AN OPEN SYSTEM RESPIROMETER FOR STUDY OF

THE GASEOUS METABOLISM OF

MICROORGANISMS

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S. E. DONOVICK AND T. D. BECKWITH
Department of Bacteriology, University of California at Los Angeles
Received for publication September 18, 1936
INTRODUCTION
Most studies of the gaseous metabolism of microorganisms
have, been performed with manometric respirometers. These
respirometers fall into three main groups: (1) the pressure is
maintained at a constant, and the change in volume is read
directly, as with the Winterstein microrespirometer; (2) the
volume is kept constant, and pressure changes are read on a
manometer, as in the Warburg form; (3) both pressure and
volume change, as in the differential Barcroft type. The closed
system type of apparatus has been developed mainly by Haldane
(1902), Barcroft (1908), and Warburg (1919). Novy, Roehm
and Soule (1925) gave a complete description of their compen-
sator manometer but stated that, "Given an aerobic organism in a
sealed tube, it is only a matter of a few hours, as a rule, for the
oxygen to disappear and to be replaced by a definite amount of
carbonic acid." They also brought out the possibility of the
effects of the carbon dioxide and other volatile substances (ace-
taldehyde, hydrogen peroxide, etc.) on both metabolism and
morphology of microbes. Winslow, Walker, and Sutermeister
(1932) made additional fundamental observations and Walker,
Winslow, and Mooney (1934) showed that the rate of production
of CO2 by Escherichia coli in peptone water was much greater
when air was bubbled through the culture than when N2 was
used. Dixon (1934) remarked that "All the methods (mano-
metric) assume that no gas other than 02 and CO2 is produced or
291
2922. E. DONOVICK AND T. D. BECKWITH

absorbed. This is generally true except in the case of bacteria.

Certain bacteria, however, are liable to give off hydrogen and
other gases and in these cases the methods fail." Howland and
Bernstein (1931), and Fenn (1935) have endeavored to obviate
certain difficulties by various alterations.
Harvey (1928) and Shoup (1929) studied the respiration of

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luminescent bacteria by both colorimetric and manometric
methods. This luminescence is dependent on the dissolved
oxygen and oxygen consumption is measured by determination
of the time necessary for a suspension of these bacteria in sea
water to exhaust the dissolved oxygen to a point where lumines-
cence dims. Harvey found that the dimming point represented
the utilization of at least 99.5 per cent of dissolved oxygen and
the concentration of oxygen decreased throughout the experi-
ment.
An ingenious method of studying gaseous exchanges of tissues
was described by Bennet-Clark (1930). The consumed oxygen
is replaced electrolytically, and the quantity of oxygen used is
determined by measuring the current utilized in electrolysis.
Here the oxygen concentration may be kept more nearly constant
than in the manometric respirometers, but again the production
of gases other than carbon dioxide may produce errors in the
apparent oxygen consumption. Walker (1932) presented a
good open system aeration train with which the carbon dioxide
and ammonia output of cultures may be studied, but no means
for studying oxygen consumption was provided.
PURPOSE
The purpose of the present work then is the development of an
open system respirometer with certain points in mind.
(1) To be able to measure oxygen intake and carbon dioxide
output of microbic cultures by a direct method as contrasted with
the indirect method of pressure changes. The production of
volatile by-products other than carbon dioxide will create no
source of error.
(2) To avoid excessive accumulation of gaseous by-products of
bacterial metabolism.
(3) The apparatus should be adaptable to the observation of
 GASEOUS METABOLISM OF MICROORGANISMS 293
certain phenomena: (a) Changes in cell count; (b) effects of
changes in culture media and other environmental conditions
upon metabolism; (c) the oxygen usage and carbon dioxide
production per cell per unit of time.
GENERAL PLAN OF APPARATUS

