Sonoma State University
Department of Biology Hanes
GILSON RESPIROMETERY
Introduction
The Gilson Respirometer is designed to measure oxygen consumption in macerated or ground tissue. It can also measure oxygen consumption in small animals. There is a side-arm on the flask that can add a chemical to the chamber during the experiment. One can add either a poison or nutrient. The limit to the size of animal is the chamber opening's size. There is also caustic in the center well, so it is possible to mistakenly burn active animals.
Purpose
Procedures
Use a minimum of 0.2g of meal worms. Weigh each group to the nearest milligram on the Mettler balance. Be sure to record which group goes into each reaction vessel. Place the larvae in the main reaction chamber.
Record the temperature of the waterbath for your data. The Gilson Respirometer requires about 1 hr to reach equilibrium, so be sure the machine is set at the temperature you desire and is turned on at the beginning of the period.
The Gilson Respirometer is a system of tubes and two chambers separated by a manometer U-tube. One of these chambers is the reaction vessel and the other (thermobarometer) acts as a control for slight temperature or pressure changes in the water bath. When a valve handle is up on the respirometer, that valve is open (air may pass through).
If gas is either used up or produced in the reaction vessel, and if valve #2 is closed, the manometer fluid will have to move in the U-tube. Turning the knurled knob will cause a metal plunger to change the volume on the reaction vessel side of the U-tube. When the fluid is moved back to its original starting point, the volume change is indicated on the dial in microliters.
Preparation of the Respirometer
As previously mentioned, the water bath requires approximately one hour to equilibrate. Set machine at least one hour before lab. The temperature of the water bath is set on a thermometer-like looking gadget. Loosen the set screw on the plastic knob on top and turn the top until the top of the plastic marker is at the desired temperature (30 deg. or 20 deg. C for the larvae). Check to see that the water comes up to the splash guard of the stir motor. Fill only with distilled water. Turn on the STIR MOTOR. If bath temperature is near or below ambient, turn on REFRIGERATION, MAIN HEATER, AND AUX. HEATER. If temperature required is above ambient, turn on only main heater and aux. heater.
Preparation of Vessels
All reaction vessels must be quite clean. All of the fittings will need to be air tight, so thin films of grease must be placed:
1. Inside rim of center well -use a toothpick . A very light coating.
2. Flask side arm connection
3. Ground glass fitting on end of manometer
Cut an accordion fold square of filter paper of a size that will just stick out of the center well and place it in the center well. Add approximately 0.2 ml of 20% KOH to the grease-rimmed center well. Check to see that all valves are open (handles up). With rubber bands secure the reaction vessels to the male ground glass fittings and the side arm stoppers to the reaction vessel.
Turn the blue flanges of the side arm stoppers to the side. Close the #1 valves of all the unused manifolds and check to see that the valve at the end of the series of manifolds is closed (down). Lower the flasks into the water bath and allow 10 minutes for temperature and pressure equalization. Set the markers (they slip up and down) to mark the bottoms of the menisci. Set the micrometer dials to a convenient number such as 50. The micrometer numbers read in microliters (ul) and each division is 0.2. When you are ready to begin timing the experiment, close the valves #2. At all times when valves #2 are closed be aware that you can MESS UP THE MACHINE by changing the temperature, pressure, or generally handling or moving the flasks. During the timing period, keep the manometer fluid level near the mark by turning the knurled knob. Set the fluid at the mark at 10 minute intervals and record the reading on the dial. NEVER turn the dial backwards from a reading of 0. It will ruin the gasket on the plunger. Continue the readings until you are satisfied that gas volume changes are constant from one 10 minute period to another (1-1/2 hrs.?)
When you are finished taking readings, FIRST OPEN VALVES #2. Disassemble the apparatus, wash and rinse and distilled water rinse the flasks and other glassware. Figure out a way to remove the animals without burning them with KOH and return the healthy animals to their home.
Record your raw data below. For your own use, plot microliters of oxygen consumed vs time on graph paper. Is this a straight line? If most of the curve is a straight line, then you may want to use only the straight part of the data to calculate the micrograms of oxygen consumed per gram minute. In the second data area record the microliters of oxygen consumed per minute for the straight part of the curve, convert that to STPD by compensating for temperature and barometric pressure (why not vapor pressure?), convert STPD to micrograms of oxygen consumed per minute by multiplying by 1.43 (Why?), and finally convert that number to micrograms of oxygen consumed per gram minute by dividing by the gram weight of the larvae.
Equipment needed
Gilson Respirometer Reaction Vessels
Glass stoppers
Grease
Rubber Bands
20% KOH
Balance
Toothpicks
Filter Paper
Scissors
Formuli needed for clalculations
Microliters to microliters (STPD) (Standard temperature and pressure, dry)
Conversion of ul (STPD) of oxygen to ug O2
GILSON
RESPIROMETRY DATA SHEET
Date________________
Respirometer Temp._________C Barometric Pressure_________Torr
Flask Larvae Gilson Readings (in microliters)
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Flask # |
Weight mg |
Minutes |
00 |
10 |
20 |
30 |
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50 |
60 |
70 |
80 |
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CALCULATE THE FOLLOWING:
Microliters O2/ min = if there is not a steady increase in the amount of oxygen used in every 10 minute period, choose a sequence of periods that are fairly consitant. Subtract the first value from the last value and divide this number by the number of minutes spanned.
Microliters (STPD)/ min = the value in the previous column times the first formula above.
Micrograms O2/min = microliters (STPD)/min times 1.43.
Micrograms O2/min g larvae = Micrograms O2/min divided by the gram weight of the larvae in the flask.
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Flask # |
microliters O2/min |
microliters (STPD)/ min |
micrograms O2/min |
micrograms O2/min g larvae |
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