Geomicrobiological investigations benefit from knowledge of geochemical and biological systems at different scales, including information about both the abiotic and the biotic components. Gathering this information requires analysis and characterization of both abiotic and biotic components of the target system. The techniques presented in this chapter were selected to cover a variety of needs in geomicrobiological studies, including general sample collection and storage, organic and inorganic compound quantification, and best practices for cultivation, observation, and analysis of microorganisms and microbial communities. In this chapter, introductions and discussions for common techniques provide the reader with a basic understanding of the technique itself, which samples can be analyzed using the technique, and how to prepare samples for analysis. Detailed methods are provided for select techniques, and citations to standard methods are provided for techniques whenever available. For techniques that are rapidly evolving, recent developments and applications are discussed.

Scanning probe microscopy (SPM) is a suite of related imaging methods, in which variations in the interaction force between a probe and a sample surface are used to generate image contrast. These instruments are incredibly sensitive; they can measure forces on the order of those required to break physical and chemical bonds, and under the most optimal conditions, atomic-scale resolution can be achieved. Although SPM is still primarily used for imaging, it is increasingly being used to measure nanoscale properties and interaction forces. This chapter serves as an introduction to the fundamentals of SPM and to the most prevalent methods needed for the investigation of mineral–microbe interactions.

Fourier-transform infrared (FTIR) spectroscopy is a technique that measures the molecular-level vibrations in a material, such as a bacterial biofilm, to get a better understanding of the chemistry of the system. This technique is best used to observe changes in a system, e.g., how bacteria protonate and deprotonate as a function of pH or how contaminants sorb to minerals/bacteria, or for tracking the precipitation of a mineral or the breakdown of a contaminant in a system. It can also be used to identify the presence of a specific contaminant in a system, e.g., the presence of bacteria on an antimicrobial surface or the presence of pesticides in water. Thewill outline the different ways in which FTIR spectroscopy may be used to analyze a variety of samples in geomicrobiology. The techniques and their applicability are detailed, from individual sample recording (via diffuse reflectance measurements) to continuous monitoring of systems (using attenuated total reflectance measurements) and spatially resolved microspectroscopic analysis (either as imaging or as determining the positions for point sampling in a heterogeneous sample), and a general strategy for data handling is given, including the basics of some multivariate techniques. We will explain how to get the best possible data using each FTIR spectroscopic method, as well as how to best treat your data before analysis. Additionally, this chapter deals with understanding how to identify the representative FTIR bands for bacteria, and how those bands can change as a function of pH.

X-ray diffraction techniques provide information regarding the formation and alteration of mineral phases that is critical for assessing geomicrobial processes. Of particular interest is the use of powder X-ray diffraction (pXRD) to identify unknown solid-state materials, determine the particle size of nanoscale mineral phases, and refine structure characteristics, such as unit cell parameters and atomic positions. The goal of this chapter is to provide practical knowledge for the successful preparation of solid mineral samples, optimal data collection strategies, and analysis of diffractograms collected from pXRD experiments. Specific uses of pXRD techniques in geomicrobiology are discussed to demonstrate the importance of diffraction in advancing our understanding of microbial communities in geologic systems.

Isothermal titration calorimetry combined with surface complexation modeling is an ideal technique to provide further characterization of microbial surface reactivity towards protons and metal ions. This technique can produce enthalpies of protonation and metal ion coordination of acidic functional groups on microbial surfaces. This information is critical for understanding the thermodynamic driving force of surface complexation and provides key information for the indirect identification of surface ligands. Topics covered in this chapter include how this technique complements traditional methods of microbial surface reactivity, necessary system characterization prior to performing calorimetric experiments, how to prepare biomass and solutions for calorimetric titrations, difficult aspects of this technique, and data analysis and interpretation.

Lipid biomarker analysis is a useful tool for characterizing microbial communities in geomicrobiology. Phospholipid fatty acids (PLFA) are major components of microbial membranes, and analysis of these markers provides insight into microbial biomass, community structure, and metabolic processes. This article reviews the methods for extraction, fractionation, derivatization, and quantification of PLFA, as well as the interpretation of PLFA patterns for microbial community analysis in natural environmental systems. The discussion centers on the development, the subsequent modifications, and the advantages and limitations of the methods. Two case studies are given to illustrate the applications of intact phospholipid profiling (IPP) and PLFA in geomicrobiology. The recent developments and future directions of microbial signature lipid analysis are also discussed.