microplate preparation for actinobacteriophage infection

Uyarlanmış Bir Optik Yoğunluk Testi Kullanarak Aktinobakteriyofaj Enfeksiyonunun Karakterize Edilmesi

Bacteriophages or phages are viruses that infect bacteria. They are the most numerous biological entities on the planet1, and it is generally accepted that bacteriophages influence bacterial evolution and ecosystem processes2,3,4. Several methods exist to describe, measure, and analyze bacteriophage host range8 and infection dynamics5,6, including agar-based methods such as the double-layer agar method7 and optical density-based microplate methods8,9,10,11,12. Each method has its own advantages and disadvantages. Due to their efficiency, plating tests using the double-layer agar method are the &#34;gold standard&#34; for host range assays, but this method is time- and labor-intensive9. Rapid microplate methods, which return results in 24 h or less, give excellent results for fast-growing bacterial hosts such as Escherichia coli9,10,11,12 but are too brief to showcase bacteriophage infection progression in slower-growing bacterial hosts such as actinomycetes7,13,14,15.
An optical density-based microplate assay designed for fast-growing bacteria cannot be run for the multiple days required to characterize infection dynamics in a slow-growing host bacterium without evaporation occurring and giving artificially high bacterial densities. Thus, obtaining comparable high-throughput data on bacteriophage infection dynamics for slow-growing bacterial species requires specialized techniques adapted for these bacteria.
The microplate method presented here reduces evaporation, thus allowing slow-growing bacteria to be co-cultured with a phage for an extended 96 h period and enabling investigations into phage infection dynamics and host range. This method also showcases the Stacy-Ceballos index16, an optical density-based metric that allows for virulence comparisons among disparate host-virus systems.

Yazar Spotlight: Aktinobakteriyofaj Enfeksiyonunu Karakterize Etmek İçin Uyarlanmış Bir Optik Yoğunluğa Dayalı Mikroplaka Testi  

Aktinobakteriyofaj enfeksiyonunu karakterize etmek için uyarlanmış optik yoğunluk tabanlı mikroplaka testi

Watch this Scientific Journal Video about Microplate Preparation for Actinobacteriophage Infection at JoVE.com

Microplate Preparation for Actinobacteriophage Infection  

Aktinobakteriyofaj enfeksiyonu için mikroplaka hazırlığı

Başlamak için, steril bir 96 kuyucuklu mikroplaka kapağının iç yüzeyine altı mililitre kapak kaplama çözeltisi ekleyin ve kenarlarından tutun. Çözümün tam olarak kapsandığından emin olmak için kapağı döndürün. Çözeltiyi dökmeden önce 20 saniye bekletin.Kapağı otoklavlanmış bir kağıt havlunun üzerine bir açıyla ters çevirin ve 35 ila 45 dakika kurumasını bekleyin. Erimiş Agarose'u 50 ila 60 santigrat derece arasında bir sıcaklığa soğutun. Daha sonra,% 0.1 Agaros çözeltisinin 100 mikrolitresini plakanın kuyucukları arasındaki boşluklara pipetin.Daha sonra 200 mikrolitre Agaros pipetini A satırı, H satırı, birinci sütun, ikinci sütun, 11 ve 12 numaralı sütunda bulunan kuyucuklara pipetin.

microplate-preparation-for-actinobacteriophage-infection

Research

JoVE Journal

Biology

Microplate Preparation for Actinobacteriophage Infection

256 Görüntüleme. Başlamak için, steril bir 96 kuyucuklu mikroplaka kapağının iç yüzeyine altı mililitre kapak kaplama çözeltisi ekleyin ve kenarlarından tutun. Çözümün tam olarak kapsandığından emin olmak için kapağı döndürün. Çözeltiyi dökmeden önce 20 saniye bekletin.Kapağı otoklavlanmış bir kağıt havlunun üzerine bir açıyla ters çevirin ve 35 ila 45 dakika kurumasını bekleyin. Erimiş Agarose'u 50 ila 60 santigrat derece arasında bir sıcaklığa soğutun. Da...

Video: Microplate Preparation for Actinobacteriophage Infection  

An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.

An optical density-based microplate method is described to quantify long-term bacterial growth in the presence of bacteriophages, suitable for actinomycetes and other slow-growing bacteria. This method includes modifications to reduce evaporation and lid condensation and R code to calculate virus infection metrics, including the area under the curve, growth maximum, and relative virulence.

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...