Current Protocols in Microbiology最新文献

Cover: Figure related to Ball and Geddes-McAlister (https://doi.org/10.1002/cpmc.94) Cryptococcus neoformans H99 wild-type cells stained with Indian ink. Image by differential interference contrast microscopy.

Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation of routine growth conditions and media, for storage of the bacteria, and for monitoring of the presence of the virulence plasmid. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Growth of S. flexneri from frozen stocks or agar stabs

Basic Protocol 2: Growth of S. flexneri in rich liquid medium

Alternate Protocol 1: Growth of S. flexneri in rich defined medium

Alternate Protocol 2: Growth of S. flexneri in minimal medium

Basic Protocol 3: Storage of S. flexneri in frozen stocks

Alternate Protocol 3: Storage of S. flexneri in agar stabs

Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells

Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay

Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID₅₀) assay

Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay

Basic Protocol 5: Storage and disinfection

Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells

Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells

Alternate Protocol 3: TCID₅₀ detection using fluorescence visualization

Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection

Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay

Support Protocol 3: Calculation of the TCID₅₀ titer by the method of Reed and Muench

Support Protocol 4: Calculation of the antibody volume for the focus-forming assay

Beer would not exist without microbes. During fermentation, yeast cells convert cereal-derived sugars into ethanol and CO₂. Yeast also produces a wide array of aroma compounds that influence beer taste and aroma. The complex interaction between all these aroma compounds results in each beer having its own distinctive palette. This article contains all protocols needed to brew beer in a standard lab environment and focuses on the use of yeast in beer brewing. More specifically, it provides protocols for yeast propagation, brewing calculations and, of course, beer brewing. At the end, we have also included protocols for analyses that can be performed on the resulting brew, with a focus on yeast-derived aroma compounds. © 2019 The Authors.

Cover: Figure related to Thesseling et al. (https://doi.org/10.1002/cpmc.91).

Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal-shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high-throughput PCR analyses of mutants in 96- or 384-well formats in a very short period of time without any concern about sample cross-contamination. This method is currently being used to validate the protein-coding gene and non-coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.

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 to 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. © 2019 The Authors.

Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. © 2019 The Authors.

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono- or poly-genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1-1 and MAT1-2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single-spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.