ABSTRACT
The instrument was designed to measure the O2 uptake of living material which consumes between 0·01 and 5·0 μl. O2/hr. Its accuracy is + 0·001 μ1., and it has the following advantages over other micro-respirometers of similar sensitivity.
It is robust, easy to handle and is suitable for routine use.
The respirometer constant is independent of the gas volume of the reaction vessel.
Its sensitivity is independent of the volume of the reaction vessel, and it can therefore be used with relatively large amounts of living material.
Experiments can be carried out over a wide range of temperatures without appreciably altering the accuracy.
It can be coupled to apparatus (described at the end of this paper), which automatically measures and records the rate of O2 uptake.
A comprehensive review of the better known micro-respirometers has been given by Tobias (1943), but to facilitate comparison with the present instrument a list of the characteristics of some of them are given in Table 1.
Principle
The principle of the instrument can best be followed from the diagram (Text-fig. 1). It consists essentially of two vessels—a reaction vessel and a compensating vessel—connected by a U-tube containing a drop of kerosene. The volume of the reaction vessel can be adjusted at will by means of a pipette. This consists of a mercury-filled reservoir from which known volumes of mercury can be displaced by means of a metal plunger actuated by a screw micrometer (the latter is not shown in Text-fig. 1). When the two vessels are cut off from the outside atmosphere, any pressure change in one relative to the other will be followed by a corresponding movement of the kerosene droplet. In order to determine the amount of gas absorbed or evolved by the experimental material, the two vessels are set up so that they differ only in the single respect that one—the reaction vessel—contains the material to be studied. The respirometer is placed in a water-bath, and after a suitable equilibration period the outlet valves are closed. After a short time the movement of the kerosene will represent changes in pressure due to the experimental material. The volume of the reaction vessel is then adjusted with the pipette until the kerosene meniscus is brought level with a fixed point on the U-tube. After a known interval the meniscus is again adjusted to the same point; the gas pressure of the whole system is thereby restored to its original value. The volume of the length of plunger introduced or withdrawn from the reservoir is equal to the volume of gas absorbed or evolved in the reaction vessel, at the temperature and pressure of the experiment.
General description of the micro-respirometer
The manometer component (Text-fig. 2) and the pipette (Text-fig. 3) are described separately. The assembled instrument is shown in Pl. 11, fig. 2.
(i) The manometer component (Text-fig. 2)
The reaction vessel and the compensating vessel (Text-fig. 2A) are both detachable, and consist of small glass cups, with ground edges (which are greased). Both vessels are clamped on to a stainless steel block (Text-fig. 2C) by means of two thumb screws (Text-fig. 2D). Sealed into the block and opening into the cavity under each vessel, is a small, fine-bore capillary U-tube (Text-fig. 2E), containing a drop of kerosene. The meniscus of the kerosene (Text-fig. 2F) is observed through a low-powered microscope (Text-fig. 4). A glass capillary (Text-fig. 2G), of slightly larger bore, connects the reaction vessel and the reservoir of the pipette (Text-fig. 2H). The metal block is clamped on to a small metal frame (Text-fig. 2I), attached to the casing of the pipette. Both vessels communicate with the outside atmosphere through holes in the block which can be closed by needle valves (Text-fig. 2J). The latter are connected to the pipette casing by rubber tubes (Pl. 11, fig. 2). Small plugs of metal or glass (Text-fig. 2L), with recessed ends fit loosely into the cavities under each vessel. The capillaries are protected by a sheet of Perspex (Text-fig. 2M).
(ii) The pipette (Text-fig. 3)
The pipette consists of a small mercury reservoir made of stainless steel (Textfig. 3 A), closed at one end by two recessed vulcanized rubber washers (Text-fig. 3 B), enclosing a drop of mercury, through which passes a silver steel plunger of known diameter (Text-fig. 3C). The reservoir and washers are held in place by a collar (Text-fig. 3D). The plunger is attached to the end of a push rod (Text-fig. 3D), held in contact with the shank of the micrometer (Text-fig. 3 G), by a spring (Textfig. 3-H). The rubber tubes leading from the needle valves on the metal block are connected to two nipples on the casing (Text-fig. 3D) which opens to the atmosphere through a hole near the top (Text-fig. 3J).
Accessory apparatus
The general arrangement of the apparatus is shown in Text-fig. 4 and Pl. 11, fig. 1. The respirometers are clamped on to the lower bar of a supporting frame so that the manometer component is completely submerged in the water. The microscope is so arranged that it can be moved along the frame and focused on each of the U-tubes in turn. The latter are illuminated by small 12 V. lamps, the light from which passes through curved Perspex rods, so that the lamps can be placed well away from the surface of the water-bath. The water-bath has a temperature differential of approximately ± 0·03° C.