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The apparatus consists of five major parts:
(1) Air-flow stabilizers which maintain gas-flow at a constant
rate, and flowmeters which measure quantitatively the
volume of air which passes through the system.
(2) Unit for sterilization of the air which is in train.
(3) Culture unit in which the aerated culture is growing.
(4) Carbon dioxide absorbers.
(5) Oxygen absorbers.
PRINCIPLE OF THE APPARATUS
The principle of the apparatus is as follows: There are two
duplicate aeration trains. The culture chamber in one train
(Test) contains 150 cc. of medium heavily inoculated with the
organism under observation; whereas in the second train (Con-
trol) an equal volume of uninoculated sterile medium occupies the
flask. The air is passed through the two systems at similar (but
not necessarily equal) rates measured by the two flowmeters.
In the control system the carbon dioxide and oxygen of the air is
absorbed, while in the test train the carbon dioxide and oxygen of
the air plus or minus any alterations which may be brought
about by the growing culture are determined. Knowing the rate
of air-flow, and the interval of aeration, it is possible easily to
determine the grams of carbon dioxide and oxygen per cubic
centimeter of air in each case. By the composition of the air
coming from the control in comparison with that from the test,
one may determine the total amount of oxygen used and the
carbon dioxide production per cell per hour may be calculated.
AIR-FLOW STABILIZERS
To assure a minimum of variation in the rate of air flow during
an experiment, not only was a delicate needle valve used, but
also several simple stabilizers were made. See A in figure 1.
294 S. E. DONOVICK AND T. D. BECKWITH

An elongated glass tube of 6 mm. diameter is placed in a tube of

2 cm. diameter and 75 to 100 cm. long. In this larger tube is
placed sufficient dibutyl phthalate for a column 50 or 75 cm.
high. The air passes from the stabilizers to the flowmeters at a
constant pressure as long as an excess of air is escaping from the
lower, inner end of the small tube of A, figure 1. The pressure is

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equal to the weight of the column of liquid displaced by the air
in bubbling out at that orifice. Thus, pressure necessary for the
gas to pass through the remainder of the aeration train may be
w Wet etespvomafty
-dp~ Itotl r e"s*#§-ef
e p

B I E C H X K.< 1, . L
FIG. 1. PLAN OF OPEN SYSTEM RESPIROMETER-OPEN TRAIN RESPIROMETER
A, airflow stabilizers; B, flowmeter; C, sterilizing unit- D, culture chamber;
E, drying tube; F, ascarite tubes; G, hydrogen stabilizer; i, gas mixing chamber;
II, first heater; 12, second heater; J1, first catalyzing chamber; J2., second cata-
lyzing chamber; K1, first weighed drying tube; K2, second weighed drying tube;
L, auxiliary drying tube.
attained by adjusting the depth of the dibutyl phthalate. The
excess air escapes through a vent at the top of the large tube.
One stabilizer with outside tube 110 cm. long and containing a
column of liquid 90 cm. deep is connected with heavy tubing to
the tank containing the compressed air. The gas coming from
this first stabilizer is divided by means of a T tube and passes
into two additional stabilizers of the same design except that the
outside tube is 75 cm. long, and the column of dibutyl phthalate
is 50 cm. high. The air from one of these last two stabilizers
 GASEOUS METABOLISM OF MICROORGANISMS 295
2

passes into the test system, whereas the air from the other
stabilizer passes into the control series.
FLOWMETERS
The drop in air pressure noted by the difference in levels in
fluid in the two sides of a flowmeter is a function of the diameter

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and length of the capillary and of the rate of air flow. By cali-
brating the flowmeters it is possible to determine accurately the
rate at which air is passing through this unit.
The capillary tube utilized in our experiments was 0.6 mm.
diameter (inside) and approximately 7.5 cm. long. The diameter
and length of the capillary used may be varied according to the
maximum and minimum rates of flow desired. The above
dimensions give satisfactory results for rates of gas flow lying
between 10 and 100 cc. per minute. The U tube of the flowmeter
was made of barometer tubing having an inside diameter of 3 mm.
The fluid used in the U tubes was dibutyl phthalate plus a small
amount of anilin red to color it. The flowmeter is incorporated
in figure 1 as B.
STERILIZATION UNIT
The air is sterilized by passing through 150 cc. of 10 per cent
H2SO4; then it is washed in 150 cc. of distilled water, and finally is
passed through sterile non absorbent cotton. See C of figure 1.
CULTURE CHAMBERS
The culture chambers are Drechsel gas-wash-bottles of 250 cc.
capacity with ground glass fittings and are included as D in
figure 1. One hundred fifty cubic centimeters of medium (sterile
in control; inoculated in test) are placed in each chamber. The
air bubbles through the culture, thus supplying oxygen and
driving the carbon dioxide or other volatile by-products out of
the culture chamber.
CARBON DIOXIDE ABSORBERS
The gas is dried by passing from the culture unit through a U
tube containing barium perchlorate which salt is used because
296 S. E. DONOVICK AND T. D. BECKWITH

