A microelectrode to measure dissolved oxygen in insect larvae

J. Ins. Physiol., 1965, Vol. 11,pp. 817 to 830. Pergamon Press Ltd.

Printed in Great Britain

A MICROELECTRODE TO MEASURE DISSOLVED OXYGEN IN INSECT LARVAE BERNARD Northern

Regional

Research

A. WEINER Laboratory,*

Peoria,

Illinois

(Received 17 November 1964)

Abstract-Variations in values for dissolved oxygen in the haemolymph of insects occur because of air exposure during collection. This fluctuation is caused by oxidation of phenolic substances and general uptake of oxygen. To determine dissolved oxygen polarographically in the larval haemocoele of Popillia japonica Newman, an indicator cathode was devised and housed in a 27-gauge hypodermic needle. Knowledge of this value was needed in the development of infective spores to control the Japanese beetle.

INTRODUCTION

THE difficulties encountered in oxygen analyses by blood collection have been noted by BUCK (1953). One method that has proved useful for the determination of oxygen in biological systems is based on polarographic principles. The theory has been given by KOLTHOFF and LINGANE (1952). Polarographic measurement of oxygen uptake by mitochondria was studied by HAGIHARA(1961). He used a rotating platinum electrode by modifying the stationary electrode system of DAVIES and BRINK (1941). Tissue respiration was investigated by STICKLAND(1960) who modified the CLARK (1955) electrode. Both types of electrode were used with biological materials in closed systems in contact with them. KREUZERet al. (1960) developed a catheter type of oxygen electrode which incorporated the platinum electrode by Clark and used it for continuous recording of blood oxygen tension in Z+ZXLWhile the diameter of their small catheter was equivalent to a 16-gauge needle (O-062 in.), they stated that considerably smaller catheter-electrodes were built. To measure the oxygen content of living insects, a small insertable electrode would be necessary. This paper describes the construction and operation of such an electrode. Our electrode system consists of a platinum cathode (indicator electrode), calomel anode, a battery to supply the polarizing voltage, and a means for determining the amount of electrical current produced by electrolysis. The anode and cathode are connected by a salt-bridge, and the indicator electrode is coated with an oxygen-permeable membrane of collodion. * This is a laboratory of the Northern Utilization Research and Development Agricultural Research Service, U.S. Department of Agriculture. 52

817

Division,

BERNARDA. WEINER

818

When a voltage of -0.7 is passed between anode and cathode, free energy is supplied so that oxygen may be reduced to hydroxyl ions. The electrical current produced, the result rather than a cause of electrode reactions, is measured in an external circuit which contains a recorder. The amount of current is directly proportional to the concentration of oxygen. MATERIALS

AND

These items were used in construction the polarographic system:* Item

METHODS

of a microelectrode

Description

and made part of

Source

Recorder

MR Recorder

E. H. Sargent and Company, Chicago, Illinois

Calomel electrode

No. 39270, 2.5 in. long

Epoxy resin

Kits supplied either as all paste or all liquid with directions for use

Platinum wire

0.001 in. diameter

Copper wire

Insulation appears to be resistant to constant immersion in salt solution and occasional exposure to steam sterilization. No. 1030E-80 This is diluted with diethyl ether, 3 volumes. The addition of 05-2 ml ethyl alcohol (95%) to 40 ml collodion-ether will clarify the slightly turbid solution (HAGIHARA,1961)

Beckman Instruments, Inc., 2500 Harbor Boulevard, Fullerton, California The Epoxylite Corporation, 10829 E. Central Avenue, El Monte, California Englehard Industries, Inc., 113 Astor Street, Newark, New Jersey Tensolite Insulated Wire Co., Tarrytown, New York

Collodion

Battery Resistors

Decade box

No. 6, 1.5 V Conventional carbon resistors with 10 per cent accuracy were selected for values close to 5000 R to serve as voltage dividers used in conjunction with the battery The decade box used as a shunt for the recorder had a range in excess of 100,000 a. lA-32-P

Does not appear to be significant

Eveready Local supplier

General Radio Co., 30 Baker Avenue, West Concord, Massachusetts

* Mention of specific equipment or firms is given as part of the exact experimental conditions and not as an endorsement of the products named over those of other manufacture.

