In the course of an investigation into the physiology of the edible snail (Helix pomatia L.), it became necessary to measure the respiration individually of large numbers of animals. A system in which CO2-free air was passed over the animals and then through baryta was tried but rejected because the snails apparently dislike currents of air, tending to withdraw into their shells. Analysis of gas samples from containers in which the animals respire was not employed, partly because the accumulation of CO2 involved may conceivably affect the respiratory exchanges, and partly because the accuracy of the method is insufficient except in very experienced hands. It was therefore decided to adopt a manometric method.
Manometrically speaking, the snail falls between two stools. It is neither small enough for the usual microrespirometers nor large enough for the devices commonly employed for such animals as small mammals. The apparatus devised to meet this situation is described in the following note, in the belief that it will be found useful by workers having to make large numbers of measurements on animals of the same order of respiration rate as snails. It should serve, for instance, for cockroaches and locusts. By making the respiration chambers smaller and the measuring tubes narrower its sensitivity could be greatly increased. Its chief limitation, in its present form, is that, not being shaken, it can only be used for air-breathing animals.
The apparatus is of the constant-volume type, i.e. it works in the same way as the Warburg microrespirometer, the CO2 given out by the animal being absorbed by KOH. The main drawback to the constant volume system is that the ratio of manometer excursion to amount of oxygen consumed varies with the gas volume in the respiration chamber, so that if, as was the case in the present investigation, measurements are made on a series of animals of different volumes the apparatus must be calibrated for each experiment. A method was, however, devised (similar to those used by Warburg (1923) and by Barcroft & Higgins (1911), for determining the volume of respirometer flasks) by means of which a calibration can be carried out very simply and quite accurately in a few seconds. The advantages of the constant volume, over other manometric systems, are great. The chief is that, as one is measuring pressure changes only, the control (thermobarometer) and experimental vessels need not have exactly the same volumes, so that the apparatus can be cheaply and easily constructed. For the same reason the Warburg system can be “polymerized”, one empty vessel being coupled as control to a whole series of experimental vessels containing animals. This results in considerable economy of space. In the constant-pressure system, as used for instance in the Haldane manometer, each experimental vessel must have its own control, and as the size of the snail necessitates the use of large vessels, the resulting lay-out would be very cumbersome.
CONSTRUCTION OF THE APPARATUS
A series of glass tubes of 4 mm. bore are mounted on a vertical board which is rigidly screwed to the bench (Fig. 1). They communicate below with each other and with a reservoir (G) whose height can be varied. It is important that the reservoir be mounted in such a way that its movements can be rapidly and exactly made ; Messrs C. F. Palmer’s adjustable screw stand, catalogue number D 12, being excellent for the purpose. The reservoir and lower ends of the tubes contain an aqueous solution of fluorescein, which gives a very clear meniscus. One of the tubes (A) is open above to the air and provided with a millimetre scale, 30 cm. long. The others (B) communicate above with the respiration chambers. These B-tubes have no scales ; instead, each of them has in front of it a glass plate on which two fine horizontal lines, 8 cm. apart, have been ruled with a diamond. The glass plates are held on to plywood blocks (shown stippled in the figure) by means of plasticine blobs (not shown) ; this method of support, while being sufficiently rigid, allows of slight adjustment of the plates at the beginning of an experiment, when the menisci are all set to the upper cross lines. To eliminate parallax in setting the menisci to the cross lines, strips of mirror (not shown) are fixed behind the B-tubes in the region of the glass plates.
The respiration chambers lie behind, immersed in a thermostatically regulated water-bath (F). Each communicates with its B-tube through the connecting tube CE, of 2 mm. bore. It can also be opened to the air through tap D. At C and E are joints of thick-walled rubber tubing. When the respiration chamber is removed from the bath at the end of an experiment it is disconnected at C. Joint E allows sufficient flexibility for this purpose, and serves also to afford a grip for a clamp, a series of which, carried on a horizontal rod running between the taps D and the thermometers and not shown in the figure, hold the respiration chambers in place in the bath.
