Current Protocols in Microbiology最新文献

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of V. fischeri, as well as recipes for useful media, are included. Specifically, this article describes procedures and considerations for growth of this microbe in complex and minimal media. It also describes assays for biofilm formation, motility, and bioluminescence, three commonly assessed phenotypes of V. fischeri. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Growth of V. fischeri from frozen stocks

Basic Protocol 2: Growth of V. fischeri in rich, undefined liquid medium

Alternate Protocol 1: Growth of V. fischeri in minimal medium

Basic Protocol 3: Storage of V. fischeri in frozen stocks

Basic Protocol 4: Biofilm assay on solid agar

Alternate Protocol 2: Biofilm assay in shaking liquid culture

Alternate Protocol 3: Biofilm assay in static liquid culture

Basic Protocol 5: Motility assay

Basic Protocol 6: Luminescence assay

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

This protocol describes the production of human papillomavirus (HPV)–derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government.

Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses

Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells

Alternate Protocol 2: Production of higher-purity quasivirus using the “Ripcord” method

Support Protocol 1: Production of HPV minicircles

Support Protocol 2: Production of recircularized HPV genomes

Support Protocol 3: Screening of fractions for viral proteins

Support Protocol 4: Screening of fractions for viral DNA

Support Protocol 5: Measuring viral titer

Support Protocol 6: Quantitation of quasivirions

Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus

Basic Protocol 3: HPV quasivirus transcription assay

Basic Protocol 4: HPV quasivirus replication assay

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Current Protocols in Microbiology published by Wiley Periodicals LLC.

Basic Protocol 1: Mammalian cell transfection and protein purification

Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2

The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5′ and 3′ ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N⁶-methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus

Support Protocol: Cell passage and plating of primary fibroblasts

Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells

Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets

This article describes common laboratory procedures that can reduce the risk of culture contamination (sepsis), collectively referred as “aseptic technique.” Two major strategies for aseptic work are described: using a Bunsen burner and using a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microorganisms on petri dishes. © 2020 by John Wiley & Sons, Inc.

Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a multicellular, filamentous hyphal form that can differentiate to produce dormant spores called conidia. These conidia are the likely infectious agent. At 37°C, T. marneffei grows as a uninucleate yeast that divides by fission. The yeast cells are the pathogenic form of this fungus. The protocols described here explain how to grow T. marneffei in the two vegetative growth forms in vitro, grow yeast cells inside mammalian macrophages, produce conidial stocks, and store strains both short and long term. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Growth of the vegetative hyphal form on solid medium

Alternate Protocol 1: Growth of the vegetative hyphal form in liquid suspension

Basic Protocol 2: Growth of the vegetative yeast form on solid medium

Alternate Protocol 2: Growth of the vegetative yeast form in liquid suspension

Basic Protocol 3: Growth for production of dormant conidia

Support Protocol: Preparation of Miracloth filter tubes

Basic Protocol 4: Growth of Talaromyces marneffei in mammalian macrophages

Basic Protocol 5: Storage of Talaromyces marneffei strains

Alternate Protocol 3: Lyophilization of Talaromyces marneffei strains

The nuclear ribosomal DNA internal transcribed spacer (ITS) is accepted as the genetic marker or barcode of choice for the identification of fungal samples. Here, we present a protocol to analyze fungal ITS data, from quality preprocessing of raw sequences to identification of operational taxonomic units (OTUs), taxonomic classification, and assignment of functional traits. The pipeline relies on well-established and manually curated data collections, namely the UNITE database and the FUNGuild script. As an example, real ITS data from culturable endophytic fungi were analyzed, providing detailed descriptions for every step, parameter, and downstream analysis, and finishing with a phylogenetic analysis of the sequences and assigned ecological roles. This article constitutes a comprehensive guide for researchers that have little familiarity with bioinformatic analysis of essential steps required in further ecological studies of fungal communities. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Raw sequencing data processing

Support Protocol: Building a BLAST database

Basic Protocol 2: Obtaining information from databases

Basic Protocol 3: Phylogenetic analysis

Cheese rind microbiomes are useful model systems for identifying the mechanisms that control microbiome diversity. Here, we describe the methods we have optimized to first deconstruct in situ cheese rind microbiome diversity and then reconstruct that diversity in laboratory environments to conduct controlled microbiome manipulations. Most cheese rind microbial species, including bacteria, yeasts, and filamentous fungi, can be easily cultured using standard lab media. Colony morphologies of taxa are diverse and can often be used to distinguish taxa at the phylum and sometimes even genus level. Through the use of cheese curd agar medium, thousands of unique community combinations or microbial interactions can be assessed. Transcriptomic experiments and transposon mutagenesis screens can pinpoint mechanisms of interactions between microbial species. Our general approach of creating a tractable synthetic microbial community from cheese can be easily applied to other fermented foods to develop other model microbiomes. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Isolation of cheese rind microbial communities

Support Protocol 1: Preparation of plate count agar with milk and salt

Basic Protocol 2: Identification of cheese rind bacterial and fungal isolates using 16S and ITS sequences

Basic Protocol 3: Preparation of experimental glycerol stocks of yeasts and bacteria

Basic Protocol 4: Preparation of experimental glycerol stocks of filamentous fungi

Basic Protocol 5: Reconstruction of cheese rind microbial communities in vitro

Support Protocol 2: Preparation of lyophilized and powdered cheese curd

Support Protocol 3: Preparation of 10% cheese curd agar plates and tubes

Basic Protocol 6: Interaction screens using responding lawns

Support Protocol 4: Preparation of liquid 2% cheese curd

Basic Protocol 7: Experimental evolution

Basic Protocol 8: Measuring community function: pH/acidification

Basic Protocol 9: Measuring community function: Pigment production

Basic Protocol 10: RNA sequencing of cheese rind biofilms

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.