Plate Dilution Frequency Assay (PDFA) data for soil samples from for 120 point locations within limestone cedar glades at Stones River National Battlefield near Murfreesboro, Tennessee

This dataset contains data collected within limestone cedar glades at Stones River National Battlefield (STRI) near Murfreesboro, Tennessee. This dataset contains information on soil microbial density (Most Probable Number of heterotrophic, culturable microbes per gram of soil) for soil samples obtained from certain quadrat locations (points) within 12 selected cedar glades. This information was obtained by performing a Plate Dilution Frequency Assay (PDFA) on a series of serial dilutions for each soil sample.For each quadrat location (point), one soil sample was obtained under sterile conditions, using a trowel wiped with methanol and rinsed with distilled water, and was placed into an autoclaved jar with a tight-fitting lid and placed on ice. Soil samples were transported to lab facilities on ice and immediately refrigerated. Within 24 hours after being removed from the field, soil samples were processed for the Plate Dilution Frequency Assay. First, for each soil sample three measurements were taken of gravimetric soil water content using a Mettler Toledo HB43 halogen moisture analyzer (Mettler Toledo, Columbus, OH, USA) and the mean of these three SWC measurements was used to calculate the 10-gram dry-weight equivalent (DWE) for each soil sample. For each soil sample, a 10-gram DWE of fresh soil was added to 90 milliliters of sterile buffer solution in a 125-milliliter plastic bottle to make the first dilution. Bottles were agitated on a wrist-action shaker for 20 minutes, and a 10-milliliter aliquot was taken from each sample using sterilized pipette tips and added to 90 milliliters of sterile buffer solution to make the second dilution. A 1-milliliter aliquot of the second dilution was transferred using a sterile pipette tip to a test tube containing 9.0 milliliters of sterile buffer solution to make the third dilution. The same process was repeated to make the fourth through seventh dilutions, for each soil sample. The six serial dilutions were inoculated onto plastic plates with pre-poured plate-count agar. Agar plates were poured at least 48 hours prior to use, covered, and inspected for contamination prior to use. Starting with the least concentrated dilution and working toward the most concentrated, eight 10-microliter aliquots were used to inoculate eight spots per dilution level onto the agar plates, with spots inoculated directly onto the agar using a sterile pipette tip within circular areas (eight per dilution level) demarcated using a permanent marker on the clear plastic bottom of each agar plate. Plates were immediately covered and incubated at 25 degrees Celcius for seven days (168 hours). After incubation, each plate was visually examined to determine the number of positive responses per dilution level. Any growth of any colony within a circular area was counted as a positive response, regardless of the size or morphology of the colony. Thus, the number of positive responses could range from zero to eight for each dilution level. For each soil sample, the total number of positive responses (the sum of positive responses for the six dilution