The Varieties of Scrubbers, Tubing, and Tubing Connectors

This chapter discusses several often-neglected areas of respirometry infrastructure. These include the correct selection of scrubber chemicals for removing water vapor and/or carbon dioxide from air streams, without undesirable interactions; chemical-free scrubbing techniques such as selective membranes, thermal condensing systems, and mathematical correction for water vapor dilution; selecting tubing for metabolic measurement; evaluating the different tubing chemistries in light of the intended application; selecting appropriate tubing diameters for the flow rates that will be used; selecting tubing connectors; maintaining connector gender conventions to minimize plumbing confusion; and other related topics.

The Varieties of Gas Analyzers

All analyzers have strengths and limitations that vary with the technology used, and directly affect their suitability for different types of metabolic rate measurement. It is important for researchers to become familiar with the characteristics of the analyzer(s) they are using. This chapter discusses the chief technologies utilized in aerial gas analyzers for O2, CO2, and water vapor, and their advantages, disadvantages, and operating characteristics. For oxygen analyzers, the single channel and differential heated zirconia cell, single channel and differential fuel cell, and paramagnetic types are described. For carbon dioxide analyzers, the single-wavelength and dual-wavelength nondispersive infrared types are discussed. For water vapor analyzers, the chilled-mirror, infrared and capacitive types are considered.

Data Analysis and Presentation

This chapter discusses ways of analyzing and presenting metabolic data while avoiding common mistakes. Topics covered include vital information often omitted from manuscripts; how to analyze the allometry of metabolic rate on mass; the mistake of reporting mass-specific or “mass-independent” metabolic rates; methods for quantifying differences between treatment groups by analysis of covariance; the importance of phylogeny in interspecific comparisons; the importance of the temperature at which measurements are made, including mammals (the thermal neutral zone); the necessity of leaving an “audit trail” from raw data through to final analysis; analyzing temperature effects such as Q10 correctly; and the proper selection of metabolic data.

Flow-through Respirometry: The Equations

This chapter demystifies respirometry equations, showing how they can be derived using a simple mental trick: focusing the analysis on the principal gas that is neither consumed nor produced by animals. The effect of dilution of oxygen by carbon dioxide, the enrichment of carbon dioxide by the consumption of oxygen, and the effects of water vapor on the concentrations of both gases are described and quantified. A system of eight equations is derived that allow oxygen consumption and carbon dioxide production to be calculated in practically any feasible flow-through respirometry system.

Flow-through Respirometry: The Basics

This chapter describes the basic theory behind the most widely used method for measuring metabolic rates: flow-through or open-system respirometry. The advantages and disadvantages of the technique are summarized and the two major types of flow-through respirometry systems are described. Recommendations are given on choosing an appropriate flow rate to compromise between speed of response and signal amplitude; on the nature and importance of the cage time-constant; on using mathematical techniques for response correction by compensating for first-order wash-out kinetics and avoiding mixing errors; the essential differences between oxygen and carbon dioxide analysis; choosing a data acquisition system; generating and measuring flow rates; removing or mathematically compensating for water vapor; important tools; and checklists for deciding on system configuration for a given investigation.

Measuring the Fire of Life: A Brief History of Metabolic Measurement

This chapter describes the evolution of respirometry from Leonardo da Vinci’s musings onwards. The works of Boyle, the brilliant and prophetic Mayow, and the well-intentioned but misguided Priestley are described. The bizarre dead-end theory of phlogiston and its apparent validity to the scientists of the day are explained in historical context. The breakthroughs of Lavoisier and Paulze, who realized the central role of oxygen and pioneered the quantitative measurement of metabolism, end the conventional historical part of the chapter, which concludes with a brief description of the deep history of the molecules most important to respirometry.

Acquiring Useful Tools and Skills

This chapter discusses useful skills and tools that can extend and amplify the reach of innovative researchers. These include programming languages; statistical packages; microcontrollers and single board computers; researcher-friendly electronic resources; circuit capture and printed circuit design packages and resources; 3D design and printing packages and resources; and laser-cutting resources. The emphasis is on open source solutions applicable to scientific research.

The Varieties of Flow Meters

This chapter discusses the most frequently used flow meters in respirometry. These include the volumetric rotameter, which is affected by ambient temperature and barometric pressure; and the mass flow meter, which directly measures the molar quantity of air passing through it, and thus automatically corrects the volume to standard temperature and pressure. The operation and calibration of each type of meter are discussed.

Flow-through Respirometry: Excurrent Flow Measurement

This chapter describes the setup, plumbing, and equations for implementing a respirometry system wherein the flow rate of the air leaving the animal chamber is known. Such systems are usually referred to as pull systems, because the air is usually pulled from a chamber or mask at a known rate, and the concentrations of incurrent and excurrent gases are alternately measured. Such systems are often the only practical way of measuring the metabolic rates of large animals. Setups and equations for oxygen-only, carbon dioxide-only, and combined oxygen and carbon dioxide systems are described. Methods for creating multiple-animal pull mode respirometry systems, for compensating flow rate, and for the automatic baselining (that is to say, measuring incurrent gas concentrations) of respirometry systems are discussed.

Constant Volume Techniques Using Gas Analysis

By using modern gas analyzers and variations of constant volume techniques described in Chapter 2, simple and high-throughput measurement of the metabolic rates of organisms ranging in size from bacteria to large insects and even small vertebrates are easily implemented. It is also possible to measure water loss rate and carbon dioxide production using only an oxygen analyzer. These respirometry techniques can be deployed in the field as well as the laboratory. Both manual and automated, computerized implementations of constant volume techniques for metabolic rate measurement are covered in full step-by-step detail, and appropriate analytical protocols for oxygen, carbon dioxide, or both oxygen and carbon dioxide analysis systems are also described in detail.