Current Protocols in Microbiology最新文献

{"title":"Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models","authors":"Marc Y. Fink,&nbsp;Danielle Shapiro,&nbsp;Steven M. Singer","doi":"10.1002/cpmc.102","DOIUrl":"10.1002/cpmc.102","url":null,"abstract":"<p><i>Giardia lamblia</i> is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. <i>Giardia</i> exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. <i>Giardia</i> was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, <i>Giardia</i> is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain <i>Giardia</i> and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: In vitro maintenance and growth of <i>Giardia</i> trophozoites</p><p><b>Basic Protocol 2</b>: In vitro encystation of <i>Giardia</i> cysts</p><p><b>Basic Protocol 3</b>: In vivo infections using <i>Giardia</i> trophozoites</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38028665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Vibrio fischeri: Laboratory Cultivation, Storage, and Common Phenotypic Assays","authors":"David G. Christensen,&nbsp;Karen L. Visick","doi":"10.1002/cpmc.103","DOIUrl":"10.1002/cpmc.103","url":null,"abstract":"<p><i>Vibrio fischeri</i> is a nonpathogenic organism related to pathogenic <i>Vibrio</i> species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of <i>V. fischeri</i>, 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 <i>V. fischeri</i>. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Growth of <i>V. fischeri</i> from frozen stocks</p><p><b>Basic Protocol 2</b>: Growth of <i>V. fischeri</i> in rich, undefined liquid medium</p><p><b>Alternate Protocol 1</b>: Growth of <i>V. fischeri</i> in minimal medium</p><p><b>Basic Protocol 3</b>: Storage of <i>V. fischeri</i> in frozen stocks</p><p><b>Basic Protocol 4</b>: Biofilm assay on solid agar</p><p><b>Alternate Protocol 2</b>: Biofilm assay in shaking liquid culture</p><p><b>Alternate Protocol 3</b>: Biofilm assay in static liquid culture</p><p><b>Basic Protocol 5</b>: Motility assay</p><p><b>Basic Protocol 6</b>: Luminescence assay</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38009387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2","authors":"Emelissa J. Mendoza,&nbsp;Kathy Manguiat,&nbsp;Heidi Wood,&nbsp;Michael Drebot","doi":"10.1002/cpmc.105","DOIUrl":"10.1002/cpmc.105","url":null,"abstract":"<p>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.</p><p><b>Basic Protocol</b>: SARS-CoV-2 plaque assay using a solid double overlay method</p><p><b>Alternate Protocol</b>: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37993304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes","authors":"Samuel S. Porter,&nbsp;Alison A. McBride","doi":"10.1002/cpmc.101","DOIUrl":"10.1002/cpmc.101","url":null,"abstract":"<p>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.</p><p><b>Basic Protocol 1</b>: Transfection, harvest, and isolation of HPV quasiviruses</p><p><b>Alternate Protocol 1</b>: Packaging HPV DNA replicated in 293TT cells</p><p><b>Alternate Protocol 2</b>: Production of higher-purity quasivirus using the “Ripcord” method</p><p><b>Support Protocol 1</b>: Production of HPV minicircles</p><p><b>Support Protocol 2</b>: Production of recircularized HPV genomes</p><p><b>Support Protocol 3</b>: Screening of fractions for viral proteins</p><p><b>Support Protocol 4</b>: Screening of fractions for viral DNA</p><p><b>Support Protocol 5</b>: Measuring viral titer</p><p><b>Support Protocol 6</b>: Quantitation of quasivirions</p><p><b>Basic Protocol 2</b>: Infection of primary human foreskin keratinocytes with quasivirus</p><p><b>Basic Protocol 3</b>: HPV quasivirus transcription assay</p><p><b>Basic Protocol 4</b>: HPV quasivirus replication assay</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.101","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37909694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup","authors":"Daniel Stadlbauer,&nbsp;Fatima Amanat,&nbsp;Veronika Chromikova,&nbsp;Kaijun Jiang,&nbsp;Shirin Strohmeier,&nbsp;Guha Asthagiri Arunkumar,&nbsp;Jessica Tan,&nbsp;Disha Bhavsar,&nbsp;Christina Capuano,&nbsp;Ericka Kirkpatrick,&nbsp;Philip Meade,&nbsp;Ruhi Nichalle Brito,&nbsp;Catherine Teo,&nbsp;Meagan McMahon,&nbsp;Viviana Simon,&nbsp;Florian Krammer","doi":"10.1002/cpmc.100","DOIUrl":"10.1002/cpmc.100","url":null,"abstract":"<p>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.