Clinical Microbiology Laboratory, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
J Clin Microbiol. 2024 Aug 14;62(8):e0074324. doi: 10.1128/jcm.00743-24. Epub 2024 Jul 29.
Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five . eight . and three . host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 10-8.3 x10 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 logPFU/mL, respectively, for all phage/bacterial species (ANOVA = 0.1-0.43), and they were highly correlated (r > 0.77, < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for phages and 6-h incubation for and phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.
双层琼脂（DLA）覆盖平板法是噬菌体计数的金标准。然而，它繁琐且耗时。鉴于人们对噬菌体治疗的浓厚兴趣，我们探索了替代噬菌体定量检测方法。总共定量了 16 种属于肌尾噬菌体科、长尾噬菌体科和短尾噬菌体科的噬菌体，使用了 5 种、8 种和 3 种宿主分离物。噬菌体通过标准 DLA 测定法（在 LB 硬琼脂 1.5%上的 LB 软琼脂 0.7%，10 毫升）和新的单层琼脂（SLA）测定法（在 LB 软琼脂 0.7%，10 毫升）进行定量，噬菌体铺展（spread）到软琼脂中或点样（spot）到软琼脂上。每种测定法的噬菌体浓度与标准测定法相关，并且计算了每种测定法与标准双层琼脂铺展之间的相对和绝对差异。用标准 DLA 测定法定量浓度为 1×10-8.3×10 PFU/mL 的噬菌体，可以用 SLA-spread、SLA-spot 和 DLA-spot 测定法进行定量，对于所有噬菌体/细菌种类，中位数（范围）相对和绝对差异均<10%和<0.98 logPFU/mL（ANOVA = 0.1-0.43），且相关性较高（r>0.77，<0.01）。此外，对于 噬菌体，可以在 37°C 孵育 4 小时后，对于 和 噬菌体，可以在 6 小时后进行噬菌斑定量，并且在 24 小时内估计浓度保持不变。与 DLA 测定法相比，SLA-spot 测定法需要的培养基更少，速度快 10 倍，并且可以当天得出结果。SLA-spot 测定法更便宜、更快、更容易操作，并且可以产生与标准 DLA-spread 测定法相似的噬菌体浓度。