its vapor pressure is very close to that of NaOH. The gas is

then passed into ascarite tubes to remove all C02, as described by
Buck (1926). The ascarite tube is made by sealing a side arm
on to a pyrex test-tube 1 cm. from its bottom. The side arms in
the present work were made 5 cm. long and consisted of the inner
cone of a No. 7 standard pyrex replaceable ground-glass connec-

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tion. In both the control and test systems two such tubes were
used in a series so that the second tube would catch any carbon
dioxide which might pass the first tube. The gain in weight in
the second tube has always been found to be negligible. These
tubes are E and F in figure 1.
OXYGEN ABSORBERS
The C02-free air leaving the second Ascarite tube passes into a
small gas-mixing chamber, H in figure 1. This chamber consists
of a low, wide-mouthed bottle of 75 to 100 cc. capacity. The
bottle is half filled with clean glass beads, and sufficient concen-
trated sulphuric acid to give a column of 2.5 cm. depth. The
C02-free air is mixed with hydrogen in this chamber. Each gas
has a separate entrance tube and controlling stopcock. In this
fashion both gases may be introduced at the same time or either
one may be stopped while the other continues to flow. The
sulphuric acid is employed primarily to fill a twofold need: (1) to
act as a trap so that the hydrogen is kept from entering the air
line; (2) to provide an excess of hydrogen which, as will be shown
later, is necessary. By watching the rate of bubbling of the
hydrogen through the acid, it is easy to judge whether the neces-
sary excess of hydrogen is being introduced. The rate of hydro-
gen flow is maintained at a constant in the two systems by means
of two additional stabilizers such as have been described pre-
viously-G in figure 1. The sulphuric acid also aids in drying
both the hydrogen and the C02free air, while the turbulence
caused by the glass beads ensures a thorough mixing of the two
gases.
The gas mixture is then completely dried by passing through a
U tube containing barium perchlorate, after which the dried
gases pass into a U tube containing 2 grams of palladinized
asbestos, I, and Is of figure 1. The palladium catalyzes the re-
 GASEOUS METABOLISM OF MICROORGANISMS 297
action between hydrogen and oxygen to form H20. Either 5 or
10 per cent commercial palladinized asbestos was found to be
satisfactory. The moisture thus formed is driven, into a weighed
drying tube containing barium perchlorate and inserted at K1
and 1K2 in figure 1. From this the air passes through a second
similar catalyzing chamber and a second weighed drying tube.
This second catalyzing chamber was found to be necessary be-

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cause, when the rate of air-flow is 20 to 25 cc. per minute, as
much as 5 per cent of the oxygen in the air remains uncombined
after passing through the first tube (I,). A third tube was not
found necessary. A small electric heater is placed under each
catalyzing tube. By heating the air around the catalyzing tubes
to approximately 250'C. several benefits are derived: (1) the
passage of the water from the catalyzing tube into the drying
tubes is hastened; (2) the catalytic action is much more complete
at this temperature than at that of the room; (3) the palladinized
asbestos appears not to become "poisoned" at this temperature.
The method then is gravimetric.
The U tubes containing the palladinized asbestos and the
weighed drying tubes were made of 11 mm. pyrex glass tubing.
The side-arms were fabricated from the inner cones of No. 7
ground glass connections as in the case of the ascarite tubes and
the outer cones of the ground glass connections were attached to
the U tubes containing the catalyzer. Thus, all parts which
had to be removed from the aeration train and weighed (e.g.,
drying and ascarite tubes) were connected to the train in their
proper places by means of standard ground glass connections.
These connections were found to be air-tight without the use of
grease or lubricant at pressures far greater than those which ever
existed in the apparatus. The ascarite tubes when charged
weighed approximately 32 grams, and the drying tubes 45 grams.
EXPERIMENTAL STUDIES
Before an experiment could be begun it was necessary to remove
all moisture and oxygen from the catalyzing chambers. This was
accomplished by driving dry hydrogen through the catalyzing and
drying series. This is done in train, unit by unit.
Preliminary to metabolic studies it was necessary first to prove
298 S. E. DONOVICK AND T. D. BECKWITH

