Learning outcomes: HSW - PowerPoint Presentation

Learning outcomes: HSW MEASURING THE RATE OF OXYGEN UPTAKE To investigate the uptake of oxygen in respiration. To measure the rate at which an organism respires. To develop practical skills. Firstly we will look at how to set up the equipment and what it is telling us Then we can consider what our independent variable could be: they can ask you any in exam.

What is a respirometer? A respirometer records the volume of oxygen taken up, in a given time , which indicates the rate of respiration It can only do this if the carbon dioxide has been removed. Look at various types of equipment: how are they going to work?

Different types of respirometer:

Respirometer Sodium/Potassium/hydroxide absorbs all CO 2 from the air in the apparatus from the beginning. It is in BOTH tubes. This allows the volume of oxygen used to be measured Living organism: 5gm of woodlice/germinating peas/maggots!Living organisms placed on wire gauze: prevents them coming into contact with soda lime which is corrosive/irritantControl tube will be set up in the same way, but without any living organisms, to ensure the results are due to respiration only (put in glass beads of same mass) The manometer tube/capillary tube allows small changes in movement of liquid to be measured and volume changes to be calculated. In more simple set up the drop of liquid allows volume changes to be seen The syringe is used to set the fluid in manometer at known level (starting point) and to return the manometer fluid levels to this point for repeats. Note starting pointsThe apparatus is left for a set period of time A manometer is an instrument for measuring the pressure acting on a column of fluid, consisting of a U-shaped tube of liquid in which a difference in the pressures acting in the two arms of the tube causes the liquid to reach different heights in the two arms.

Respirometer http://www.phschool.com/science/biology_place/labbench/lab5/respwork.html Why does the liquid move? During the time left: Oxygen molecules are absorbed by the organism and used in respiration. The same number of carbon dioxide molecules are released but these are absorbed by the soda lime.(KOH) This reduces the volume of air in the test tube with living organismThis reduces the pressure inside the test tube Atmospheric pressure pushes the liquid along the tube, (towards the tube with living organism) until the pressure in and outside the tube is equal. The distance moved by the liquid in a given time is measured. If your tube does not have volumes marked onto it you will need to convert the distance moved into volume of oxygen used. (Remember the volume used = π r² (3.14) × distance moved, where r = the radius of the hole in the pipette.) This value can then be used to calculate the volume of oxygen taken in by living organism per minuteYou are collecting a mean range of readings for oxygen uptake

To prevent temperature fluctuations a water bath should be used:

Temperature: If you are simply  comparing  the rates of respiration at different temperatures, then you do not need to convert the distance moved by the manometer fluid to a volume. You could just plot distance moved on the y-axis of your graph and time on the x-axis. The rate of respiration is represented by the gradient of the graph.

Using small organisms the rate of aerobic respiration can be determined by measuring the uptake of oxygen using a respirometer Place 5g of organism into the tube and replace the bung. Introduce a drop of dye into the glass tube. (depending on respirometer used)Open the connection (three-way tap) to the syringe and move the fluid to a convenient place on the pipette (i.e. towards the end of the scale that is furthest from the test tube). Mark the starting position of the fluid on the pipette tube with a permanent OHT pen. Isolate the respirometer by closing the connection to the syringe and the atmosphere and immediately start the stop clock. Mark the position of the fluid on the pipette at 1 minute intervals for 5 minutes. At the end of 5 minutes open the connection to the outside air. Measure the distance travelled by the liquid during each minute (the distance from one mark to the next on your pipette). If your tube does not have volumes marked onto it you will need to convert the distance moved into volume of oxygen used. (Remember the volume used = πr² × distance moved, where r = the radius of the hole in the pipette.) Record your results in a suitable table. Calculate the mean rate of oxygen uptake during the 5 minutesCollect mean rate of oxygen uptake results from other groups.

