Workhorse of TdO Labs

This design was developed during the COVID-19 lockdown and published in 2022. This page provides an overview of the respirometer’s function. If you are interested in the details of construction, check out the Build Your Own Respirometer page.

Big Picture

The respirometer consists of:

• A chamber where the measurement happens
• A controller, which acquires the data and sends it to a computer
• A computer, which logs and analyzes the information.

The chamber and controller will be described in detail below, whereas there is nothing special about the computer.

For simplicity, a few things have been omitted from the diagram above. First, temperature must be held constant, so the chamber would be contained in an environmental chamber or water bath. The specific method of maintaining temperature is not important, as long as temperature is stable within a few tenths of a degree Celsius. Also, only one channel is shown, but multiple channels of data are normally acquired during each experiment. The system is modular, and is expanded by simply plugging more chambers and controllers into the computer.

Chamber

The chamber is a gastight container that contains the organism, a pressure sensor, material to absorb exhaled carbon dioxide, and an electrolytic oxygen generator.

The main body of the chamber is a Schlenk reaction flask, at left in above photo. It has a tapered, ground glass joint (1a) for access , and a stopcock valve (1b) to equilibrate pressure. The larger flask with a 24/40 joint works well for larger beetles (>500 mg), whereas a smaller Schlenk tube with a 19/22 joint fits smaller insects.

The sensor plug (upper right) fits into the joint of the chamber. It contains the Bosch BME 280 pressure/temperature/humidity sensor (2b), a connector for the oxygen generator (2c), both of which connect to a cable from the controller via a six-pin connector (2a).

The oxygen generator consists of a screwcap tube, with the cap (3a) drilled to accommodate a coaxial jack with the platinum anode and copper cathode wires. The tube (3b) is filled with saturated copper sulfate solution.

A small perforated tube (4) is filled with soda lime to absorb carbon dioxide exhaled by the animal.

Controller

The controller has three primary functions

1. Powering and reading the pressure sensor through an I²C connection.
2. Sending electric current to the oxygen generator when pressure inside the chamber decreases to a pre-set threshold.
3. Uploading data to a computer.

All of these functions are handled by a controller designed around an Arduino Nano Every.

We are also using a seven-channel version (above), which combines seven controllers, a digital thermometer (left connection), and a USB hub to carry the data to the computer.

Setup

For each experiment, the glass joints are cleaned, the oxygen generators and soda lime tubes are filled, the beetles added to the chambers and the chambers are assembled. During this period the stopcocks are left open to keep the chambers at ambient pressure.

The assembled chambers are placed into the temperature control device (usually a water bath), connected to the cables going to the controllers, and allowed to equilibrate with the stopcocks open. During this time, data acquisition starts. The stopcocks are then closed, and the chambers equilibrate for another hour.

Once the chambers have fully equilibrated and temperatures are stable, the oxygen generators are activated to pressurize the chambers to slightly above ambient.

Data Acquisition and Structure

Once the experiment starts, pressure inside the chamber decreases as the organism consumes oxygen and the exhaled carbon dioxide is absorbed by the soda lime. When the pressure reaches the threshold, the controller sends electric current through the oxygen generator, which re-pressurizes the chamber. This cycle continues for a period of a few hours.

The above graph shows a typical experiment from start to finish. The black line shows the pressure inside the chamber, which starts at the ambient pressure of ~800 hPa (note that the lab is at about 2190 m elevation). The stopcock is closed at the time indicated by the white arrow, sealing the chamber. Because the organism inside is consuming oxygen, and the exhaled carbon dioxide is removed by the soda lime, the pressure decreases steadily. At the black arrow, the current to the oxygen generator (red line) is switched on. As oxygen is produced, the pressure increases, until it reaches the pre-set threshold of 812 hPa. At 812 hPa, the controller turns off the current (note red line returning to zero mA). The organism continues to consume oxygen, so the pressure drops until it reaches 810 hPa, at which time current turns on again. The process repeats, with pressure alternating between 810 and 812 hPa as oxygen is consumed by the organism and replaced by the oxygen generator.

Data Analysis

During an experiment, the data look like the screenshot above. Data from each controller is logged using the program PuTTY, with each window representing one channel of data.

These data can then be imported into a program such as Excel for further analysis. Each current pulse can then be integrated to determine the amount of charge that passed through the electrolyte, and from that the amount of oxygen generated can be calculated.

For More Information

Details regarding construction and programming can be found at the Build Your Own Respirometer page.

A video tutorial showing the use of a coulometric respirometer to measure oxygen consumption in the fruit fly Drosophila melanogaster can be found at the Journal of Visualized Experiments here. Unfortunately, the site is paywalled, so your institution will need a subscription or you will need to know someone who does.