that the described aeration train could analyze accurately a

flowing stream of air. In the first series of experiments, then, no
cultures were used. One hundred fifty cubic centimeters sterile
medium of the composition to be used in the later work were
placed in both culture chambers. Air was bubbled through both
systems at constant similar rates measured by the flowmeters.

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The duration of the average experiment was two hours. The
total water and carbon dioxide absorbed in each system was
determined. The rate of flow and the time interval of flowing was
known; therefore, the total number of cubic centimeters of air
passing through each system was easily computed. Then,
comparisons were made of the grams of carbon dioxide and oxy-
gen per cubic centimeter of air as determined in successive
experiments. The following is a sample calculation:
Experiment number = Series I, 1.
Rate of air flow = 11.9 cc. per minute.
Time interval = 120 minutes.
Grams H20 absorbed = 0.5907.
01.9x120 X 32* = 3.68 X 10-4 grams 02 per cubic centimeter air.
*
32/36 = conversion factor HsO to 02.
During an experiment the hydrogen must be flowing at a rate
equal to or a little greater than that of the air in order that there
may be available an excess of hydrogen in the catalyzing cham-
bers. Only one-fifth of the air consists of oxygen, and the use of
hydrogen must be governed accordingly.
In order to have a supply of air which should be constant in
composition for a number of experiments, air was compressed in a
tank to a pressure of 75 to 80 pounds per square inch.
Since there are many satisfactory methods of absorbing quanti-
tatively the carbon dioxide from a flowing stream of air, this
factor was considered of less importance in the present work.
The chief emphasis has been placed upon developing a method of
determining accurately the amount of oxygen in a moving body of
air.
In table 1 are the results obtained for grams of 02 per cubic
 lC=P]RIMNT
NUMBER

Series I:
X
XI
XII

XI
XIII

XIVa
b
XV a
b
GASEOUS METABOLISM OF MICROORGANISMS

centimeter of air using the described apparatus. There were

conducted three series of experiments which were alike except
that a new supply of air had been placed in the tank for each
series. Since the amount of CO2 per cubic centimeter of air is so
negligible (approximately 5.4 X 10-7 grams per cubic centimeter
at atmospheric pressure and room temperature) there were only
slight increases in the weights of the ascarite tubes and these
were well within the limits of experimental error of weighing.
Therefore the CO2 results are omitted.

ATICOF AIRL-
EXPERIMNT
RAT
FLOW
AI-

cc. per

11.9
15.8
19.8

10.6
14.4

21.4
19.2
18.6
23.8
minute
TABLE 1
TIME INTERVAL
TIMbtDNTERVA
FLOWING

minutes

120
120
120

120
120

106
97
123
123
WEQUIVALENT
WATER
ABSORBED

grams

0.5907
o.7827
1.0248
Average.................................................
Series II:
0.4648
0.6252
Average.................................................
Series III:
0.8071
0.6683
0.8120
1.0195
OXYGEN
PER CUBIC CENTIMETER
OF AIR

grams

3.68 X 10-4
3.67 X 10-'
3.75 X 10-4
3.70 X 10-4

3.25 X 10-4
3.22 X 10-4
3.24 X 10-4

3.16
3.19
3.16
3.10
X 10-4
X 10-4
X 10-4
X 10-4
299

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Average................................................. 3.15 X 10-4