A MICROELECTRODE

OXYGEN IN INSECT LARVAE

Description

Item Shielded

TO MEASURE DISSOLVED

cable

Nitrogen

Gas flow rate device

Polarographic current was carried from the needle to the recorder and back to the calomel electrode by means of shielded wire Standard tank

06-l

5 tubes

819

Source The Belden cable was obtained from local suppliers

National Cylinder Gas Division of Chemetron Corporation, Chicago, Illinois Fisher-Porter

Construction of needle electrode The need for diminutive equipment because of the size of the grub led to the use of a 27-gauge hypodermic needle for insertion into the haemocoele of larvae_ Small electrodes mean decreased areas of exposure for platinum wire. Since the amount of electrical current produced is proportional to the area of exposed platinum, a sensitive recorder is used to amplify the small currents. In this case, 60 mpA causes full-scale deflexion of the recorder. The anode consists of a slightly modified calomel electrode on which an epoxy resin is applied to the point from which wire emerges from the Bakelite head and at the junctions of plastic and glass. The cathode consists of platinum wire insulated and housed in a conventional 27-gauge hypodermic needle with a 0.75 in. shaft (Fig. 1). Glass insulation for the platinum wire was fabricated from 5 mm Pyrex glass tubing, drawn in two steps: the initial diameter was about 1 mm and the final diameter (O.D.) was 0.006 in. Platinum wire, 0.001 in. in diameter, was fed into the glass capillary with sharply tipped tweezers. This step was one of many done in construction under a dissecting microscope (10 x ). The glass-insulated wire consisted of about 1.5 in. of glass capillary and 3 in. of platinum wire, which extended slightly beyond one end of the glass tubing. This end was fed carefully into the needle and out through the hub. The wire was then drawn out of the glass tube at the hub end so that 0.25 in. protruded from the glass tube at the tip of the needle. The glass tube itself extended O-5 in. beyond the tip of the needle. At this point, the glass-platinum junction at the tip of the needle was sealed over a cold, small, yellow flame. The sealed wire was drawn into the needle shaft to expose the bevel. A layer of epoxy paste was applied to the inside of the bevel (see Fig. 1). The paste was cured at 65-70°C for 20-30 min. The ‘bed’ of epoxy plastic was made thin enough so that the glass tube could be redrawn O-75 in. out of the tip of the needle. Epoxy paste was then applied to the hub end of the bevel of the needle while drawing the tubing slowly back into the needle. When the glass tube had been drawn just into the shaft of the needle, it was cured once again in the oven. The wire was cut to a length just short of the end of the needle and the tip of the wire was embedded in epoxy paste. A ‘wall’

820

BERNARDA. WEINER

was built to protect the bare platinum wire from abrasion during insertion into the insect by using the bevel edges of the needle as ‘footing’. The needle was fastened with rubber bands to a suitable stand such as a 50 ml graduated cylinder. The platinum wire, which extended from the glass tube at the hub end of the needle,

Insulated

Copper

Wire ,.*’

_/

,/*

_/’

__.‘-

I’ :

Top View

/*

:

/=

: 1’ :

__________L~__ ....___.__j_,_.

Platinum

Wire

,

Bare

Side View Cross Section

Glass Tube,

sealed

FIG. 1. Diagrammatic

to Pt. Wire

views of the oxygen

lmbedded

Tip of Pt. Wire

cathode housed in a hypodermic

(-)

needle.

Cathode; needle

(t) Calomel anode

FIG. 2.

The electrical

circuit used with a recording of the microelectrode.

potentiometer

in the operation

FIG. 3. Electrodes and t h e r m o s t a t i c cradle in use.

