A technique to assess the ability of distinct Candida strains to efflux substrates, as well as to compare the effectiveness of efflux inhibitors, is important for analysis of antifungal drug resistance mechanisms and the mode of action of antifungals. We describe a method that measures the ability of Candida species to extrude the fluorescent dye Nile red as an output for efflux activity. This involves exposing cells to Nile red and using flow cytometry to quantify cellular fluorescence, enabling numerous samples to be processed in a limited time frame. This protocol provides a simple, yet effective method for quantifying efflux in drug-resistant Candida species. © 2020 Wiley Periodicals LLC Basic Protocol 1: Growth and sample preparation of stained Candida Basic Protocol 2: Quantitative measurement of fluorescence by flow cytometry Alternate Protocol: Qualitative determination of fluorescence using microscopy.

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has been identified as the causal agent of COronaVIrus Disease‐19 (COVID‐19), an atypical pneumonia‐like syndrome that emerged in December 2019. While SARS‐CoV‐2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS‐CoV‐2 can be achieved by tissue culture infectious dose−50 (TCID50), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS‐CoV‐2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication‐competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS‐CoV‐2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS‐CoV‐2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS‐CoV‐2 plaque assay using a solid double overlay method Alternate Protocol: SARS‐CoV‐2 plaque assay using a liquid overlay and fixation‐staining method

Figure related to Ball and Geddes

Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10-20 Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative stain method.

Cover: In Frazer et al. (https://doi.org/10.1002/cpmc.76), Alternative media for white-opaque switching assays. (A) On YPD + phloxine B opaque cells take up the dye and show pink colonies after incubation for 7 days at room temperature. Scale bar, 2 mm. (B) White and opaque colony phenotypes on CHROMagar Candida medium. Scale bar, 1 mm. W, white; O, opaque.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter has a significant impact on human health. In the developed world, most campylobacteriosis cases are attributed to the consumption of undercooked, contaminated poultry; however, it has been shown that Campylobacter can be transmitted to humans through contaminated water and other types of food, including beef and milk. As such, high-resolution microbial source-tracking is essential for health department officials to determine the source(s) of Campylobacter outbreaks. For these reasons, this protocol provides the techniques needed for isolation of Campylobacter from agricultural and environmental sources, as well as human clinical specimens. Additionally, we describe a simple method for preparing high-quality genomic DNA that can be used for whole-genome sequencing and downstream bioinformatics analyses of Campylobacter genotypes. © 2018 by John Wiley & Sons, Inc.

Phylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies. In this unit, we offer one specific method to construct a Maximum Likelihood phylogenetic tree. We outline how to identify homologous sequences and construct a multiple sequence alignment. Following alignment, sequences are screened for potentially confounding factors such as recombination and genetic saturation. Finally, a Maximum Likelihood phylogenetic tree can be constructed implementing a rigorously tested model of evolution. The workflow outlined in this unit provides sufficient background for inferring a robust phylogenetic tree starting from a particular gene of interest. © 2018 by John Wiley & Sons, Inc.

Coxiella burnetii is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C. burnetii. In this unit, the media currently utilized for axenic culture of C. burnetii are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for C. burnetii axenic growth under control conditions are provided as a reference. © 2018 by John Wiley & Sons, Inc.