Chapter Outline: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography with fluorescence in situ hybridization (FISH)

Chapter Outline: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography with fluorescence in situ hybridization (FISH)

Proposed title: Assessment of prokaryotic biological activity, at the single cell level, by combining microautoradiography and fluorescence in situ hybridization (FISH) 

Chapter no.: _________

Author(s):      Cleber C. Ouverney (PhD)

                        Department of Biological Sciences

                        San Jose State University

                        San Jose, CA 95192-0100

                        Phone:             +1(408) 924-4806

Fax:                 +1(408) 924-4840

                        E-mail:             cleber.ouverney@sjsu.edu

Notes:

·       Some materials in this chapter may overlay with those covered in the Molecular Detection of Target Organisms and Sequences as well as chapters covering Physiological Detection of microorganisms.

Proposed topics

1)    A culture-independent method to render function of the 99% microbes in most environmental sites.

2)    A brief overview of in situ detection of biological activity by environmental microbes.

3)    How do MAR-FISH, STARFISH, Micro-FISH methods work to assess microbial metabolic activity in situ at the single cell level.

4)    Advantages to the approach, specifically the ability to simultaneously detect specific microbes using fluorescently labeled oligonucleotide probes and detect the capacity of microbes to uptake specific dissolved nutrient.

5)    What are the pros and contras of such approach?

6)    How do the microautoradiography-FISH methods compare to similar approaches?

7)    What substrates are used to render what type of metabolic activity?

8)    Suggestions on how to increase fluorescence signal to detect slow growing organisms.

9)    Will such laborious methods withstand the era of fast genomic sequencing?

10) Broad implications from studies applying such methods in the understanding of biogeochemical cycling of dissolved organic and inorganic nutrients in aquatic systems.

11) What other discoveries in the microbial world have been made from these techniques?

Chapter Highlights

The following concepts will be conveyed in this chapter:

1. How metabolic activity can be assessed from uncultured microbes in natural microbial communities.

2. Is Autoradiography-FISH technique right for my application? What do I need to know to make an intelligent decision?

3. Can genomic sequence data be tested/validated using this approach? How?

Chapter Outline: Gold-tagged in situ hybridization for whole cell detection in environmental samples

Chapter Outline: Gold-tagged in situ hybridization for whole cell detection in environmental samples

Proposed title: Gold-tagged in situ hybridization for whole cell detection in environmental samples                                                                      

Chapter no.: _________

Authors:         Hannes Schmidt (MS) and Thilo Eickhorst (PhD)

                        University of Bremen

                        Leobener Str., UFT

                        28359 Bremen, Germany

                        Phone:             +49(421) 218-63446

Fax:                 +49(421) 218-9863446

                        E-mail: eickh@uni-bremen.de

Notes:

·      TBD

Proposed chapters

In situ detection of microbes in environmental microbiology

Aim

Methods available and their limitations

-       Detection of single microbial cells

-       Identification and visualization in situ

-       Quantification of absolute and relative abundances

-       Top-to-bottom approach

Need for combinative approaches and higher resolution

-       Correlative in situ detection of microbes and living conditions

-       Surface structure and substrate conditions

-       Combination with high resolution microscopy techniques

-       Quantification and localization on different microscopic scales

NanoGold as a marker for in situ detection of microbes

State of the art

-       Nanogold-labeled substrates

-       Immunological approaches

-       ISH-approaches (Table)

-       Autometallography (Silver/Gold)

Problems/drawbacks

-       Review of previously documented problems for ISH techniques

-       General problems in environmental samples

-       Need for increased specificity and signal intensity

Gold-FISH – microscopy/detection of fluorescent and gold signals

Development of the new Gold-FISH protocol

-       Goal: simultaneous application/deposition of fluorescent and gold markers

-       Signal amplification for both markers

-       Enhancement of nanogold particles for EM applications

Results/Applications so far

-       pure and mixed bacterial cultures

-       soil and sediment

-       plant roots

Technical aspects/hints /troubleshooting for the application of Gold-FISH to environmental samples

-       Cell wall permeabilization

-       Autometallography

-       Surface charge

-       Computer-aided analysis

Potential for gold-based detection techniques in Environmental microbiology

-       Correlative microcopy of Gold-FISH (e.g. FM and SEM-EDS)

-       Combination with nanoSIMS

-       Potential for non-invasive microscopy (e.g. X-ray CT)

Chapter Highlights

The following concepts will be conveyed in this chapter:

1. Novel approach for a simultaneous labeling of microorganisms with a fluorescent dye and nanogold
2. Optimization of specific nanogold deposition by tyramide signal amplification
3. Possible application of different microscopy techniques for the analysis of gold-FISH labeled single cells
4. Microorganism interactions on different surface morphologies and microenvironments in terms of structure and biogeochemical conditions
5. Element-microorganism interactions

Chapter outline: Sampling of Wastewater and Biosolids for Bacteria and Viruses

Proposed title:  Sampling of Wastewater and Biosolids for Bacteria and Viruses

Chapter no.: _________

Author(s):       John Scott Meschke

                        Department of Environmental and Occupational Health Sciences

University of Washington

                        4225 Roosevelt Way NE, suite 2338

                        Seattle, WA 98105-6099

Notes:

·          Material in this chapter may overlap with other planned chapters on Detection of Pathogens in Sludges, etc. (Judy Blackbeard) and Assessing the Efficacy of Wastewater Treatment (Wesley Pipes). 