Safety/risks/minimise/Ethics Soda lime is corrosive, but much less of a hazard than solutions of potassium or sodium hydroxide. Eye protection is needed when handling the soda lime. Do not handle directly; use a spatula and wear disposable gloves. Avoid exposing invertebrates to corrosive soda lime dust. ETHICS:Handle live animals with care to avoid harming themSmall, plastic spoons and soft brushes can work well for both maggots and woodlice; germinating seeds need no special measures apart from gentle handling, though disposable gloves may be used. After the investigation, effective hand-washing procedures should be used. Living animals should be returned to the environment that they were taken from.

Comparing respirometers: Simple respirometer Disadvantage: Does not allow you to reset; It needs a control tube used alongside it; No scale so measurements likely to be less accurate. Advantage:Very simple to set up Minimal number of connections makes a good seal easier to obtain.Simple respirometer 2Disadvantage:Needs a control tube alongsideAdvantage: Fairly simple to set up Can be reset using syringe Scale allows for more accurate measurements U-tube respirometerDisadvantageTendency for the connections to leak in elderly school/college models (making the equipment useless); More expensive.Size=difficult to use in water bathAdvantageDoes not need to have an additional control as the second tube balances out the effects of changes in temperature or atmospheric pressure; The syringe allows you to move the liquid in the U to reset the apparatus.

Questions: What will be your control? If using germinating peas, you could have a tube with dried peas (not germinating), glass beads etc. ALL same mass

The Control tube: What could cause a movement of liquid in the control tube towards the respirometer? drop in temp inside tube increased atmospheric pressure What could cause a movement of the liquid in the control tube away from respirometer? increase in temperature inside tube decreased atmospheric pressureWhat could you do to correct your estimate of oxygen uptake if the liquid in the control has moved?The distance moved by the liquid in the control in each minute towards the boiling tube should be subtracted from the distance that the liquid in the experimental respirometer moved Movement of the liquid in the control away from the boiling tube should be added to the distance in the experimental tube.

What are your control variables: How/Why? Environmental temperature – could be controlled via a water bath or monitored to check for any change.WHY ? Number of organisms – controlled by careful counting/double-checking numbers.Mass of organisms – controlled by weighing. To keep both number and mass the same, using organisms of similar size will be helpful.Mass of soda lime – this should be weighed out.Activity and age of organisms – partly controlled by choosing organisms of the same age (days germinated/same length of maggot, etc.) Species – there may be variation in metabolic rates in organisms of different species, so the same species organisms should be used.

Results: Suggest why a mean range of oxygen uptake readings should be taken? A mean range of readings should be taken because: USING LIVING ORGANISMS Organisms have different metabolic rates There may be different environmental temperatures/fluctuations Difficult to obtain the same mass of organisms Equilibrium time: Organisms may have been left for different times before starting to take readings.

Why a mean was calculated and the significance of any variation within the data from which the mean was calculated Taking several readings over five minutes is equivalent to carrying out repeated measurements and that using a mean value is more accurate than individual values. Each reading within the five minutes may be different; this could be due to the organism still acclimatising The apparatus still equilibrating Changes in the activity of the organism, etc.

Suggest what could be done to reduce the range of results, making the results more precise. So to reduce the range of results and make them more precise: Use similar organisms: same size and age place respirometers into a water bath to maintain temperature Standard equilibration time Same person carrying out the same task to minimise random errorRe-using same equipment to control any systematic error

Results Draw a graph that shows the mean rate of oxygen taken up against the variable you have investigated, for example the mass (or numbers) of organisms or temperature. Take care to use appropriate units. A scatter diagram may be the best choice to present your data; you should be able to justify why this might be the best choice. Use a scatter diagram to identify a correlation between two variables Decide if any correlation, positive or negative, is evident between your two variables and use a statistical test to determine if the correlation is significant.

What IV shall we change? Temperature If temperature is used as the independent variable then the range 5–40 °C is suggested as suitable With 5 °C increments Ideally 3 repeats at each temperature What science do we include??

