Computational protocol: Oxygen Relieves the CO2 and Acetate Dependency of Lactobacillus johnsonii NCC 533

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Protocol publication

[…] The Anopore™ method (inoculation, incubation, imaging) was carried out as described before by den Besten et al. . Multiple Anopore™ inorganic membranes (Anodisc™) were placed on MRS-agar plates and four dilutions (102–105) of an overnight MRS culture of L. johnsonii NCC 533 were spotted per slide (to ensure that colonies would be physically separated to allow individual quantification). The plates were incubated at 37°C in Oxoid jars that were filled with a defined gas mixture by vacuuming, followed by replacement with the gas mixture of choice, and repeated 3 times at the start of the experiment as well as after every opening of the jar for sampling. Single Anopore™ slides were removed at different time points and transferred to a microscopic slide with a pronarose layer that contained the cell permeable SYTO9 stain and the cell impermeable propidium iodide stain (baclight live/dead staining, Molecular Probes, Invitrogen). SYTO9 enters all cells but is replaced by propidium iodide whenever the membrane integrity is compromised.For every time point and condition between 20 and 147 microcolonies were randomly selected from one slide and imaged directly without the use of a cover slip or immersion oil. Photographs were taken through both red and green filters with a cooled charge-coupled device camera (Princeton Instruments, SARL, Utrecht, The Netherlands) mounted on an Olympus BX-60 fluorescence microscope. A threshold was applied to create a binary image of the image intensity plots and these were superimposed using ImageJ. An example of such an image is shown in Supplementary materials, in which images of several colonies are combined. The colony size and viability were quantified using the ImageJ. The ImageJ plugin ObjectJ was employed to facilitate colony selection. […]

Pipeline specifications

Software tools ImageJ, ObjectJ
Application Microscopic phenotype analysis
Organisms Lactobacillus johnsonii NCC 533
Chemicals Acetyl Coenzyme A, Oxygen, Pyruvic Acid