Notes on the design
(I) The metal block and reaction vessel
The reaction vessel and compensating vessel are placed as close together as possible in order to avoid temperature differences between them. The metal block, besides providing a rigid base for the vessels, tends to smooth out temperature variations of short duration.
The vessels can be of any size, but the diameter of the base in contact with the metal block must be as small as possible. When the diameter is large, small movements of the vessel relative to the block, which occur when the grease seal is gradually expressed, give rise to oscillations of the manometer fluid. For general use, 4 mm. is a convenient diameter for the base of the vessel ; should larger vessels be required, they must be made with bulging, rather than parallel sides.
(2) Capillaries
The bore of the capillary U-tube is between 0·1 and 0·2 mm. Theoretically, a finer bore would increase the sensitivity but, in practice, it is found that even the best manometer fluids tend to stick and move erratically in capillaries of less than 0·1 mm. unless they are frequently cleaned. The use of a microscope with two-third objective and × 8 ocular makes it possible to adjust the meniscus to within 0·01 mm. of a given position. This represents a volume change of about 0·0006 μl. in a 0·1 mm. diameter capillary which is less than the smallest volume that can be measured with the pipette.
(3) Zero mark
A scratch on a very small droplet of black wax is the most satisfactory mark; engraved marks weaken the capillaries.
(4) Manometer fluid
The most satisfactory manometer fluid is a light kerosene marketed under the name of ‘odourless distillate’ which has a low viscosity and does not evaporate easily. Petroleum ether is too volatile.
(5) Needle valves
The needle valves require careful construction. The needle (Text-fig. 2N) fits loosely in the sleeve, and it is important that when it makes contact with its seating it should come to rest. (The needles are allowed to make their own seating and are not ground in.) Before the instrument is assembled the valves are tested with a vacuum pump. A properly constructed valve should hold a vacuum of 3 mm. mercury for over an hour, but it is sometimes necessary to grease the needle lightly before it will pass this test.
(6) Pipette
The pipette is the central support of the instrument. It is larger than the rest of the respirometer, but only the mercury contained in the reservoir is liable to give rise to errors when the temperature varies. These errors, however, are not important if the volume of mercury does not exceed 10 µl. No error is introduced by the thermal expansion of the metal push rod because it is compensated by a corresponding expansion of the metal casing of the pipette.
(7) Pipette plunger
The terminal half inch of the plunger, which is used to displace mercury in the reservoir, must have a uniform diameter. The smallest diameter that can be used conveniently is . because smaller plungers bend and give rise to inaccuracies. The accuracy of the pipette depends on the accuracy with which the plunger displaces the mercury, for this reason the mercury in the reservoir must be free of air bubbles. Their presence can be detected by applying a vacuum to the end of the pipette capillary and observing the Hg meniscus.
Experimental procedure
(1) Equilibration
The time that must be allowed for the instruments to equilibrate depends on the difference between the bath and room temperature. In order to determine the length of equilibration period required, the instruments are set up with both chambers empty. The valves are closed as soon as they have been placed in the water-bath, and the volume changes are measured. A series of typical equilibration curves are shown in Text-fig. 5. An equilibration period of half an hour is adequate for a temperature difference of 8° C. ; this can be reduced to 20 min. for a temperature difference of 1°.
(2) The measurement of O2 uptake
The reaction vessel may be set up in one of two ways depending on the type of experimental material. Solid objects, such as insect eggs, are placed in the recessed end of the metal plug (Text-fig. 2L); suspensions and material in a liquid medium are placed in a hanging drop in the roof of the reaction chamber. The CO2 absorbent (M/10 KOH) is placed on a disk of no. 40 Whatman filter-paper either in the top of the chamber or on the recessed plug. When the O2 uptake of suspensions is being measured, the volume of the drop must be chosen carefully. There is no stirring in these instruments, and therefore the volume of the drop in relation to its surface must be such that the rate of diffusion of O2 does not become a limiting factor.
After they have been filled, four small drops of ‘Lubriseal’ grease are placed on the ground surfaces of the vessels, and they are then clamped on to the metal block with the thumb screws. The instrument is placed in the water-bath. The rubber tubes attached to the needle valves are gently squeezed to make sure the capillaries are not blocked. After an appropriate equilibration period, the valves are closed and tested by bending and squeezing the rubber tubes. Five minutes later the meniscus is adjusted to the zero point on the U-tube. This adjustment is repeated at intervals. The actual volume changes are obtained by multiplying the difference between successive readings of the micrometer by the volume per unit length of the plunger. A series of respiration experiments are described in Appendix I.