The respiration chamber itself consists of a 5 in. length of glass tubing of in. internal diameter, closed above and below by rubber bungs. The lower bung is permanently cemented into position ; when the apparatus is being set up for an experiment, thin Canada balsam is run round the upper to eliminate leaks and slip. Within the chamber is a wide-mouthed dish containing 15 ml. of 20 per cent KOH, and guarded above by a disk of perforated zinc (dotted line). The animal is placed above the disk. It is of course important, while handling the respiration chamber, to avoid splashing KOH on to the animal, which would be injured, or on to the zinc, which would evolve hydrogen. If the KOH is coloured with thymol blue it is easily watched and there is no danger of its going astray. Vaselining the inside of the upper part of the KOH vessel is also helpful. If KOH comes in contact with a snail the fact can be detected at once, as it causes a brilliant yellow discoloration of the mucus.
There is no theoretical limit to the number of B-tubes, and of respiration chambers, which can be put together on one apparatus. The writer finds that four or five tubes make a convenient assembly. As it is easy to run two or three such apparatuses at the same time, the respirations of a dozen animals can be measured simultaneously by a single worker.
METHOD OF OPERATION
One of the respiration chambers contains no animal and serves as control (thermobarometer). The rest contain animals.
After setting up the respiration chambers, they are left for hours with the taps open to come into temperature equilibrium with the bath. The menisci are then all levelled to the upper cross lines by means of reservoir G and the taps are closed (the lower cross lines are used only in calibration—see below). Thereafter the pressure changes in the chambers containing the animals are measured at intervals. This is done by levelling the meniscus in the control tube to the upper line and reading A, then immediately levelling one of the others to the upper cross line and again reading A. The difference between the two readings gives the pressure difference caused by the oxygen consumption of that particular snail since the taps were closed. Any fluctuations in temperature or barometric pressure will affect all the chambers equally. The writer’s practice, with snails, is to read each tube (by comparison with the control tube) every 10 min. or so for an hour.
It is easy to calculate G1 from the barometric pressure at the moment of closing the tap, the temperature indicated by a thermometer whose bulb is placed behind the glass plate carrying the cross lines, and the volume of the tube between the cross lines. The latter quantity can be determined once and for all, by setting up an empty respiration chamber and comparing the result of a calibration carried out as just described with that of a Wünzer and Neumann calibration.
SENSITIVITY AND ACCURACY
The sensitivity of the apparatus is inversely proportional to the volume of the respiration chambers. As here described, it gives a scale difference of about 4 cm. per ml. oxygen consumed by the animal.
The chief factor limiting accuracy is the calibration method. In order to have a definite standard of accuracy the scale-tube A is always read to the nearest millimetre. A worker used to handling the apparatus and setting the meniscus gets figures which are identical or differ only in 1 mm. in consecutive calibrations. With the dimensions as described above, A2—A1 in the calibration equation is over 60 mm. so the calibrations are consistent to within 2 per cent. In reading the actual respirations the scale is again read to the nearest millimetre and the results are repeatable, so the measurements may be taken safely as good to within 4 per cent.
If the thermostat bath is well stirred and accurately regulated (the writer uses two mechanical stirrers and an ordinary mercury-toluol thermoregulator controlling a gas jet) and if plenty of time is allowed for temperature equilibrium to be attained before beginning the measurements, no significant differences appear between the various members of a series of empty respirometer chambers set up on the apparatus.
EFFECTIVENESS OF CO2 ABSORPTION
The great importance of this factor as a potential source of error was stressed by Dixon & Elliott (1930) in a discussion of the use of Barcroft differential manometers as microrespirometers. If the animal in the respiration chamber is giving off CO2 at a constant rate, and if the rate of absorption of the CO2 is proportional to its concentration in the chamber, the result will be that a steady state is ultimately attained when the rate of evolution of CO2 is equal to the rate of absorption. If the absorption is inadequate, i.e. if the ratio of absorption rate to CO2 concentration is too low, then two disturbing factors may arise. The first is that the concentration of CO2 present when the steady state is reached may be high enough to affect the respiration of the animals. The second is that the steady state may only be reached very slowly, and may be incompletely attained at the time when the taps are closed and readings are begun. In the latter case, besides the respiration of the animals, the manometer will record a change in volume due to the gradual attainment of the steady state. Dixon & Elliott showed that considerable errors may arise from this cause if, in the Barcroft apparatus, the KOH is present as solution in the centre tube of the bulb. In view of these results, the rapidity of CO2 absorption in the respiration chambers of the present apparatus was critically investigated. The treatment which follows is modified from that of Dixon & Elliott.
x = volume of CO2 in the respiration chamber at any moment, in ml.
c = absorption constant, so that rate of absorption of CO2 by the KOH is cx ml. per min.