A MICROELECTRODE

TO MEASURE DISSOLVED

OXYGEN IN INSECT LARVAE

821

was soldered to insulated copper wire, A conventional solder joint was formed as close to the needle hub as possible. The solder joint was washed with carbon tetrachloride, and care was taken to prev-ent the entrance of solvent into the glass capillary. Finally, enough epoxy liquid was poured into the hub of the needle to cover the solder joint and the plastic cured again to complete construction of the microelectrode. The electrical circuit for the polarographic system is shown in Fig. 2, and the various components are described in the tabular listing. The microelectrodes were coated with collodion by slipping them five to ten times into the dropper from a dropping bottle containing collodion solution and connected into the circuit overnight. Diffusion current decreased quickly from 2 to 3 PA to 0.1 PA. The tracing was kept on the chart by use of the range selector dial and variation of shunt resistance. Maintenance of constant temperature To avoid current variation owing to temperature changes, two thermostatic devices were fabricated. First, a jacketed cylinder was built 5 x 45 cm in size internally and fitted with glass tubing that allowed samples to be drawn from the bottom of the inside cylinder. Water from a constant temperature reservoir was pumped at the rate of 1 l/min through the jacket to maintain a uniform temperature. The inner cylinder was filled with 0.25 M potassium chloride solution, chosen because its ions have nearly equal transference numbers. The salt concentration used was an attempt to duplicate the colligative properties of the haemolymph, which have an effect on the transfer of electrical current. The colligative properties of the blood were based on a freezing-point depression as reported by LUDWIG (195 1). Gases were introduced near the bottom of the cylinder through a sintered glass sparger. The platinum needle electrode was allowed to rest on the surface of the sparger, while the calomei electrode was suspended in the salt solution. The second thermostatic device was a cradle to hold the grub in a fixed position at constant temperature while measuring the dissolved oxygen of its haemolymph (Fig. 3). The cradle was fashioned out of an aluminium block 7 x 10 x 2.5 cm. A 2 cm hole was drilled through the longitudinal axis forming a channel which allowed a flow of water at 25°C at the rate of 1.3 l/min. Three small blocks were machined and fastened to the main block by small bolts (Fig. 4). One block, shaped like a hollowed half-cylinder, served as the bed for the grub. Another was drilled to accommodate the calomel electrode. The third, shaped like a hollow oblong, served to immobilize the microelectrode by means of a knurled bolt which was tightened against a brass insert after the needle was in place. A Teflon sheet, 0.001 in. thick, was placed between the grub and the cradle to prevent electrical shorts. Also, the electrodes were wrapped with Teflon sheeting to insulate them from the aluminium housing. This intimate contact of small blocks to the thermostatic metal assured excellent heat transfer, and both larvae and electrodes quickly assumed the temperature of the cradle. A salt bridge between the grub and the calomel electrode was made by rolling dry tissue paper which was then soaked in a saturated potassium chloride solution.

BERNARD A. WEINER

822

Finally, a rectangular Lucite box, which had inlet and outlet gas tubes and a hole lined with a rubber grommet to hold a thermometer, was fitted over the top of the cradle to enclose the larva completely. This compartment allowed changing the gas environment of the grub. Side

__

Appjoximate

Top

View -.

View ~--

___~

$calelL

FIG. 4. Construction diagrams for the thermostatic cradle: (1) needle cathode, (2) Teflon sheet to insulate needle from metal, (3) brass shim to protect Teflon from turning bolt in needle housing, (4) needle housing, (5) Teflon sheet to insulate larva from metal cradle, (6) aluminium block to house inner metal cradle, (7) inner metal cradle, one of several trough sizes, (8) thermometer, (9) Lucite housing for calomel electrode, (10) calomel electrode, (11) gas-inIet tube in Lucite enclosure, (12) aluminium block, (13) channel in aluminium block through which water flows to maintain constant temperature, (14) wire fasteners for rubber tubing, and (15) bolt to immobilize inner metal cradle.

