A Resource Page for Microrespirometers

(A resource page)

Brad Williamson

Introduction:

The AP-Biology Listserv community (click here to subscribe) is a superb reference and resource for all biology teachers. Traditionally one of the most discussed laboratories is Lab 5 on cellular respiration. Measuring the rate of respiration is more technically challenging than many lab procedures. There is simply too many places for potential error with the respirometer's construction and use. There's a lot of tweaking and fussing with the equipment to get it to work right--how's a novice to know when to tweak and when to collect data? Several years ago, Richard E. Lee, published an article on the use of microrespirometers to study small insect metabolism in The American Biology Teacher.

Lee's design

Lee, Richard E. (1995). Using Microrespirometers to Measure O2 Consumption by Insects and Small Invertebrates. The American Biology Teachers, vol. 57, no. 5, 284-5.

Lee's microrespirometer design offered some unique advantages for high school laboratories that investigate cellular respiration.

They are cheap--each respirometer cost less than $0.25.
They have adjustable volumes.
They work quickly.
There small size means that their temperature equlibrate rapidly in water baths.
Generally, they don't leak.

I've modified Lee's design to work with high school students. In particular, I chose not to use the KOH as a manometer fluid, instead placing the KOH in a cotton plug at the bottom of the syringe. These respirometers work particularly well for the AP Lab 5. Give them a try I think you'll find that more of your students successfully complete investigations that investigate cellular respiration.

The biology behind the prodedure:

The general or summary equation for cellular respiration is:

C6H12O6 + 6O2 --> 6 CO2 + 6 H2O.

From this equation, if glucose is the energy source then for every one molecule of oygen consumed one molecule of carbon dioxide is produced. In the microrespirometers the gas molecules are sealed by the liquid manometer fluid plug. Gaseous carbon dioxided is absorbed and combined with the potassium hydroxide to form a solid compound removing carbon dioxide molecules from the interior atmosphere of the microrespirometer. Since oxygen is consumed by the process of cellular respiration the overall gas volume decreases. Outside air pressure is therefore greater and the greater air pressure forces the manometer fluid plug into the capillary.

How a respirometer works

Respiration can then me measured by how fast the manometer fluid plug moves along the small capillary tube.

Drawing of working setup

Materials:

• goggles
• hot-glue gun
• 3–4 one-quarter inch flat metal washers
• permanent glass-marking pen
• thin-stem dropping pipettes
• absorbent cotton
• nonabsorbent cotton
• germinating Fast Plant® seeds (or other material or organism)
• glass beads (or dry, baked Fast Plant seeds)
• Celsius thermometer
• centimeter ruler
• manometer fluid (soapy water with red food coloring)
• constant-temperature water baths
• 2 1-mL tuberculin syringes without needles
• 40-μl plastic, capillary tubes
• 15% solution of KOH, potassium hydroxide solution

Materials

I'm using NaOH in Drano in this demo instead of KOH because it is easier to get--it works also.

Constructing a microrespirometer:

Plug in the hot-glue gun and allow it to heat up.
Carefully insert a 40-μl plastic capillary into the syringe where the needle normally would be. Insert it as far as the plunger tip but no further. This will help to prevent the capillary from becoming plugged with glue.
While holding the capillary tube straight up, add a small amount of hot glue around its base (where it meets the syringe). This seals the capillary to the syringe. Keep the capillary pointed straight up until the glue cools—this should not take long. If needed, add a bit more glue to ensure an airtight seal between the capillary and the syringe.
After the glue has cooled, pull back on the plunger and make sure that the glue has not plugged the capillary. If the capillary is plugged, carefully remove the glue and the capillary and start over.

Setting up the microrespirometer:

Draw a small quantity of manometer fluid into the full length of the microrespirometer’s capillary tube. Then eject the fluid back out of the capillary. This coats the inside of the tube with a thin soapy film that helps prevent the manometer fluid from sticking.
Carefully insert a small plug of absorbent cotton into the barrel of the microrespirometer, all the way into the “0” mL or cc mark. You can pack the cotton to the end with the barrel of a clean thin-stem pipette.
I've added scales to each capillary tube with double stick tape. You can find a pdf of the scales that you can print out here.
Add 1 small drop of 15% KOH to the cotton in the microrespirometer. Be careful to no add too much. CAUTION: Make sure you protect your eyes with goggles because KOH is caustic.
Add a small plug of nonabsorbant cotton on top of the absorbent cotton plug already inside the barrel of the microrespirometer. You can pack the cotton to the end with the barrel of a clean thin-stem pipette. This nonabsorbant plug is optional with seed but it does protect the seeds from the caustic KOH.
Slowly reinsert the syringe plunger. CAUTION: Be careful to point the capillary tip into a sink or container. There may be excess KOH in the syringe that might squirt from the end of the capillary. Push the plunger in until it reaches the cotton so that any excess KOH is removed. Remove the plunger to add seeds.
Add the 0.5 mL germinating seeds to the microrespirometer. Push the plunger in to the “1.0 ml” mark. This creates a sealed microrespirometer chamber with a 1-milliliter volume.
Place 3–4 washers around the barrel of the microrespirometer. The washers provide weight so that the microrespirometer will sink.
Place the microrespirometer assembly in a room temperature (about 20°C) water bath. Adjust the level of the water bath so the capillary tube is sticking out of the water while the barrel of the microrespirometer is completely submerged. In order to easily read the capillary tube, it must be out of the water. Make sure the capillary tube is open (not sealed).
Repeat steps 1–8 for preparing additional experimental microrespirometers in warm (about 40°C) or cold (about 0°C) water baths.

Controls:

Because a microrespirometer is so sensitive to changes in gas volume, it is also extremely sensitive to changes in temperature and air pressure. For this reason, you may need a control microrespirometer to measure and correct for temperature and pressure changes that occur while using the microrespirometer. The thermobarometer (control) is set up just like the microrespirometer except that it contains nonliving matter instead of germinating seeds.

Add the 0.5 mL of beads or baked seeds to a second microrespirometer. Reinsert the syringe plunger and push it in to the “1 mL” mark. This seals the chamber and creates a chamber that has the same volume as the experimental microrespirometer.
Place 3–4 washers around the barrel of the control.
Place the assembled control in the water bath next to the experimental microrespirometer. Adjust the level of the water bath so the capillary tube is sticking out of the water while the barrel of the control is completely submerged. In order to easily read the capillary tube, it must be out of the water. Make sure the capillary tube is open (not sealed).

Data Collection and Analysis

Place the thermometer into the water bath along with the microrespirometer and the control.Wait 5 minutes to allow the temperature in the microrespirometers to equalize.
Use a dropping pipette to add one small drop of manometer fluid to the tip of each capillary tube. If everything is working properly, the drop will be sucked down into the capillary tube. The manometer fluid will seal the chamber of the microrespirometers. (You should use the plunger on the control microrespirometer to get the manometer fluid into the capillary. Pull on the plunger until the manometer drop is about half-way down the capillary.)
As oxygen is consumed, the manometer fluid plug will move toward the chamber. Record the starting position of each plug by marking its position on the capillary with a marker. Be sure to mark the bottom edge of the plug. These are your Time 0 marks. Begin timing once you have made the Time 0 marks.
At one-minute to five-minute intervals (depending on how fast respiration is taking place, measure the temperature of each water bath and mark the position of the manometer fluid for each capillary tube. Be sure to mark the bottom edge of the fluid plug. Continue marking the positions until the fluid in the microrespirometers has traveled the entire length of the capillary, or until 25 minutes has passed. Record the temperature reading in a data table.
Lab write-up

Extended investigation