(3) Gas mixtures
The vessels can be filled with any desired gas mixture, but the plugs which normally hold the experimental material cannot be used. If a holder is required it must have a hole down the centre so that the gas mixture can reach the upper part of the chamber.
The vessels are greased and placed in position but are not clamped down ; so that the gas can escape between the droplets of grease. The gas supply is connected to the rubber tube attached to the needle valve.
When the gas has been turned on the kerosene droplet is observed to ensure that an adequate pressure is maintained in the vessel, and to assist ventilation the micrometer is screwed up and down at intervals. The gas supply is then disconnected ; the chambers are clamped down on to the block and the needle valves are closed.
(4) Mixing reagents during an experiment
The inner surface of the reaction vessel is either waxed or covered with a thin layer of ‘Silicone’ grease. The reagent with the larger volume is placed in the top of the vessel, and the other is placed in a droplet on the side. A small iron-filled glass bead is placed in the latter and the reagents are mixed by drawing the two droplets together by means of a magnet.
The automatic setting and recording apparatus
The apparatus is designed to interfere as little as possible with the normal arrangement of the respirometer. The only addition to the instrument itself is a large gear wheel attached to the micrometer head (Text-fig. 4, 2) which is coupled by a system of gears, and a flexible drive to an electric motor by means of which the micrometer is adjusted. The motor is controlled by a photocell attached to the microscope.
The microscope is focused on the capillary U-tube in the usual way, but the eyepiece is replaced by an opaque screen, in which is a slit cut to fit the image of the kerosene meniscus (Text-fig. 6B). The photocell, carried in a hinged housing so that it can be swung into place without disturbing the microscope (Text-fig. 4, 1), is illuminated by light passing through the slit in the screen. The amplifier connected to the photocell is so arranged that as long as the field of the slit is occupied by kerosene (Text-fig. 6B) sufficient current is passed by the cell to operate the relay which switches on the electric motor coupled to the micrometer, but as soon as the meniscus enters the field and reduces the light intensity below a certain level (Text-fig. 6C) the relay opens and the motor is switched off. In this way the device is able to set the meniscus to a fixed point whenever it is switched on. The present instrument uses an intermittent light source and an a.c. amplifier (as described in Appendix III) and is able to set the meniscus as accurately as it can be set by hand.
In order to get a sufficiently high intensity of light at the cathode of the photocell, the standard curved Perspex rod is replaced by a straight one with convex ends and a small mirror to reflect the light up on to the U-tube. In this way a strong beam of light from a 12 V. ribbon filament projector lamp is focused on the capillary.
The device records intermittently. The amplifier circuit and light source are switched on by a time switch at intervals varying from one to six times per hour. If the volume of gas in the reaction chamber has decreased in the interval, then the meniscus will have moved up past the control slit so that enough light will fall on the photocell to operate the motor switch; the motor then screws the micrometer head down until the image of the meniscus reaches its zero position. The relay then opens and the motor stops.
The number of revolutions of the motor required to restore the-meniscus to its zero position each time the amplifier and light are switched on is recorded automatically. In the early instruments the record was a series of vertical lines on the smoked drum of a kymograph, the interval between each being the interval between successive settings of the meniscus, the height representing the number of revolutions of the motor and hence the amount of oxygen consumed in each interval. A group of recordings are shown in Text-fig. 7. This method of recording has now been replaced by a revolution counter which prints the number of revolutions on a paper strip.
Summary
I wish to thank Prof. J. Gray, F.R.S., in whose department the instrument was developed, and also all those members of the staff who have helped me with the design and construction of the instrument, in particular Dr Ralph Brown. I should also like to thank Mr S. Falloon of the Cavendish Laboratory for designing the amplifier which has made the automatic attachment possible, and Lord Rothschild for helpful criticism of the manuscript.
The instrument was designed while I was receiving a research grant from the Agricultural Research Council, and The Royal Society kindly made a grant for the construction of the automatic attachment.
Appendix I
Experimental results
The stability of the new micro-respirometers is demonstrated by the results of three experiments on the O2 uptake of Rhodnius prolixus eggs, shown in Text-fig. 8, in which the O2 uptake calculated from successive readings of the micrometer are plotted cumulatively.