R = rate of evolution of CO2 by the animal, in ml. per min. It will be assumed that this is constant, which is reasonably true for snails.
If therefore the taps are closed, and readings are begun, t1 min. after setting up the apparatus, the manometer will record, in addition to the oxygen consumption of the animal, a volume change equal to expression (3) above.
In order to assess the value of x at the steady state and the error due to incomplete attainment of that state when the taps are closed, it is necessary to measure the constant c. This is done by setting up a respiration chamber with KOH but no animal, liberating CO2 into it, and following manometrically the absorption of the gas.
Dixon & Elliott liberated CO2 by the action of oxalic acid on sodium bicarbonate when testing Barcroft manometers. As in their respiration experiments they studied tissues bathed in saline and the CO2 was therefore liberated into a fluid phase ; their method of determining c corresponds to the conditions of their respiration experiments. In the apparatus here described, however, the animals liberate gaseous CO2 and in studying CO2 absorption it therefore gives a truer approximation to the respiration conditions if gaseous CO2 is injected into the respiration chamber.
For injecting CO2, the pipette shown in Fig. 3 was used. It is inserted into the upper bung of a respiration chamber in place of the thermometer. The manipulation of the pipette is as follows : mercury is poured in to the level X. The instrument is then held in the horizontal position, the mercury flowing into the bulb, and CO2 is led in from a cylinder through A, and out through a rubber tube of which one end is under water and the other attached to B. After allowing sufficient time for the pipette to be filled with pure CO2, tap C is closed and the pipette turned to the vertical position. The mercury fills the right-hand limb of the U-tube, imprisoning CO2 in the left. The pipette is now set up on a respiration chamber, both the chamber and the U-tube of the pipette being immersed in the bath. A second respiration chamber is set up to serve as thermobarometer. After sufficient time has been allowed for temperature equilibration, the taps of the respiration chambers are closed and the menisci levelled to the upper cross lines. The tap on the pipette is then opened and quickly closed again, CO2 being thus injected into the respiration chamber. A baffle-plate of cellophane, D, carried on a silver wire, removes the suspicion that the CO2 is being shot straight down on to the surface of the absorbent ; it can be bent aside while the pipette is being filled with CO2. After the injection, the difference in reading between the two chambers is taken in the usual way at intervals of 2 min., and thus the absorption of the gas is followed.
As c has the dimensions “ratio per unit time ”, it is not necessary to calibrate the apparatus, or to know the amount of CO2 injected (=a) in the tests. Neither factor will affect the slope of the graph. It is only necessary to plot the logarithm of the manometer reading against time in minutes.
The results of three such tests are shown in Fig. 4, the graph giving a value for c of 0·156 per min. Using this result, we can now evaluate the terms in equations (2) and (3) above.
The amount of CO2 present in the steady state will be . In the case of snails, basing our estimate on oxygen consumption figures and assuming a respiratory quotient of 1, R runs near to but does not exceed 0·1 ml. per min., so the volume of CO2 present will be anything up to 0·645 ml., depending on the activity of the animal. As the total volume of the respiration chamber is about 250 ml., this means a steady state of anything up to 0·26 per cent CO2. It is very unlikely that this is enough seriously to affect the respiratory rate.
The possibility may here be pointed out of controlling the humidity within the respiration chambers over a wide range. The value of c found above is unnecessarily good and the strength of KOH could probably be widely varied without lowering the absorption constant to an inefficient level. In this case the humidity in the respiration chamber can be regulated according to the graphs given by Buxton (1931). If 20 per cent KOH is used, as described above, the relative humidity will be about 85 per cent.
A manometric apparatus for studying the oxygen consumption of large land invertebrates is described. Essentially it is a “polymerized” Warburg manometer. Simplicity of construction and manipulation have been aimed at, the apparatus being intended for use where large numbers of routine measurements have to be made. The effectiveness of CO2 absorption is experimentally investigated, and factors limiting the sensitivity and accuracy of the apparatus are discussed.