研究方法探讨
王世梅,周立祥.提高氧化亚铁硫杆菌和氧化硫硫杆菌平板检出率的方法:双层平板法[J].环境科学学报,2005,25(10):1418-1420
提高氧化亚铁硫杆菌和氧化硫硫杆菌平板检出率的方法:双层平板法
- A renovated approach for increasing colony count efficiency of Thiobacillus ferrooxidans and Thiobacillus thiooxidans: Double-layer Plates
- 基金项目：国家自然科学基金(No.20377023);江苏省自然科学基金重点项目(No.BK2004213);新世纪优秀人才支持计划(No.NCET-04-0405)
- 作者
- 单位
- 王世梅
- 南京农业大学资源与环境科学学院环境工程系, 南京 210095
- 周立祥
- 南京农业大学资源与环境科学学院环境工程系, 南京 210095
- 摘要：用2%的水琼脂作底层平板,上面涂布异养菌(酵母R30)菌液,再倒入分离硫杆菌的固体培养基作上层平板,涂布一定稀释度的含硫杆菌的菌悬液,30℃下倒置培养.结果显示,氧化硫硫杆菌菌落在双层平板上检出的时间比单层平板提早5 d,检出率提高3.6倍,氧化亚铁硫杆菌菌落在双层平板上,检出率提高2.1倍.琼脂经高压灭菌后产生较高浓度的对化能自养菌有毒的水溶性有机物可能是影响单层平板检出率的重要原因,而双层平板下层的异养菌因能消耗上层的水溶性有机物从而可大幅度提高硫杆菌的检出效率.
- Abstract：A double-layer plate approach was developed to increase the colony count efficiency of chemoautotrophic Acidithiobacilli bacteria. 0.1ml of heterotrophic yeast strain Rhodotorula sp. R30 solution was spread onto the bottom layer made of 2% of agar in plate, and then, the mineral-salt medium containing 3% of agar powder for Thiobacillus thiooxidans or 0.4% agarose for Thiobacillus ferrooxidans were poured on it as upper layer. After the plates were cold, 0.1ml of the bacterial suspension containing T. thiooxidans or T. ferrooxidans was spread evenly on the superstratum. The double-layer plate was cultured at 30℃. The colonies of T. thiooxidans appeared on the double plates for five days earlier than on the single plate. Furthermore, the colonies of T. thiooxidans formed on the double-layer plate were 3.6 times as many as that on the single-layer plate. Likewise, for T. ferrooxidans strain, the plating efficiency was enhanced by 2.1 folds. The high concentration of dissolved organic matter, which is toxic to chemoautotrophic Acidithiobacilli bacteria, released from agar with autoclave were responsible for the lower colony count efficiency for single-layer plate. On contrast, the yeast strain lived in underlayer consumed a lot of dissolved organic matter in medium, resulting in a higher colony count efficiency for double-layer plate.
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