levels) was compared to a chart derived from Harris and Sommers (1968) to produce an estimate of the most probable number (MPN) of organisms per milliliter of suspension according to Cochran (1950). This same chart also provided 95 percent confidence interval upper and lower bounds on the MPN estimates for each sample. The values contained in the fields of this dataset represent the upper and lower bounds of these 95 percent confidence intervals for MPN. For example, 052512_LB contains the lower bounds of the 95 percent confidence intervals for MPN for soil samples collected on May 25, 2012, and 052512_UB contains the upper bounds of the same confidence intervals. Detailed descriptions of experimental design, field data collection procedures, laboratory procedures, and data analysis are presented in Cartwright (2014).References:Cartwright, J. (2014). Soil ecology of a rock outcrop ecosystem: abiotic stresses, soil respiration, and microbial community profiles in limestone cedar glades. Ph.D. dissertation, Tennessee State University.Cochran, W. (1950). Estimation of Bacterial Densities by Means of the “Most Probable Number.” Biometrics, 6, 105–116.Cofer, M., Walck, J., and Hidayati, S. (2008). Species richness and exotic species invasion in Middle Tennessee cedar glades in relation to abiotic and biotic factors. The Journal of the Torrey Botanical Society, 135(4), 540–553.Harris, R., & Sommers, L. (1968). Plate-dilution frequency technique for assay of microbial ecology. Applied Microbiology, 16(2), 330–334.本数据集采集自田纳西州默弗里斯伯勒附近的斯通河国家战场（Stones River National Battlefield, STRI）内的石灰岩雪松裸地（limestone cedar glades）。本数据集包含从12处选定的雪松裸地的样方点位（采样点）获取的土壤样品的土壤微生物密度信息——即每克土壤中可培养异养微生物的最大概率数（Most Probable Number, MPN）。该信息通过对每份土壤样品的一系列梯度稀释液开展平板稀释频率测定（Plate Dilution Frequency Assay, PDFA）获得。 每份样方点位（采样点）均在无菌条件下采集一份土壤样品：使用经甲醇擦拭、蒸馏水冲洗的抹刀取样，将样品装入带密封盖的高压灭菌罐中并置于冰上。土壤样品全程冰运至实验室后立即冷藏。自野外采样起24小时内，即对土壤样品开展平板稀释频率测定实验。 首先，针对每份土壤样品，使用梅特勒托利多HB43卤素水分分析仪（Mettler Toledo, Columbus, OH, USA）测定3次重量法土壤含水量（gravimetric soil water content, SWC），取三次测定的平均值计算每份样品的10克干重当量（dry-weight equivalent, DWE）。每份样品取10克干重当量的新鲜土壤，加入125毫升塑料瓶中的90毫升无菌缓冲液，制备第一级稀释液。将塑料瓶置于手腕式摇床上振荡20分钟，使用灭菌吸头吸取10毫升悬浮液至含90毫升无菌缓冲液的容器中，制备第二级稀释液。使用无菌吸头吸取1毫升第二级稀释液至含9.0毫升无菌缓冲液的试管中，制备第三级稀释液。重复上述操作，依次制备第四至第七级稀释液，最终每份样品共获得6级梯度稀释液。 将6级梯度稀释液接种至预先配制好的平板计数琼脂（plate-count agar）培养皿中。培养皿需提前至少48小时制备，制备后加盖封存，使用前需检查是否存在污染。从最低浓度稀释液开始，依次向最高浓度稀释液，每份稀释液取8份10微升的悬浮液，接种至培养皿琼脂表面预先用记号笔在塑料底面上圈定的8个圆形区域内，每个圆形区域接种一个点样。接种完成后立即加盖培养皿，置于25摄氏度条件下恒温培养7天（168小时）。 培养结束后，目视检查每个培养皿，统计每个稀释液级别对应的阳性反应点数。只要圆形区域内出现任何菌落生长，无论菌落大小或形态，均记为1次阳性反应。因此每个稀释液级别的阳性反应数范围为0至8。针对每份土壤样品，将6个稀释液级别的阳性反应总次数与Harris和Sommers（1968）推导的对照表进行比对，根据Cochran（1950）的方法计算每毫升悬浮液中微生物的最大概率数（MPN）估计值。该对照表同时给出了每份样品MPN估计值的95%置信区间上下限。本数据集各字段的数值即为上述MPN估计值的95%置信区间上下限。例如，`052512_LB`代表2012年5月25日采集的土壤样品的MPN估计值95%置信区间下限，`052512_UB`则代表同批次样品的95%置信区间上限。 实验设计、野外数据采集流程、实验室操作流程及数据分析的详细说明详见Cartwright（2014）的研究。 参考文献： 1. Cartwright, J. (2014). 岩石露头生态系统的土壤生态学：石灰岩雪松裸地中的非生物胁迫、土壤呼吸及微生物群落特征. 博士学位论文, 田纳西州立大学. 2. Cochran, W. (1950). 基于“最大概率数”估算细菌密度. 生物统计学（Biometrics）, 6, 105–116. 3. Cofer, M., Walck, J., & Hidayati, S. (2008). 田纳西州中部雪松裸地的物种丰富度与外来物种入侵及其与非生物和生物因子的关联. 托里植物学会杂志（The Journal of the Torrey Botanical Society）, 135(4), 540–553. 4. Harris, R., & Sommers, L. (1968). 微生物生态学测定的平板稀释频率技术. 应用微生物学（Applied Microbiology）, 16(2), 330–334.