A MICROELECTRODE

TO MEASURE DISSOLVED

OXYGEN IN INSECT LARVAE

823

Standardization Standardization of the platinum-calomel system for the determination of dissolved oxygen was done by chemical determination and reference to the literature. Sulphite oxidation values (COOPER et al., 1944) gave an average saturation value of 0.215 mM 0,/l at 25°C. The Pomeroy-Kirschman-Alsterberg modification of Winkler’s method (American Public Health Association, 1960) resulted in 0.228 + 0.002 mM/l. MC_qRTHUR (1916) reported a value of 0.237 mM for the same volume, temperature, and salt solution. Standardization was accomplished by mixing nitrogen and air in a suction flask equipped with gas flow-rate devices while maintaining a source pressure of 10 lb/in2 for both air from a house line and nitrogen or oxygen from a tank. With this source pressure, constant gas percentages were more easily achieved although occasional adjustment was still needed. The

80 -

0

4

8

12

16

20

FIG. 5. Response of platinum-calomel polarographic system to oxygen in salt _ __ _. _ _ solution (O-0) and in the haemocoele of the Japanese beetle larvae (.-.-.?

mixture was then led to the fritted glass sparger in the standardization cylinder containing 0.25 M KC1 solution at 25°C. Fig. 5 shows that the diffusion current is proportional to partial pressure of 0,. Nitrogen was used to establish the point of residual current level (zero per cent oxygen). This value varied from O-4 to 5 per cent of the current produced by a salt solution saturated with air. In most cases, residual current greater than 1 per cent could be traced to electrical shorts in the needle and improper grounding of apparatus. When nitrogen was scrubbed by sodium pyrogallate solution and compared to untreated gas, no difference in residual current could be detected. Electrode metals Two silver wires, one mil in diameter, were coated with Lucite, installed in a 27-gauge needle, and packed (by a vacuum technique) with epoxy resin. While it

824

BERNARDA. WEINER

was possible to obtain electrical current from oxygen reduction, the minuteness of the exposed silver electrodes and the high degree of polarization obscured the data. PHILLIPS and JOHNSON (1961), however, made electrodes of silver and obtained satisfactory results when probes were used instead of needles. RESULTS

AND DISCUSSION

E#ect of membrane thickness and temperature KINSEY and BOTTOMLEY (1963) reported that the diffusion coefficient varies with membrane thickness. If different types of membranes were used or if strict control of their thickness could not be maintained, changes must be made in the compensating network involving thermistors. Reports that diffusion current varied according to the thickness of semipermeable membranes used (SAWYER et al., 1959; HAGIHARA, 1961; KINSEY and BOTTOMLEY, 1963) were supported by additional data obtained on collodion membranes experimentally prepared (Table 1). The membranes were formed by dipping into a collodion solution. TABLE ~-EFFECT OF MEMBRANE THICKNESSON DIFFUSIONCURRENT

Description

Diffusion current, m@

Bare electrode (platinum) One layer of 25% collodion Three layers of 25% collodion Ten layers of 25% collodion

118 77 66 38

“/6 decrease in current

34 44 68

An amperage change of 3 per cent per degree of Centigrade occurs with our system. This change compares to 4 per cent in the electrode (Pt-Ag) used by KINSEY and BOTTOMLEY (1963). SAWYER et al. (1959) report a temperature coefficient of 5 per cent and CHARLTON also provides a most useful list of temperature (1961), of 4 per cent. Charlton coefficients for given temperature ranges as a function of membrane type and thickness in both moving and still liquids. Variation in diffusion current was logarithmic with temperature (Fig. 6). Our results are in contrast to those by CARRITT and KANWISHER (1959) who report temperature coefficients of 4 per cent per degree at 10°C to about 8.5 per cent per degree at 25°C. As noted, temperature coefficients of the platinum-calomel system were determined. In the following experiment the site of temperature dependence in our Separate containers were connected by an agar-saltsystem was also investigated. When the vessel containing the bridge and brought to varying temperatures. platinum electrode was maintained at 25°C and when the vessel containing the calomel electrode was dropped to 5.3 + 0.7”C, no change in diffusion current

A MICROELECTRODE

TO MEASUREDISSOLVED

OXYGEN

825

IN INSECTLARVAE

occurred. If the electrodes were interchanged, platinum exposed to 5.3 f 0*7”C, and calomel held at the higher temperature, a 50 per cent drop in diffusion current resulted (temperature coefficient was 2 per cent per degree).

