Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates
Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates
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DOI：
10.1128/aem.66.6.2641-2646.2000
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发表时间：
2000-06-01
影响因子：
4.4
通讯作者：
Witholt, B
中科院分区：
文献类型：
--
作者：
Duetz, WA;Rüedi, L;Witholt, B
用于异质培养物保藏的小型化生长系统不仅在减少对培养空间和培养基的需求方面具有吸引力，而且还使大量菌株的并行处理更加可行。我们在此报告了深孔微量滴定板中氧转移速率的优化以及复制系统的开发，该系统允许对 96 种冷冻甘油储备培养物进行同时和可重复采样，同时剩余培养物体积保持冷冻状态。氧转移速率源自恶臭假单胞菌的生长曲线和由于亚硫酸盐的钴催化氧化而导致的氧消失速率。最大氧转移速率（38mmol升（-1）h（-1），对应于188h（-1）的传质系数）是在300rpm的轨道振荡、振荡直径5cm和培养物体积0.5ml的条件下测量的。 2.5 厘米的振动直径导致数值降低三倍。这些高氧转移率使得恶臭假单胞菌在葡萄糖矿物质培养基上以高达 1 ml 的培养体积生长期间达到约 9 g（干重）升(-1) 的细胞密度。使用矿物培养基和富培养基对由需氧菌株（主要来自假单胞菌属、红球菌属和产碱菌属）组成的培养物保藏库评估生长和复制系统。通过在微量滴定板顶部使用海绵硅胶和脱脂棉的夹层盖，充分防止了剧烈曝气期间的交叉污染和过度蒸发。
Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable, We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite, Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.