The consistency of individual instruments is illustrated by a series of controls from experiments on the O2 uptake of the eggs of R. prolixus. The O2 uptake of each batch of eggs was measured twice in the same respirometer. The determinations lasted i hr., and the eggs were removed from the respirometers between each.
The results obtained for the rate of O2 uptake of yeast suspensions by two of the new micro-respirometers and a set of standard Warburg manometers are shown in Table 3.
The 5 μl. samples were measured with a Linderstrom-Lang type-constriction pipette. Pipetting errors probably account for the variations in these experiments.
Appendix II
Assembling the apparatus
I. Filling pipette
Measure diameter of plunger (taking care not to bend it). Examine under binocular microscope to see that there are no sharp edges that might scratch the vulcanite washers.
Place vulcanite washers on plunger. Lightly grease with Lubriseal and place a small drop of Hg in the recess. Place reservoir over plunger. Screw down collar.
Take a suitable length of capillary and fit into end of reservoir. Warm and place Apiezon wax ‘W’ on joint. Allow to cool.
- Place Hg filler over reservoir and capillary and fill with Hg as shown (Text-fig. 9). Evacuate. Test to see that there is no leak in reservoir. Then cover end of capillary with Hg from side arm and remove from vacuum pump. The Hg will then be forced into the reservoir.Text-fig. 9.
Test again by turning on pump and observe mercury meniscus in capillary.
Remove from filler and screw down micrometer to expel the Hg from the capillary, until, when the plunger is again withdrawn, the mercury can just be seen at the joint between the glass and the reservoir.
II. Assembling manometer component
Slide the manometer frame into position and clamp the stainless steel block on to it.
Bend the pipette capillary with a microflame until open end drops into the centre hole in stainless steel block.
Remove the stainless steel block.
Warm the ends of the capillary U-tube and melt on a drop of Apiezon ‘W’. Do the same to the open end of the pipette capillary.
Replace the block and heat it with the flame until the wax on the pipette capillary begins to flow over the metal. Then place, the U-tube in position. Heat the block until wax ‘melts ‘on to the surface.
Allow to cool. Place Perspex guard in position, and fit rubber tubing to nipples on the valves and pipette casing.
With a bent capillary place a drop of odourless kerosene in the U-tube which should have been carefully cleaned before assembling.
Inspection before use
Screw micrometer up and down and ensure that the mercury is moving smoothly.
Examine the valve seatings to see that the edges of the holes are not scratched.
Examine pointed ends of the needles. Warm in a flame and grease lightly.
Test ground surfaces of vessels. A small drop of water sufficient to cover the ground surface when it is pressed on to the slide should be enough to make the vessel stick to the slide.
Tilt instrument and observe kerosene to ensure that it moves freely.
Caution
Care should be taken not to allow kerosene to touch the waxed joints. A small roll of filter-paper in the cavities in the block when adding kerosene is a useful safeguard.
Appendix III
The Amplifier
The circuit shown in Text-fig. 10 forms the link in the servo mechanism which controls the setting motor.
To avoid difficulties of d.c. amplifier operation, the light source is chopped at 500 c.p.s. and is focused as described on to the photocell (‘Cintel’ V.A. 31 made by Messrs Cine Television Ltd.)—a vacuum-type photocell with a sensitivity of about 10 μA/lumen. This is followed by a cathode follower stage with an effective input impedance of 25-30 MΩ. The cathode follower output is fed to a two-stage amplifier with a maximum gain of 10,000: gain is controlled by a variable negative feedback. The output from the amplifier stage at 500 c.p.s. is rectified in a voltage doubler circuit. The positive going d.c. output from the rectifier is connected directly to the grid of the final valve, which is arranged normally to have a standing bias of—15 V., under these conditions it is cut off and the motor relay therefore opens ; the relay operates at a grid voltage of −3, i.e. for a rectified signal voltage of 12 V. At maximum gain the system should thus be operative for a light input in the order of 10-4 lumens, in practice about 10-3 is available at the photcell.
The circuit has been thoroughly decoupled, particular care being taken to isolate the relay operating surges from the common h.t. supply. Battery operation is used throughout for both the h.t. and l.t.
SUMMARY
A new micro-respirometer is described, capable of measuring rates of volume change from 5 μl./hr. down to 0·01 μl./hr. to the nearest 0·001 μl. It is a constant-pressure nul-reading instrument and the actual volumes of gas absorbed or evolved are obtained from readings of the instrument by a simple multiplication.
A device is described which sets the instrument automatically and records the volume changes at regular intervals.