7 log Diffusion

Current,

millimicroamperes

FIG. 6. The effect of temperature

on diffusion currents of -0.7 V vs. S.C.E.

at a constant

potential

Data from Table 2 were used to plot an estimate of the energy of activation (Ea) for oxygen diffusion. The Arrhenius equation was used in the form log K = (&z/2-3R)(lJT)+C. A reaction rate constant, K, was given by diffusion

TABLE

~-ENERGY

Temperature “C 14.78 20.67 24.83 29.83 40.50

OF ACTIVATION

FOR THEDIFFUSION

OF OXYGEN

IN O-25 M KC1

Diffusion current, i, m@ 17.7 21.5 25.5 30.0 46.9

Log i 1.248 1.332 1.465 1.477 1,671

Ai/APC

0.65 0.84 1.00 1.58

l/T”

x 103 3.50 3.41 3.36 3.31 3.19

current, i, found at absolute temperature, T, where R was the gas constant and C a constant of integration. Energy of activation for the diffusion of oxygen in

826

BERNARDA. WEINER

0.25 M KC1 was the slope of the line obtained by plotting log i against reciprocal of absolute temperature. A value of 460 Cal/mole was obtained.

the

Response time A response time for the needle to oxygen while in standard salt solution was measured by noting the time required for initial deflexion of the recorder pen to changes in gas. When air was replaced by N,, the response time was 6-10 sec. Approximately 4 min was required for the diffusion current to decrease to 5 per cent of the maximum level. When nitrogen was replaced by air, lo-15 set elapsed before oxygen reduction was recorded. Of course the response time will vary with the thickness of the collodion membrane, a thin membrane giving the fastest response. When oxygen content was measured in larvae, response times of 1-3 set were observed with the polarographic system. An inanimate system simulating the grub was then prepared in which diffusion was the primary method of gas transport. This system allowed comparison of gas transport in the larvae to an in vitro system. The synthetic conditions were set by the use of 0.2 ml of 0.25 M KC1 solution in a 8/32 Visking casing in which a small glass rod was placed to simulate the gut of the grub. The synthetic tube was also placed in the larval cradle in contact with a paper salt-bridge. The indicator electrode needle was injected laterally into the

I

I

100

200

:

Time, set FIG. 7. Rate of diffusion of oxygen through larval skin and into the haemocoele of the Japanese beetle grub. I@ = 0.01 sec.

Visking-casing cavity and the calomel electrode placed in contact with the saltbridge. A response time of 2 set to N, or air was indicated on the recorder. This strong similarity in speed of response between larvae and a physical system supports the contention that transport of oxygen in Japanese beetle larvae is largely controlled by diffusion (WIGGLESWORTH, 1956) and is quite rapid. Diffusion is zero