In the above results, it is evident that oxygen in a flowing

stream of air may be determined with accuracy by the method
employed. Results in both trains of the apparatus were closely
similar.
It remained then to show that this respirometer not only gave
constant results for the oxygen composition of a given body of
air but that it could measure the oxygen usage and the carbon
300 S. E. DONOVICK AND T. D. BECKWITH

dioxide production of a microbic culture. To do this a number of

experiments were run, using Saccharomyces cerevisiae as the
organism to be studied.
PROCEDURE IN THE METABOLIC STUDIES
The yeast was grown on a large slant of Sabouraud's agar for

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several days at 270C. Forty-eight hours previous to the experi-
ment, 10 cc. of liquid medium in which the yeast was to be
growing during the run was added to the slant, and the culture
was incubated for an additional 24 hours. The heavy growth
thus obtained in the 10 cc. of medium then was poured into the
culture chamber of the apparatus, 140 cc. of fresh medium were
added and incubated at 270C. until the time of the experiment.
In the first two of our experiments, this final incubation period
was 36 hours but in all others it was 24 hours. Two hours before
the actual run was begun, the culture chamber was removed from
the incubator and connected into the aeration train. Air was
bubbled through the culture chamber in order to drive out the
accumulated carbon dioxide and to bring the liquid to equilibrium
with the external atmosphere. The experiment itself usually
continued for approximately two hours and except for the fact
that a growing culture was occupying one of the culture chambers,
the procedure was the same as that described previously. The
air was bubbled through both systems at measured rates. Since
the rates of flow do not remain entirely constant throughout a
run, flowmeter readings were taken every minute until both
trains of the respirometer had come to equilibrium, and then
every ten minutes until the end of the test. At the same time the
H2-flow was adjusted to supply an excess. The air then was
turned off and hydrogen continued to pass through the catalyzing
chambers until all the water produced from the hydrogen and
oxygen was driven over into the drying tubes.
Immediately before and after each run, Sabouraud's agar pour
plates were made of the culture. The cell counts always were
found to be increased only slightly after the two-hour period.
Therefore, to determine the average number of living cells present
during the experiment, the arithmetical mean of the "before" and
 GASEOUS METABOLISM OF MICROORGANISMS 301
"after" counts was computed. It was possible to. obtain counts
which checked satisfactorily if care was taken with mixing dilu-
tions. The pour plates were made in quadruplicate of the
cultures diluted to 10-5, 10-6, and 10-7. The plates were incu-
bated at 270C. for 48 hours before counting.
After the experimental run was completed, and when all of the
water was driven from the catalyzing chambers into the drying

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tubes, the ascarite and the drying tubes were carefully stoppered
and rubber policemen were fitted over the side arms. These
tubes then were placed in a desiccator for several hours before
the weighings were made in order to remove any moisture from
external surfaces.
TABLE 2
EXPERIMENT
NcUMBER | MOLS 02 USED
BY CULTURE PER
AVERAGE NUMBER
OF LIVING YEAST MOLS
PER
02 USED
CELL PER HOR
MOLS C0S
PRODUCED PER CELL
NUMBER
~HOUR CELIS PRESENT PRCLPEHOR PER HOUR

XVI* 0.53 X 10-2 1.78 X 1010 0.30 X 10-12 0.16 X 10-12

XVII* 0.45 X 10-2 1.75 X 1010 0.26 X 10-12 0.21 X 10-12
Average .0.28 X 10-12
XIX 0.24 X 10-2 1.29 X 1010 0.19 X 10-12 0.23 X 10-12
XX 0.18 X 10-2 1.02 X 1010 0.17 X 10-12 0.28 X 10-12
XXI 0.21 X 10-2 1.09 X 1010 0.20 X 10-12 0.30 X 10-12
XXIII 0.11 X 10-2 0.673 X 1010 0.16 X 10-12
XXIV 0.10 X 10-2 0.530 X 1010 0.18 X 10-12
Average .0.18 X 10-12
* Final
incubation period of 36 hours duration; all others, 24 hours.
Table 2 is a composite of the results obtained in the present
studies. As mentioned earlier, chief emphasis has been placed
upon the oxygen usage since there are many satisfactory methods
of studying carbon dioxide production. Consequently, a particu-
lar precaution which must be taken for the accurate study of
carbon dioxide production, but which does not affect the study
of oxygen consumption, was ignored here. This was an analysis
of the culture medium before and after each experiment for
"bound" carbon dioxide (carbonates).
The apparent high carbon dioxide production per cell per hour
302 B. E. DONOVICK AND T. D. BECKWITH 0