</p><p><b>Basic Protocol 1</b>: Mammalian cell transfection and protein purification</p><p><b>Basic Protocol 2</b>: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37844282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using Direct RNA Nanopore Sequencing to Deconvolute Viral Transcriptomes","authors":"Daniel P. Depledge,&nbsp;Angus C. Wilson","doi":"10.1002/cpmc.99","DOIUrl":"10.1002/cpmc.99","url":null,"abstract":"<p>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 <i>N</i>⁶-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 &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Productive infection of primary fibroblasts with herpes simplex virus</p><p><b>Support Protocol</b>: Cell passage and plating of primary fibroblasts</p><p><b>Basic Protocol 2</b>: Preparation and sequencing of dRNA-seq libraries from virus-infected cells</p><p><b>Basic Protocol 3</b>: Processing, alignment, and analysis of dRNA-seq datasets</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"57 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.99","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37807656","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aseptic Technique","authors":"Tomasz Bykowski,&nbsp;Brian Stevenson","doi":"10.1002/cpmc.98","DOIUrl":"10.1002/cpmc.98","url":null,"abstract":"<p>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 &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"56 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.98","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37719502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Laboratory Maintenance and Growth of Talaromyces marneffei","authors":"Alex Andrianopoulos","doi":"10.1002/cpmc.97","DOIUrl":"10.1002/cpmc.97","url":null,"abstract":"<p><i>Talaromyces marneffei</i> 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, <i>T. marneffei</i> 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, <i>T. marneffei</i> 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 <i>T. marneffei</i> 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 &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Growth of the vegetative hyphal form on solid medium</p><p><b>Alternate Protocol 1</b>: Growth of the vegetative hyphal form in liquid suspension</p><p><b>Basic Protocol 2</b>: Growth of the vegetative yeast form on solid medium</p><p><b>Alternate Protocol 2</b>: Growth of the vegetative yeast form in liquid suspension</p><p><b>Basic Protocol 3</b>: Growth for production of dormant conidia</p><p><b>Support Protocol</b>: Preparation of Miracloth filter tubes</p><p><b>Basic Protocol 4</b>: Growth of <i>Talaromyces marneffei</i> in mammalian macrophages</p><p><b>Basic Protocol 5</b>: Storage of <i>Talaromyces marneffei</i> strains</p><p><b>Alternate Protocol 3</b>: Lyophilization of <i>Talaromyces marneffei</i> strains</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"56 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.97","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37628806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Step-by-Step Pipeline for the Ecological Analysis of Endophytic Fungi using ITS nrDNA Data","authors":"Maripaz Montero-Vargas,&nbsp;Efraín Escudero-Leyva,&nbsp;Stefani Díaz-Valerio,&nbsp;Priscila Chaverri","doi":"10.1002/cpmc.96","DOIUrl":"10.1002/cpmc.96","url":null,"abstract":"<p>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 &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Raw sequencing data processing</p><p><b>Support Protocol</b>: Building a BLAST database</p><p><b>Basic Protocol 2</b>: Obtaining information from databases</p><p><b>Basic Protocol 3</b>: Phylogenetic analysis</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"56 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.96","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37520159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution","authors":"Casey M. Cosetta,&nbsp;Benjamin E. Wolfe","doi":"10.1002/cpmc.95","DOIUrl":"10.1002/cpmc.95","url":null,"abstract":"<p>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 &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Isolation of cheese rind microbial communities</p><p><b>Support Protocol 1</b>: Preparation of plate count agar with milk and salt</p><p><b>Basic Protocol 2</b>: Identification of cheese rind bacterial and fungal isolates using 16S and ITS sequences</p><p><b>Basic Protocol 3</b>: Preparation of experimental glycerol stocks of yeasts and bacteria</p><p><b>Basic Protocol 4</b>: Preparation of experimental glycerol stocks of filamentous fungi</p><p><b>Basic Protocol 5</b>: Reconstruction of cheese rind microbial communities in vitro</p><p><b>Support Protocol 2</b>: Preparation of lyophilized and powdered cheese curd</p><p><b>Support Protocol 3</b>: Preparation of 10% cheese curd agar plates and tubes</p><p><b>Basic Protocol 6</b>: Interaction screens using responding lawns</p><p><b>Support Protocol 4</b>: Preparation of liquid 2% cheese curd</p><p><b>Basic Protocol 7</b>: Experimental evolution</p><p><b>Basic Protocol 8</b>: Measuring community function: pH/acidification</p><p><b>Basic Protocol 9</b>: Measuring community function: Pigment production</p><p><b>Basic Protocol 10</b>: RNA sequencing of cheese rind biofilms</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"56 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.95","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37503528","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}