Overlap and the errors of plaque counting: I. The overlap biases of observed counts and their correction

Howes, D. W.

doi:10.1017/s0022172400041723

Cited by 6 publications

(4 citation statements)

References 12 publications

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“…All tubes were stored upright in the dark at 4°C in between measurements. A correction factor was applied to the observed count of lysis plaques to compensate for overlap bias (Howes, 1969; Howes and Fazekas de St Groth, 1969) (see Data S1).…”

Section: Methodsmentioning

confidence: 99%

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

O’Connell

Roupioz

Marcoux

2022

Journal of Applied Microbiology

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Aims: To measure the infectious titre (IT) decay rate for various bacteriophages as a function of storage container material. Additionally, parallel light scattering and infectious titre measurements reveal distinct mechanisms for IT loss, depending on phage. Methods and Results: Suspensions of bacteriophages 44AHJD, P68 and gh-1 were stored in various labware. IT of each suspension was repeatedly measured over the course of 2 weeks. Large variability in IT decay was observed, with >4 log 10 loss in glass and low-binding polypropylene. Incubation of polymer containers with Bovine Serum Albumin (BSA) resulted in a consistent reduction in IT decay. Aggregation state of phage suspensions was studied by nanoparticle tracking analysis (NTA), revealing highest aggregation in glass-stored suspensions and lowest after storage in BSA-treated containers. Conclusions: Glass and 'low-binding' containers may aggravate IT decay while BSA treatment may present an easy mitigation strategy. IT versus NTA titre diagrams highlight the importance of phage inactivation in combination with aggregation.Significance and impact of the study: Container material is a significant determinant of bacteriophage IT decay. It is therefore essential to confirm IT following storage and tailor choice of phage storage containers accordingly. Aggregation of phages and adsorption onto labware surfaces are not only the mechanisms accounting for IT loss but also biological instability.

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Section: Methodsmentioning

confidence: 99%

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

O’Connell

Roupioz

Marcoux

2022

Journal of Applied Microbiology

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“…Traditional plaque assay is recognized as the golden standard method for measuring viral titers, including for HSV [ 5 ]. However, there are significant limitations of this method, such as (1) long hands-on time during the experiment and the waiting time for the results (usually 3~7 days), (2) cumbersome procedures with a low throughput when titrating multiple clones, and (3) the inherent subjectivity and possible errors during the plaque counting [ 6 ]. 50% cell culture infectious dose (CCID 50 ) is also widely used for virus titration [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Development and Preliminary Application of a Droplet Digital PCR Assay for Quantifying the Oncolytic Herpes Simplex Virus Type 1 in the Clinical-Grade Production

Guo

Deng

Liang

et al. 2023

Viruses

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Oncolytic herpes simplex virus (oHSV) is a type of virus that selectively targets and kills cancer cells, leaving normal cells unharmed. Accurate viral titer is of great importance for the production and application of oHSV products. Droplet digital PCR (ddPCR) is known for having good reproducibility, not requiring a standard curve, not being affected by inhibitors, and being precise even in the detection of low copies. In the present study, we developed a droplet digital PCR assay for the quantification of HSV-1 and applied it in the oHSV production. The established ddPCR showed good specificity, linearity, a low limit of quantification, great reproducibility, and accuracy. The quantification result was well-associated with that of plaque assay and CCID50. Amplification of the purified virus without DNA extraction by ddPCR presented similar results to that from the extracted DNA, confirming the good resistance against PCR inhibitors. With the ddPCR, viral titer could be monitored in real time during the production of oHSV; the optimal harvest time was determined for the best virus yield in each batch. The ddPCR can be used as a useful tool for the quantification of oHSV and greatly facilitate the manufacturing process of oHSV products.

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“…Procedures for correcting overlap bias of plaque counts, described in the preceding paper (Howes, 1969), yield more reliable estimates of the numbers of plaque-forming units actually present in samples. However, to take full advantage of the method, it is also necessary to specify the errors associated with plaque counting.…”

Section: Introductionmentioning

confidence: 99%

Overlap and the errors of plaque counting: II. The bias of the variance and the concealment of errors

Howes

Groth

1969

J. Hyg.

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SUMMARYThe overlapping of plaques compresses their distributions and reduces observed variances. For a given distribution the reduction of the variance is substantially larger than that of the mean. To derive the error of a plaque assay from the assumption that the variance equals the mean may therefore lead to serious over-estimation of the precision of the assay.Procedures for estimating the true error of plaque assays are developed, and their use is illustrated on experimental material.

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Overlap and the errors of plaque counting: I. The overlap biases of observed counts and their correction

Cited by 6 publications

References 12 publications

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

Development and Preliminary Application of a Droplet Digital PCR Assay for Quantifying the Oncolytic Herpes Simplex Virus Type 1 in the Clinical-Grade Production

Overlap and the errors of plaque counting: II. The bias of the variance and the concealment of errors

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