A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage

• Published in: Journal of virological methods 2021-08, Vol.294, p.114177-114177, Article 114177
• Main Authors: Benala, Manikantha, Vaiyapuri, Murugadas, Visnuvinayagam, Sivam, George, Joshy Chalil, Raveendran, Karthika, George, Iris, Mothadaka, Mukteswar Prasad, Badireddy, Madhusudana Rao
• Format: Article
• Language: English
• Subjects: 2-Step microtiter plate assay; Coliphage; Multiplicity of infection; Vibriophage
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2021.114177

•The 2-step microtiter plate assay tests nine MOIs ranging between 0.0001 and 10000 in the 1st step.•Optimum in vitro MOI obtained in the 2nd step helps the user to employ minimum quantity of bacteriophage for phage therapy.•The assay was tested using coliphage-Φ5 active on AMR E.coli and vibriophage-ΦLV6 active on luminescent Vibrio harveyi.•The sequential 2-step microtiter plate assay finds application for optimizing MOI of phages selected for phage therapy. A 2-step microtiter plate assay was developed to simultaneously check wide values of MOIs of bacteriophages, ranging between MOI-0.0001 and MOI-10000 in the first step and optimize the most suitable MOI (lowest quantity of phage) for inhibiting the growth of the target bacteria in the second step. The results of the first step revealed that the effective MOI of coliphage-ɸ5 for controlling the growth of antimicrobial resistant (AMR) E. coli was between 4.36 and 43.6 for E.coli-EC-3; between 38.2 and 382 for E.coli-EC-7 and between 81.5 and 815 for E.coli-EC-11. The optimum MOI of coliphage-ɸ5 determined in the second step was 17.44, 191 and 326 for controlling the growth of E.coli-EC-3; E.coli-EC-7 and E.coli-EC-11, respectively. The effective MOI of vibriophage-ɸLV6 for controlling luminescent Vibrio harveyi in the first step was found to be between 18.3 and 183 and the optimum MOI as determined in the second step was 79. The sequential 2-step microtiter plate method yielded faster optimization of MOI and was economical compared to the conventional flask method. The measurement of OD values at 550 nm and 600 nm showed similar trend and replicate data from 5-wells and 3-wells yielded identical pattern indicating that the measuring absorbance data in 3-replicate wells at either OD550 or OD600 is sufficient to generate quantifiable phage lysis data. The 2-step microtiter plate assay finds application in phage therapy in human health care, agriculture and animal agriculture for determining the optimum MOIs for selected bacteriophages.