A MICROELECTRODE

TO

MEASURE

DISSOLVED

OXYGEN

IN

INSECT

LARVAE

827

order for the initial quarter minute and first order (K = O*Ol/sec) until equilibrium with air is reached as shown in Fig. 7. Stability of needle The stability of a platinum-calomel system with time has varied from zero to 0.3 per cent in terms of decrease in diffusion current per hour. Decrease of diffusion current has been termed polarization and this phenomenon appeared to be linear with time. HAGIHARA(1961) discussed this problem and showed that negligible polarization occurred in a 10 min recording after initial exposure to protein solution. Reduction of diffusion current was said to occur because of ‘reduced activity of the active surface of the platinum electrode due to interference by the components of the mitochondrial suspension, mainly proteins’. Electrolyte concentration and pH Constant temperature and air-flow rates were maintained while increments of 4 M KC1 were added to the jacketed cylinder containing distilled water, the needle, and the calomel electrode. Diffusion current held constant while molarity of the salt solution increased from 0.02 to O-61. Further addition of 4 M salt solution to a concentration of 1.2 M caused a 7 per cent drop in current. The drop was expected since solubility of oxygen decreases with increase in ionic strength (MCARTHUR, 1916). Dilute acid and base (0.05 M) were added to an air-saturated salt solution (O-25 M KCl) in the standard cylinder. The pH varied from 1.8 to 12.8, but the diffusion current remained constant. Velocity of liquid past collodion membrane The effect of rate of air flow into the standard cylinder on diffusion current was also investigated. The needle was fastened 2-3 mm from the end of a glass rod and the rod allowed to rest on the sintered-glass sparger. Air-flow rate was varied from 2-O to O-1 l/min and a 2.3 per cent drop in electrolytic current was noted. This decrease agrees with work done by KINSEY and BOTTOMLEY (1963). A short reaction time by the needle to the presence of air was evident from the increasing range of oscillation of the recorder pen at low rates of air flow. Thus, there was no oscillation down to an air flow rate of 0.9 l/min, but 4 per cent of total excursion of the pen at air saturation was seen at O-1 l/min. Finally, when air sparging was stopped and the solution became still, a drop in diffusion current of 8-10 per cent occurred. KREUZER et al. (1960) stated that a minimum velocity of l-2 cm/set was necessary if little or no change in diffusion current was desired. During efforts to estimate oxygen content of larvae, the effect of velocity of the solution past the needle tip was considered. Grubs were pierced by the polarographic needle while they were under the influence of ether. Anaesthesized grubs were easier to manipulate and lessened the chance for haemorrhage when the needle was inserted. The grub was then ‘glued’ in place by perforated, clear, adhesive tape and exposed to an air flow of 2 l/min. Oxygen levels were taken only

828

BERNARDA. WEINER

when the grub resumed movement several minutes later. The undulation of the trapped grub should provide the velocity of liquid past the cathode so that comparison to standard conditions in salt solution could be made. Performance of needle in larvae More than 100 larvae of the Japanese beetle, third instar, normal in appearance, were used to establish dissolved oxygen content. The needle and calomel electrode were allowed to equilibrate in 0.25 M KC1 so that a constant value was obtained on the recorder. Each grub was anaesthesized by a paper roll saturated with ether in a closed Petri dish. The grub was then fastened into the thermostatic cradle. The electrodes were quickly removed from the salt solution. After the calomel electrode was placed in contact with the salt-bridge, the needle was inserted into the haemocoele. The insertion was made as close to the skin surface as possible to prevent rupture of the gut. An average oxygen content of 37 per cent of air saturation with a standard deviation of 16.9 was found for a temperature of 25°C. Residual current was determined by passing nitrogen into the Lucite chamber of the thermostatic cradle. Dissolved oxygen content was then calculated in the following fashion: An arithmetic fraction was formed by subtracting values for residual current from diffusion current found in the grub and dividing by the difference of residual and diffusion currents in the standard salt solution. This fraction equalled per cent of air saturation. Technique of injection The anaesthesized grub is straightened out from its typical U-shaped posture and placed in the cradle with its back up. Two, half-inch wide, perforated, adhesive tapes then ‘glued’ the grub in place by application to anterior and posterior portions, leaving the mid-section bare to insert the needle. The grub was not placed under pressure with the tape to avoid excessive haemorrhage when the needle was inserted. The needle was thrust into the lateral aspect of the grub while a tweezer held the skin steady. This site was preferred to a dorsal injection so that main blood vessels and hearts located in the dorsal area were avoided. The knurled knob of the needle slot was tightened against the insulated hub of the needle. Finally, the Lucite top was placed in position and air flow started. DISCUSSION Frequently, grubs ceased movement while the needle was in the haemocoele. This resulted in a decreased, steady-state value. Then, if movement occurred spontaneously or by tactile stimulation, a drop in diffusion current was observed. This result was surprising since a moving liquid resulted in X-10 per cent increase in amperage as described earlier (CHARLTON, 1961). The drop that occurred was attributed to increased use of oxygen resulting in lower oxygen content. Apparently the increased metabolism of violent muscular exertion produced an effect great enough to override the variable of liquid movement.

