Methods
The Orphan Lab has developed a number of foundational methods that have been used across the field of microbial ecology. From the nanoscale to the ecosystem scale, the lab aspires to push the boundaries of what can be seen and known about microbial life and its biogeochemical effects.
Fractional abundance of 15-N (A), 13-C (B), and 2-H (C) reveals anabolic activity patterns within G. sulfurreducens biofilms
NanoSIMS
The Orphan Lab has pioneered the application of nanoscale secondary ion mass spectrometry (nanoSIMS) to problems in microbial ecology. Using stable isotopes as probes, and using nanoSIMS to quantitatively measure isotopic abundances within intact microbial samples with nanometer resolution, we answer questions such as, "How active are microbes within their native environments? How does the spatial organization of microbes affect their activity? Which nutrients do microbes metabolize and cycle through the ecosystem?" There are currently only about 50 nanoSIMS instruments around the world, one of which is housed here at the Caltech Microanalysis Facility.
Caltech Microanalysis Facility
FISH (Fluorescent in-situ hybridization)
FISH (using fluorescent probes to target the 16S region of microbial genomes) has enabled major advances in the visualization of microbes within environmental samples. Combined with NanoSIMS, FISH allows us to identify not only which microbial cells are active in a given habitat, but also to identify the taxonomic family to which they belong. Our lab is expanding beyond conventional FISH, using seqFISH to target specific mRNA molecules to identify transcriptionally active cells.
Biological Imaging Center
Symbiotic aggregate of archaeal cells (red) surrounded by bacterial cells (blue), identified by FISH.
Visualising nucleic acids (blue; DAPI) and newly synthesized proteins (green; BONCAT) in AOM consortia (scale bar: 10 microns)
BONCAT
Bio-orthogonal non-canonical amino acid tagging (BONCAT) was collaboratively developed in the laboratories of Erin Schuman (Max Planck Institute for Brain Research) and David Tirrell (Caltech); (Dietrich et al. 2006, PNAS) and adapted by our lab to enable rapid and inexpensive fluorescence-based identification of translationally active uncultured microbes (Hatzenpichler et al 2014, Environ. Microbiol and Hatzenpichler et al 2016 PNAS) and newly produced viruses (Pasulka et al 2018 Environ. Microbiol; and Martinez-Hernandez et al. in prep) directly in diverse environmental samples. BONCAT is now used routinely in microbial ecology and geobiology labs globally. Here, a non-canonical methionine surrogate is introduced into environmental samples and incorporated into newly synthesized proteins using the native translational machinery of the cell. After incubation, translationally active cells and viruses can be fluorescently labeled through click chemistry and viewed by microscopy or alternatively recovered for downstream sequencing using fluorescence activated cell sorting. This approach is also compatible with environmental proteomics.
SEM and TEM
High resolution scanning electron (SEM) and transmission electron microscopy (TEM) allows for detailed structural images at sub-micron resolution that enable visualization of: 1) the physical nature of the cell-mineral and cell-cell interfaces, 2) intracellular structures, and 3) when coupled with energy dispersive spectroscopy can yield semi-quantitative elemental maps. When coupled with serial block face imaging such as SEM 3View, high resolution three dimensional volumes can be constructed, with single slices preserved for further downstream analyses. We also use X-ray microtomography (XRM) to build non-destructive cross sections of physical samples to reconstruct three-dimensional models to resolve detailed 3-dimensional microstructures.
National Center for Microscopy and Imaging Research
TEM imaging of individual viruses
Sulfur K-edge XRF map of microbial aggregates at 1 µm spatial resolution, collected at SSRL beamline 14-3
Synchrotron-based Imaging
The intense light generated by synchrotrons enables the examination of materials and organisms at resolutions and in modes that are otherwise unattainable. We use X-Ray Fluorescence (XRF) and X-ray Absorption Near Edge Spectroscopy (XANES) to analyze the elements and species within microbial cells and their habitats in order to answer fundamental questions about how cells store, share, and cycle nutrients.
Stanford Synchrotron Radiation Lightsource (SSRL)
Metagenomics and 16S Amplicon Sequencing
Only a tiny fraction of microbes can be cultured in a laboratory; the rest remain uncultured, and their taxonomic identities, physiological capacities, and community functions can only be inferred from genomic sequence data. Our lab collects and sequences large numbers of microbes from largely unexplored marine and deep biosphere environments. Our server, Ocean, enables the rapid computation required to analyze these large datasets to make sense of the huge diversity of microbial life teeming in unexplored marine and deep biosphere environments.
Metagenomic binning using Anvi’o (left) and annotated concatenated marker gene phylogeny of bins of interest and reference sequences (right)
ASM-clust representation of diversity of proteins in the molydopterin oxidoreductase superfamily
Proteomics
The Orphan Lab employs a proteomic approach to better understand the relationship between genes and function in microbial communities. Using techniques such as stable-isotope labeling and BONCAT, we identify actively synthesized proteins in energy-constrained systems. Through these approaches, we can determine proteins and pathways that are critical for community homeostasis and response to environmental stimuli.
Geochemistry
In order to provide environmental context for the sediment samples we collect in the field, we have access to dual channel ion chromatography and ion chromatography with mass spectrometry systems at the Water and Environment Lab. These instruments allow us to quantify the concentration of major monovalent and divalent ionic species and small organic acids. We can also query the isotopic abundances of those analytes. This gives us insight into the metabolic processes occurring in situ as well as in laboratory incubations.
Resnick Water and Environment Lab
Microelectrodes measuring sulfur species in a microbial mat
High pressure vessels to mimic in-situ pressure observed by marine subsurface microbes
High Pressure Incubations
Incubating microbes at high pressure requires specialized equipment. In the Orphan Lab, portable 1.4 L high-pressure vessels are used. These aluminum-alloy cylindric containers are capable of creating hydrostatic pressure up to 6000 psi (about 410 atm or 41 MPa) using an oil reservoir and hydraulic pump to compress the sample within the vessel. Temperature is controlled by placing the vessel within a cold room to mimic in situ temperature conditions.
Microbial Electrochemistry
Microbial electrochemistry focuses on studying the electron transfer between microbial cells and conducting surfaces such as electrodes and minerals. Beyond characterizing the electron transfer pathway in the two model organisms (Geobacter and Shewanella) for biotechnological applications, this tool has been extensively used to cultivate subsurface (marine and terrestrial) microorganisms. The Orphan lab houses two multichannel potentiostats: Biologic VSP 300 and Admiral Squidstat Prime, available to probe intriguing questions still unexplored in the field of microbial electrochemistry including ecophysiology of structured microbial communities inhabiting redox-active surfaces.
Manual for VSP 300
Electrochemical cells in water bath
Scanning electron image of Geobacter biofilms grown on a poised electrode
Other Methods
The Orphan Lab also uses a variety of other resources on campus for analysis and imaging of microbial and mineral samples. These methods include infrared and Raman microscopy, bulk X-ray fluorescence spectroscopy, SEM, benchtop XRF imaging, and X-ray powder diffraction. More information about these instruments and resources can be found below.
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