A NEW FORM OF MICRO-RESPIROMETER; WITHA NOTE ON THE EFFECT OF CLEAVAGE

ON THE RESPIRATION OF THE EGGOF RANA

BY ALBERTO STEFANELLI

From the sub-Department of Experimental Zoology, Cambridge

{Received 7 October 1936)

(With Eight Text-figures)

IN order to observe the respiration of single egg-cells it seemed desirable to constructa respirometer of greater sensitivity than those designed by Barcroft (1908) orWarburg (1926), or than that of any of the various modifications of these two instru-ments (Novy et al. 1925; Thunberg, 1905; Winterstein, 1911; Fenn, 1927-8; Fraps,1930; and Kriiger, 1934).

The type of manometer recently described by Duryee (1936) can, in its mostsensitive form, record the respiration of a frog's blastula but not that of a singleundivided egg-cell. Duryee's instrument, apart from its relative complexity, doesnot permit of close inspection of the egg whilst the latter is enclosed in the apparatus.In order to overcome these difficulties I have constructed the simple but extremelysensitive manometer which is described below.

The instrument, in its simplest form, is shown in Fig. 1. Two small cylinders(a, b) are attached to a flat glass plate (4x9 cm.) by means of Canada balsam. Eachcylinder has an internal diameter of 4 mm., is approximately 6 mm. in length, andhas therefore a volume of about 80 mm.3 Into each cylinder opens the end of acapillary manometer (approximately 0-002 mm. in diameter). The other arms ofthe manometers are attached to the upper end of the glass plate and are open to theair. Each cylindrical container is connected to another capillary by a closing device(c). The latter consists of a small L-shaped tube containing mercury, the height ofwhich can be regulated by a thumb-screw. By means of the screw the ends ofthe capillaries leading from the cylindrical containers can be closed by means of themercury. It was found, in practice, that the pressure inside the containers did notvary with variations in the degree of pressure exerted by the mercury on the endsof the capillaries. The free rim of each container is carefully polished so that thecontainer can be sealed by means of a small coverslip attached by a film of vaseline.The manometers were usually filled with paraffin oil coloured with Sudan III andthe level of the fluid determined by means of a scale graduated into half millimetres.

172 ALBERTO STEFANELLI

The manometers are set up as follows. Strips of filter paper (3 mm. wide and2 cm. long) are curled into cylinders and one is inserted by means of forceps intoeach of the containers (a and b). These strips are then very carefully saturated by10 or 20 per cent KOH solution by means of a micro-pipette, and they then adhereclosely to the inner surface of each container. Great care must be exercised toprevent any of the KOH solution entering the manometer capillaries. The tissue,whose respiration is to be recorded, is then placed on the centre of a small glass

Fig. 2

Fig. 1

Fig. 1. Simple form of reapirometer. (a) container with tissue; (b) thermo-barometer; (c) mercurytap with operating screw.Fig. 2. Section of container (a) in Fig. i. (a) capillary leading to mercury tap; (6) manometer capil-lary; (c) wall of container; {d) coverslip; (<) filter paper with KOH; (/) egg or tissue.

coverslip, and the latter attached by means of vaseline to the free rim of one of thecontainers as in Fig. 2, after which the other container is similarly closed by acoverslip without tissue but bearing a small drop of water. During these operationsthe tap (c) is left open so that the containers are open to the atmosphere. The wholeapparatus is then immersed in a thermostat to a level just below the screw (c) of thetap; by placing the respirometer in a suitable position it is easy to observe both thetissue and the level of the oil in the manometer without disturbing the apparatus.Usually about 15 min. are required for the instrument to acquire a steady tempera-ture, after which the tap (c) can be closed and readings can be taken.

A New Form of Micro-respirometer 173

It will be noted that one side of this instrument acts as a thermo-barometer. Therecording manometer can be calibrated by the method devised by Dixon (1934), butit was found in practice that the volume of gas absorbed by the tissue correspondedalmost exactly to the volume occupied by the difference in height of the oil in themanometer. During the course of a prolonged experiment it is desirable to open thetap (c) periodically so as to record changes over the same length of the manometer

arm

Water"level

Fig. 3. Differential manometer; note the unequal lengths of the capillaries entering the mercury tapat (c). This arrangement enables the instrument to be used either as a simple or a differential mano-meter.

