Reviewer
Elías Barquero-Calvo
  • Research associate, Universidad Nacional
Research fields
  • Immunology, Microbiology
Bacterial Growth Curve Measurements with a Multimode Microplate Reader
Authors:  Ariel T. Rogers, Kaitlin R. Bullard, Akash C. Dod and Yong Wang, date: 05/05/2022, view: 5615, Q&A: 0

Bacterial studies based on growth curves are common in microbiology and related fields. Compared to the standard photometer and cuvette based protocols, bacterial growth curve measurements with microplate readers provide better temporal resolution, higher efficiency, and are less laborious, while analysis and interpretation of the microplate-based measurements are less straightforward. Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. Here, we describe a detailed protocol for this development and provide the homemade program for the new analysis method.

Determination of Fungal Tolerance Index to Heavy Metals and Heavy Metal Resistance Tests
Author:  Rosa Paulina Calvillo-Medina, date: 11/05/2021, view: 1816, Q&A: 0

Fungal metallo-tolerance has been described in different species and plays an important role in bioremediation of contaminated environments. Metallo-tolerance is mainly documented by microdilution assays and agar well diffusion methods using equipment that can be expensive. The tolerance index can be calculated to determine the efficiency of a fungus to degrade and resist heavy metals. The present protocol is based on analyzing the tolerance index and minimum inhibitory concentration of the metallo-tolerance potential of culturable fungi on solid media. This can be calculated by daily measurements of colony size on agar supplemented with different concentrations of heavy metals. This method is an easy approach to determine fungal heavy metal resistance using simple laboratory equipment without spectroscopy.

Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
Authors:  Johannes Thoma and Björn M. Burmann, date: 12/20/2020, view: 3262, Q&A: 0

Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (2H and 15N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic components in-situ clears the way to reaching an in-depth understanding of the functional and mechanistic details of this unique cellular compartment.

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