Department of Plant and Soil Sciences, University of Massachusetts, 01003, Amherst, MA, USA.
Microb Ecol. 1994 Jul;28(1):79-99. doi: 10.1007/BF00170249.
The microbial ecology of karstic ground water is largely unknown. The density, activity, and diversity of bacteria indigenous to subsurface karstic material in Mammoth Cave National Park, Mammoth Cave, Kentucky were studied using minimally disruptive, on-site procedures. Two sites, located 100 m below the surface and consisting of saturated fine to coarse sand in pooled water, were examined. Samples were taken aseptically using modified, sterile 60-cc syringes. Total cell and total respiring cell densities were determined using an acridine orange/p-iodonitrotetrazolium violet (AO/INT) staining procedure. Cells in selected cores were stained with INT and incubated in the cave for 4 h prior to fixing with glutaraldehyde and subsequent transport to the laboratory. Cells were stained with AO in the laboratory. Low- and high-nutrient media were used to determine viable cell counts. Plates were incubated in the cave for 1 day at ambient temperature prior to transportation to the laboratory in an insulated cooler. Viable cell counts ranged from 1.0 × 106 to 8.1 × 106 cells wet g(-1) of sediment. Total direct counts were 3.9 × 106 and 1.4 × 107 cells wet g(-1) for the Olivia's Dome and the Catherine's Dome sites, respectively. Viable cell counts were highly similar to respiring cell counts at both sites. At the Olivia's Dome site, viable cell counts represented 26-31% of the direct cell counts, while 58% of the total cell count were actively respiring. At the Catherine's Dome site, viable cell counts represented 11-58% of the direct counts, while 53% of the cells were actively respiring. A total of 237 strains recovered from low- and high-nutrient media at both Olivia's and Catherine's Domes, and 10 reference strains were examined for 117 morphological, biochemical, and physiological characteristics. Results were coded in a binary fashion and analyzed using numerical taxonomic techniques. Similarity values were calculated using a simple matching coefficient. Fifty-two clusters, ranging in size from 2 to 13 members, were defined at the 80-85% similarity level with the weighted pair-group mathematical average algorithm (WPGMA). The matrix was examined using the Jaccard coefficient and WPGMA clustering to control for distortion due to negative matches and varying group size. Presumptively identified genera include, Arthrobacter, Brevibacterium, Bacillus, Cornyebacterium, Actinomyces, Aureobacterium, Chromobacterium, and Mycobacterium. Pseudomonas spp. were not recovered. Fifty percent of the clustered operational taxonomic units (OTUs) were not identified. Thirty percent of the clustered OTUs were irregular, asporogenous, Gram-positive rods. The bacterial communities varied between sites, and isolation medium had a strong influence on the strains recovered. The bacterial community in the karstic sediments sampled exhibits a high degree of diversity having no dominant strain or strains.
喀斯特地下水的微生物生态学在很大程度上是未知的。肯塔基州猛犸洞穴国家公园地下喀斯特物质中土著细菌的密度、活性和多样性使用最小干扰的现场程序进行了研究。两个地点位于地表以下 100 米处，由充满水的细到粗沙组成，进行了检查。使用改良的无菌 60 毫升注射器无菌采集样本。使用吖啶橙/碘化硝基四唑紫（AO/INT）染色程序测定总细胞和总呼吸细胞密度。用 INT 对选定核心中的细胞进行染色，并在洞穴中孵育 4 小时，然后用戊二醛固定，随后运送到实验室。在实验室中用 AO 对细胞进行染色。使用低营养和高营养培养基来确定活菌计数。将平板在洞穴中孵育 1 天，在环境温度下，然后在隔热冷却器中运输到实验室。活细胞计数范围为 1.0×106 至 8.1×106 个细胞湿克(-1)沉积物。Olivia's Dome 和 Catherine's Dome 站点的总直接计数分别为 3.9×106 和 1.4×107 个细胞湿克(-1)。Olivia's Dome 站点的活菌计数与呼吸细胞计数高度相似。在 Olivia's Dome 站点，活菌计数占直接细胞计数的 26-31%，而总细胞数的 58%为活跃呼吸。在 Catherine's Dome 站点，活菌计数占直接计数的 11-58%，而 53%的细胞为活跃呼吸。从 Olivia's 和 Catherine's 穹顶的低营养和高营养培养基中总共回收了 237 株菌，并对 10 株参考菌株进行了 117 种形态、生化和生理特性的检查。结果以二进制形式编码，并使用数值分类技术进行分析。使用简单匹配系数计算相似性值。使用加权对组数学平均算法（WPGMA）在 80-85%相似性水平上定义了大小从 2 到 13 个成员的 52 个聚类。使用 Jaccard 系数和 WPGMA 聚类检查矩阵，以控制阴性匹配和不同群体大小引起的失真。推定的属包括节杆菌、短杆菌、芽孢杆菌、科恩氏杆菌、放线菌、金杆菌、Chromobacterium 和分枝杆菌。未回收假单胞菌。聚类的操作分类单元（OTU）的 50%未被识别。聚类的 30%OTU 为不规则、无孢子、革兰氏阳性棒状。不同地点的细菌群落不同，分离培养基对回收的菌株有很大影响。采样的喀斯特沉积物中的细菌群落表现出高度的多样性，没有优势菌株或菌株。
