双层琼脂(DLA)覆盖噬斑测定是噬菌体计数的黄金标准。然而,它既麻烦又耗时。鉴于对噬菌体疗法的极大兴趣,我们探索了噬菌体定量的替代方法。共有16种不同的噬菌体属于Myoviridae,硅藻科,并对五种肺炎克雷伯菌进行了定量,八个铜绿假单胞菌,和三个鲍曼不动杆菌宿主分离株。用标准DLA测定(10mL的LB软琼脂0.7%,LB硬琼脂1.5%)和新的单层琼脂(SLA)测定(10mL的LB软琼脂0.7%)对噬菌体进行定量,其中噬菌体铺展(铺展)到软琼脂中或点样(斑点)到软琼脂上。每个测定的噬菌体浓度与标准测定相关,并计算每个测定与标准双层琼脂铺展之间的相对和绝对差异。用标准DLA测定的噬菌体浓度1×104-8.3×1012PFU/mL用SLA扩散定量,SLA点,和DLA斑点测定,中位数(范围)相对和绝对差异分别为<10%和<0.98log10PFU/mL,分别,对于所有噬菌体/细菌物种(方差分析P=0.1-0.43),两者呈高度相关(r>0.77,P<0.01)。此外,肺炎克雷伯菌噬菌体孵育4小时后,铜绿假单胞菌和鲍曼不动杆菌噬菌体孵育6小时后,可以在37°C下定量斑块,估计浓度在24小时内保持不变。与DLA测定相比,SLA斑点测定需要更少的培养基,快了10倍,并产生当天的结果。SLA斑点测定法更便宜,更快,更容易执行,并产生与标准DLA扩散测定相似的噬菌体浓度。
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 K. pneumoniae, eight P. aeruginosa, and three A. baumannii 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 × 104-8.3 x1012 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 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii 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.