间延长而增加，8～10d下降。因肝、脾和肾脏组织中微生物ATP含量检测结果无统计学差异性，故合并三个器官的数据为内脏组。PMI在0～9d内与肌肉组织中微生物ATP含量拟合最佳，以PMI为自变量的三次多项式回归方程为：y=0．02x~3-0．166x~2-0．666x+13．412(R~2=0．989，P＜0．01)；在0～10d内与内脏组织中微生物ATP拟合最佳，以PMI为自变量的三次多项式回归方程为：y=0．016x~3-0．127x~2-0．809x+13．324(R~2=0．986，P＜0．01)。
3．大鼠死后3天起，肌肉、肝脏、脾脏和肾脏组织中均有细菌生长，菌种并不单一，可见G~-、G~+杆菌、G~+球菌等，但四种组织匀浆液培养的细菌中始终有一种G~-小型杆菌存在，在有氧和无氧条件下均可生长。死后7～9d，四种组织中先后出现一种G~+的大杆菌，至死后10d，四种组织有氧和厌氧培养均可见。对优势菌种进行菌种分析，鉴定结果为奇异变形杆菌。
4．在1 mg／L～10 mg／L范围内，光密度(OD)值与TMA-N浓度呈现良好线性关系，一元线性方程为y=0．053x+0．001 8(y为OD值，x为TMA-N浓度)，R~2=0．9991，P＜0．001。平均日内检测偏差为2．37％，平均日间检测偏差为3．2％。平均加样回收率为98．4％。
5．肌肉、肝脏和肾脏组织中TMA-N含量随死亡时间延长而增加，肌肉组织中TMA-N含量于死后第7d，肝脏和肾脏组织于死后第8d达到浓度高峰，随后开始下降，于死后第10d再次升高；因肝、肾组织内TMA-N含量无统计学差异性，故合并两个器官的数据进行回归分析；PMI在2～7d内与肌肉中TMA-N浓度变化拟合最优，以PMI为自变量的三次多项式回归方程为：y=-0．457x~3+6．51 9x~2—24．574x+27．207(R~2=0．969)；在3～8d内与肝肾中TMA-N浓度变化拟合最优，以PMI为自变量的三次多项式回归方程为：y=0．509x~3-9．153x~2+55．727x-95．819(R~2=0．953)。
[结论]
1．本实验方法可灵敏、准确、快速的检测尸体组织中的微生物ATP含量，填补了利用生物发光法检测尸体组织内微生物ATP含量方法的空白。
2．尸体组织中微生物ATP含量变化可用于PMI推断，高度腐败或不完整的尸体仍可以取材检测，可大大拓宽PMI推断研究的时间范畴。
3．尸体组织腐败的优势菌种为奇异变形杆菌，死后7～10d组织中出现一种新的G~+大杆菌，其出现时间与微生物ATP、TMA-N含量变化趋势发生波动的时间范围一致。
4．分光光度法检测死后尸体组织中的TMA-N灵敏、准确、稳定、重复性好，可用于进一步检测尸体组织内TMA-N含量变化与PMI推断的研究。
5．腐败产物TMA-N含量可以用于推断PMI，为晚期PMI推断提供了新思路和新指标。
[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~(-12)mol/L to 10~(-6)mol/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=0.02x~3-0.166x~2-0.666x+13.412 (R~2=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=0.016x~3-0.127x~2-0.809x+13.324 (R~2=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 tissuse
4.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 R~2 was y=-0.457x~3 +6.519x~2-24.574x+27.207 (R~2=0.969),whichcorresponding 2-7d range of PMI;for group liver-kidney,the equation with highest R~2was y=0.509x3 - 9.153x~2+55.727x - 95.819 (R~2=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.
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