By appropriate changes in design the simple type of manometer can be convertedinto one of the differential type. The latter is shown in Fig. 3. The two containers{a and b) being of equal volume, the movement of the manometer is half thatcharacteristic of a manometer open to the air. By making the ends of the capillarieswhich open into the closing device of unequal length (see Fig. 3) the differentialinstrument can be converted into the non-differential form by raising the level ofthe mercury in the trap to the level required to close only one of the capillaries. Fig. 4shows the results obtained from a neurula of Triton using the manometer first in thenon-differential form and subsequently in the differential form. Since the sensitivity

ALBERTO STEFANELLI

0-006

g

lpti

§

Ioft

0-0-

0-

0-

0

005004

003

002

•001

0

Simple manometer Differential manometer

•-0-005

'-0-OQt

10 20 30 40 50 60 70Time in minutes

60 90 100 110

Fig. 4. Graphs showing rate of respiration of an embryo of Triton alpatris (open neural tube stage).On the left is shown a record obtained with the apparatus when used in the non-differential form;on the right a similar record of the same material when the apparatus was used as a differentialmanometer.

Fig. s Fig. 6

Fig. 5. Complete apparatus showing attachment of calibrated micro-pipette.Fig. 6. Figure showing details of construction of micro-pipette: (a) milled head of plunger screw,(6) fine glass rod attached to plunger screw by rubber collar (c). Note the expanded end of the glassplunger rod.

A New Form of Micro-respirometer 175

of the differential instrument is less than that of the simple type, it is sometimesconvenient (as suggested by Duryee) to increase the volume of the control container;this can be done by connecting it by an aperture through the glass plate to a Petridish cemented firmly on to the back of the plate. In this way the instrument acquiresalmost the same sensitivity as the simpler form, but longer time must be left for theinstrument to reach thermal equilibrium.

When it is desirable to ensure very accurate calibration it is convenient to use thetype of manometer shown in Fig. 5 in which one container is permanently attachedto a calibrated micro-pipette (/) attached to an adjustable pressure device (d). Whencalibrating, the capillary (g), but not capillary (h), is closed by means of the tap (c) anda known volume of gas is removed from the container (a) by rotating the screw (d) ofthe micro-pipette and the difference in the height of the oil in the capillary (e) isobserved; this gives a direct calibration of the instrument. If the same operationbe performed with both capillaries (g and h) closed, and the volumes of the twocontainers (a and b) are equal, the displacement in the level of the oil in capillary(e) is one-half of what is observed when only capillary (g) is closed. If container(b) has a much larger volume than container (a) the sensitivity of the apparatus isapproximately the same whether capillary (h) be closed or not.

The advantages of this instrument are threefold, (i) Owing to the small volumeof the containers the sensitivity is very great, for 0-003 mm.3 can readily be recordedwith a little experience, (ii) It is very easy to construct, (iii) It is possible to observethe tissue at all stages of the observations.

The instrument, described above, was designed in order to measure the rate ofoxygen consumption of a single egg when it is undergoing normal cleavage. Therespiration of dividing cells has been studied by numerous workers, but there is nounanimity of opinion concerning the type of changes in oxygen consumption, if any,which occur during the process of division. It seems not unlikely that some of thosediscrepancies are due to the fact that comparatively large numbers of eggs havebeen observed simultaneously, so that unless the material is extremely homogeneousany change in the level of respiration which is of short duration might be un-observable. The respirometer described in this paper is capable of measuring therespiration of a single egg of Ranafusca every 5 min. or less, so that a large number ofobservations can be made during the period of one division of the egg. Twentyexperiments of this type have been carried out during the three first divisions andthe results indicate that the rate of respiration of a dividing egg is very much morevariable than that of the fertilized egg (see Figs. 7 and 8). In the case of a dividingegg, peaks (as defined by two or more consecutively high determinations) occur inthe graph which are almost certainly beyond the limits of experimental error. Thesepeaks appear to be closely related to particular phases of each cleavage (see Fig. 7).It is obvious that a very considerable number of observations would be necessarybefore it is possible to correlate the observed increases in respiration with definitephases of cell division, but I hope to be able to reach a conclusion on this point inthe near future.

A New Form of Micro-respirometer 177

REFERENCES

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(1928). Amer.J. Pkytiol. 84, n o .FRAPS, M. (1930). Pkytiol. Zool. 3, 242.GERARD, R. W. (1931). Amer.J. Pkytiol. 97, 522.GERARD, R. W. & HARTLINE, H. K. (1934). J. cell. comp. Phytiol. 97, 141.HOWLAND, R. B. & BERNSTEIN, A. (1931). J. gen. Pkytiol. 14, 339.KALAMUS, H. (1928). Z. vergl. Pkytiol. 7, 304.KROGBR, F. (1934). Z. vergl. Pkytiol. 21, 249.Now, F. G., ROCHEN, H. R. & SAULE, M. H. (1925). J. infect. Dit. 36, 109.SCHMIDT, R. O. (1933). Amer.J. Pkytiol. 104, 303.THUNBBRG, T. (1905). Skand. Arch. Phytiol. 17, 74.WARBURG, O. (1926). Vber den Stoffioechtel der Tumoren. Berlin: Julius Springer.WINTBRSTEIN, H. (1912). Biochem. Z. 46, 440.

(1911). Z. Biol. Techn. 3, 246.