What IV shall we change? Mass of organisms? How could we change? What science?

Linking to the science: Conclusion Oxygen is the final electron acceptor, and it eventually combines with hydrogen to make water. The carbon dioxide comes from the carbon dioxide released in the link reaction and the Krebs cycle as the carbohydrate is broken down.

Describe how this apparatus could be used to find the mean rate of respiration: 6 marks* 1. reference to constant temperature ; 2. use of water bath / eq ; 3. reference to {suitable / stated / fixed time / eq} ; 4. Reference to measuring {volume / distance} ; 5. description of how to obtain volume ; 6. calculation of rate described / eq ; 7. reference to replicates ; 8. description of control e.g. no woodlice ; 9. idea of welfare of animals important ; 10. reference to {mass / eq} of woodlice ;

What can be measured using a respirometer? The rate of uptake of oxygen: As the organisms respire, they take oxygen from the air around them and give out carbon dioxide. The removal of oxygen from the air inside the tube reduces the volume and pressure, causing the manometer fluid to move towards the organisms. The carbon dioxide given out is absorbed by the soda lime. The distance moved by the fluid is therefore affected only by the oxygen taken up and not by the carbon dioxide given out. You would not expect the manometer fluid in the tube with no organisms to move, but it may do so because of temperature changes. This allows you to control for this variable, by subtracting the distance moved by the fluid in the control manometer from the distance moved in the experimental manometer (connected to the living organisms), to give you an adjusted distance moved. Calculate the mean (adjusted) distance moved by the manometer fluid per minute. If you know the diameter of the capillary tube, you can convert the distance moved to a volume: volume of liquid in a tube - length x πr2 This gives you a value for the volume of oxygen absorbed by the organisms per minute

Using a respirometer to measure RQ/Respiratory quotients It is sometimes useful to be able to deduce which substrate is being used in a person’s metabolism at a specific time. This can be done if the volume of oxygen taken in, and the volume of carbon dioxide given out are measured. The respiratory quotient (RQ) is the ratio of the volume of oxygen absorbed and carbon dioxide given off in respiration RQ = Volume of carbon dioxide given off Volume of oxygen used per unit of time

Calculate RQ Calculate the RQ for the aerobic respiration of Glucose. Carbohydrates have RQ of 1 Calculate the RQ for the fatty acid oleic acid, when respired aerobically. C 18H34O2+ 25.5O2  18 CO 2 + 17 H2O 0.71 This is the usual RQ value for lipids

Calculating RQ for Anaerobic respiration C 6 H 12O6  2C2H 5OH + 2CO2 A high RQ value suggests that anaerobic respiration is taking place. No RQ can be calculated for the lactate pathway as no carbon dioxide is given off.

Respiratory Quotients (RQ) The respiratory quotients of different respiratory substrates are well documented from previous investigations. Carbohydrate 1.0 Protein 0.9 Fat 0.7 It is possible to deduce which substrate is being used by the metabolism at a specific time. NB if a mixture of substrates is being used then the figure will be different from those above.

Using a respirometer to measure RQ For this, we need to know both how much oxygen is taken in , and how much carbon dioxide is given out .Set up two respirometers. The second respirometer should also contain the same mass of live maggots (or whatever organism you are investigating) but should not contain soda lime. You could put some inert material into the tube (for example, the beads) instead of soda lime. The mass and volume of the inert material should be the same as the mass and volume of the soda lime. This second tube is therefore just like the first one except that it does not contain soda lime. The carbon dioxide given out by the respiring organisms is therefore not absorbed.The difference between the distance moved by the manometer fluid in the experimental tube and the distance moved in the control tube is therefore due to the carbon dioxide given out For example, if the respiratory substrate is carbohydrate, then the amount of carbon dioxide given out will equal the amount of oxygen taken in. The fluid in the control tube will not move, so y=O.

Exam questions An Investigation 11 marks The apparatus ..10 marks