Hockett Kevin L, Baltrus David A
School of Plant Sciences, University of Arizona;
School of Plant Sciences, University of Arizona.
J Vis Exp. 2017 Jan 14(119):55064. doi: 10.3791/55064.
The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.
软琼脂覆盖技术最初是在70多年前开发的，已广泛应用于微生物学研究的多个领域，包括与噬菌体和细菌素（蛋白质类抗菌剂）相关的研究。这种方法相对便宜，资源需求 minimal。该技术包括将供体菌株（可能含有一种或多种有毒化合物）的上清液点样到接种了细菌测试菌株（可能对有毒化合物敏感）的凝固软琼脂覆盖层上。我们利用这种技术筛选丁香假单胞菌菌株库中的种内杀伤作用。通过将这种方法与沉淀步骤和靶向基因缺失相结合，可以区分同一菌株产生的多种有毒化合物。使用这种技术通常回收的两种拮抗剂是噬菌体和细菌素。这两种剂可以通过另外两个简单的测试来区分。对含有噬菌体的上清液进行连续稀释会导致单个噬菌斑数量随着稀释倍数的增加而减少，而对含有细菌素的上清液进行连续稀释会导致澄清区随着稀释倍数的增加而变得均匀更浑浊。此外，当将噬菌体点样到接种了相同菌株的新鲜软琼脂覆盖层上时会产生澄清区，而由于细菌素被稀释，当将细菌素转移到新鲜软琼脂平板上时不会产生澄清区。