A MICROELECTRODE TO MEASUREDISSOLVEDOXYGENIN INSECTLARVAE

829

A curious phenomenon was seen when the needle was removed from the haemocoele of the grub and transferred to the standardization cylinder. Instead of achieving the same pen-tracing level on the recorder, the level was 5-15 per cent lower. In the space of half an hour the original level was again achieved. This phenomenon may have occurred because of a deposition of protein film which later was washed away by the salt solution. In contrast, HAGIHARA (1961) reported negligible decreases in diffusion current because of mitochondrial protein. While his experiments showed time intervals of only several minutes, our work recorded oxygen contents over a period of several hours. It appeared that time of exposure of the collodion membrane to protein may have been a factor when absorption and accompanying decrease in diffusion current were considered. An additional variable was the use of ether anaesthesia which was described by LUDWIG (1951).

Ether

would tend to prevent

the coating

of collodion

by haemo-

lymph. In spite of this property of antigelation by ether, larval blood formed films on collodion membranes, a characteristic apparently not shared by mitochondrial

protein.

-4s a final note, from Faraday’s produced

the amount

of oxygen

removed

by electrolysis

was calculated

law to be 5 rnp moles of oxygen per hour when the diffusion

by the reduction

current

of this gas was 0.05 PA.

REFERENCES AMERICANPUBLIC HEALTH ASSOCIATION(1960) Standard Methods for the Examination of Water and Wastewater (11th Ed.), Part II, Pomeroy-Kirschman-Alsterberg Modification,

p. 316.

Boyd

Printing

Co.,

Inc.,

Albany,

New York.

BUCK J. B. (1953) Physical properties and chemical composition of insect blood. In Insect Physiology (Ed. ROEDER K. D.), p. 163. John Wiley and Sons, Inc., New York. CARRITT D. E. and KANWISHERJ. W. (1959) An electrode system for measuring dissolved oxygen. Anal. Chem. 31, 5-9. CHARLTON G. (1961) A microelectrode for determination of dissolved oxygen in tissue. Appl. Physiol. 16, 729-733. CLARK L. C. Jr. (1955) Monitor and control of blood and tissue oxygen tensions. Trans. Amer. Sot. Artif. Internal Organs 21, 41-57. COOPER C. M., FERNSTROMG. A., and MILLER S. A. (1944) Performance of agitated gasliquid contactors. Ind.Eng. Chem. 36, 504-509. DAVIES P. W. and BRINK F. Jr. (1941) Microelectrodes for measuring local oxygen tension in animal tissues. Rev. Sci. Instr. 13, 524-533. HACIHARABUNJI (1961) Techniques for the application of polarography to mitochondrial respiration. Biochim. biophys. Actu 46, 134-142. KINSEY D. W. and BOTTOMLEY R. A. (1963) Improved electrode system for determination of oxygen tension in industrial applications. J. Inst. Brewing 69, 164-173. KOLTHOFP I. M. and LINCANE J. L. (1952) PoZarogruphy (2nd Ed.). Interscience Publishers,

Inc.,

New York.

KREUZER F., HARRIS E. D. Jr., and NESSLER C. G. Jr. (1960) A method for continuous recording in vivo of blood oxygen tension. J. uppl. Physiol. 15, 77-82. LUDWIG D. (1934) Studies on the metamorphosis of the Japanese beetle (Popilliu japonica Newman). Ann. ent. Sot. Amer. 27, 429-434.

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LUDWIGD. (1951) Composition of the blood of Japanese beetle (Popillia japonica Newman) larvae. Physiol. Zoiil. 24, 329-334. MCARTHURC. G. (1916) Solubility of oxygen in salt solutions and the hydrates of these salts. r. Phys. Chem. 20, 495-502. PHILLIPS D. H. and JOHNSONM. J. (1961) Aeration in fermentations. J. Biochem. Microbial. tech. Eng. 3, 277-309. SAWYER D. T., GEORGER. S., and RHODESR. C. (1959) Polarography of gases. Quantitative studies of oxygen and sulfur dioxide. Anal. Chem. 31, 2-5. STICKLANDR. G. (1960) Polarographic measurement of oxygen uptake by pea-root mitochondria. Biochem. J. 77, $36-640. WIGGLESWORTH V. B. (1956) Insect Physiology (5th Ed.), p. 16. John Wiley and Sons, Inc., New York. (Methuen’s biological monographs.)