·          Content covered in this chapter will include microbiological sampling considerations, primary and secondary concentration techniques, and separation/purification processes for target microbes in raw waste water, primary, secondary, and final effluent, and biosolids.

·          Techniques relevant to viruses, bacteria, and eukaryotic microbes will be covered. Application to both pathogenic and non-pathogenic targets will be discussed.

·          Culture and other detection techniques will not be covered in detail (rather they will only be referred to as they related to the upstream sampling, concentration, and purification techniques).

Proposed topics

1)     Introduction: overview of wastewater treatment (purpose, processes, communities involved, etc.), matrix description/characterization (raw wastewater, primary effluent, secondary effluent, final/tertiary effluents, biosolids), etc.

2)     Relevant sampling schemes:  purpose/rationale for sampling, types of sampling schemes and appropriate uses, relevant regulation, qualitative and quantitative approaches, etc.

3)     Methods for Sampling of Viruses

a.      Viral Targets: pathogens and phage

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Sample Collection: sample volumes, sample preservation, time to processing, etc.

                                                  ii.     Concentration Methods:  filtration, precipitation, acid adsorption/elution and centrifugal methods

                                                iii.     Separation/Purification Techniques: phase separation, organic extraction, serial filtration, gradient centrifugation, etc.

c.      Biosolids

                                                    i.     Sample Collection: sample mass, preservation, time to processing, etc.

                                                  ii.     Elution/Extraction Techniques

                                                iii.     Separation/Purification Techniques: phase separation, organic extraction, serial filtration, gradient centrifugation, etc.

d.     Nucleic Acid Purification

4)     Methods for Sampling of Bacteria

a.      Bacterial Targets: indicator bacteria, pathogens, process bacteria (active and nuisance)

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Concentration and Enrichment Methods

                                                  ii.     Separation Methods

c.      Biosolids

                                                    i.     Elution/Enrichment Methods

                                                  ii.     Separation Methods

d.     Nucleic Acid Purification

5)     Methods for Sampling of Eukaryotic Microbes

a.      Eukaryotic Targets: algae, fungi, protozoa, helminth ova

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Concentration Methods

                                                  ii.     Separation Methods

c.      Biosolids

                                                    i.     Elution Methods

                                                  ii.     Separation Methods

d.     Nucleic Acid Purification

6)     Use of Sampling and Recovery Controls

Chapter Highlights

The following concepts will be conveyed in this chapter:

1.  Matrix effects of sampling methods

2.  Impact of target on sampling methods

3.  Sampling for whole cell or culture based detection versus nucleic acid based detection.

Chapter outline: Animal Gut Microbiomes

Animal Gut Microbiomes--Marchesi

Focus on livestock and companion animals

Introduction

Focus and benefits of studying animal gut microbiota:

·     Comparison with other animals and humans – relating to phylogeny and dietary differences

·     Understanding and improving feed conversion and nutrition (diet)

·     Controlling methane production in ruminants

·     Understanding enteric disease (infection > imbalance)

·     Public Health Microbiology (carriage of zoonotic agents)

·     Novel pathogen discovery (bacterial and viral?)

·     Impact of antibiotics, vaccines, etc

·     Sample sizes (number of replicates) can be much larger than human studies

Sampling considerations

·     Faecal samples are readily available but exposure to air means they may not be completely representative of communities in the gut.

·     Individual gut sections and/or luminal contents can be sampled during post-mortem examinations or via surgical fistulations – different regions will harbour different microbial communities and will have different physicochemical conditions.

·     Methods for selective recovery of planktonic and adherent microbial communities from digesta.

Detection of specific microorganisms of interest (e.g. enteric pathogens)

·     Bacteria (e.g. Salmonella, Campylobacter, etc)

·     Viruses and bacteriophage

·     Protozoa

·     Archaea

·     Yeasts and Fungi

Microbial community analysis

·     Nucleic acid extraction methods

·     Metagenome shotgun sequencing (including library construction and screening)

·     Targeted amplicon sequencing (e.g. 16S, Fungal ITS, AMR genes, etc)

·     RNA extraction, enrichment and analysis methods

·     Data analysis and interpretation

Metabolism of the gut microbiome

·     Methanogenesis and other hydrogen sinks in the GI tract

·     Role of hydrogen consuming organisms in the rumen

·     Microbial groups which utilise hydrogen

·     Approaches and methods for characterisation of the different functional groups

·     Biotransformation and biodegradation of phytochemicals (that toxify and detoxify)

·     Significance of microorganisms that metabolise phytochemicals.

Metaproteomics

·     Extraction methods

    Analysis