may be explained upon the basis that the yeast was growing in a
medium rich in glucose content, and anaerobic fermentation may
have been proceeding at the same time as oxidation. However,
since no studies were made of the changes in composition of the
medium in so far as sugar content was concerned, no definite
conclusions on this point may be deduced. A point of interest

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here was that demonstrated by Meyerhof (1925). This author
found that one mol of respired oxygen causes the disappearance of
1.5 to 2 mols of fermented carbon dioxide, that is, oxidation of one
mol of glucose protects 4 to 6 mols of glucose from being fer-
mented. Meyerhof found further that under aerobic conditions,
Brewer's yeast ferments 50 mols of glucose for every mol oxidized;
but after 15 hours in a glucose-phosphate solution the ratio drops
to 4 mols fermented to 1 oxidized. For pressed yeast this ratio
was from 2 to 4 fermented to 1 oxidized, and for wild yeast, 0.3: 1.
It seems apparent, then, that to report the respiratory quotient
merely in terms of carbon dioxide produced and oxygen used, as
is done frequently, may be of little significance unless careful
studies are made of the simultaneous changes occurring in the
chemical composition of the culture medium. Pulley and
Greaves (1932) determined that the amount of carbon dioxide
produced by yeasts under varying conditions is not proportional
to the increase in the number of cells present.
CULTURE MEDIUM
In all of the experiments the medium used was similar to that
listed as No. 193 in the compilation of culture media by Levine
and Schoenlein (1930). However, glucose was substituted for
sucrose, and 0.5 per cent Difco peptone was added. This medium
consisted of: water, 1000 cc.; NH4Cl, 1.88 gram; glucose, 100
grams; K2HP04, 1 gram; peptone, 5 grams. Sterilization was
by means of a Berkefeld filter. However, the Arnold steamer
may be used.
DISCUSSION
In table 2 it will be tfoted that the average yeast cell consumes
approximately 0.20 X 10-12 mols of oxygen per hour in the rich
 GASEOUS METABOLISM OF MICROORGANISMS 303
medium used in our work. It is apparent that the results for
the oxygen consumption in the various experiments check very
satisfactorily. The possibility of errors in the cell counts would
permit of greater variation.
In spite of the great abundance of studies of yeast metabolism
reported in the literature, few papers present the data in such

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TABLE 3
ORGANISM REPORTED BY EXPERIMENTAL MOL Os PER CELL
CONDITIONS PER HOUR

Lactobacillus pentoaceti- Hunt (1933) 0.36 X 10-15

cus to
0.85 X 10-15
Sarcina lutea Gerard and Falk In water 0.28 X 10-15
(1931) In glucose 0.28 X 10-1l
to
0.84 X 10-12
Photobacterium phos- Harvey (1928) 150C. 0.56 X 10-16
phoreacens 21.50C. 1.33 X 10-16
Commercial yeast Field, Martin, and pH 6.0 0.6 X 10-12
Field (1935) pH 6.8 0.7 X 10-13
Yeast (species?) Harvey (1928)* 0.85 X 10-13
Saccharomyces cerevisiae Field (1935) pH 5.8 0.14 X 10-18
pH 6.0 0.13 X 10-13
pH 6.8 0.14 X 10-13
pH 7.8 0.06 X 10-13
Saccharomyces cerevisiae Author (1936) Aerated in 0.18 X 10-12
glucose
Arbacia eggs Tang (1931) Unfertilized 1.3 X 10-12
Arbacia eggs Tang and Gerard Fertilized 4.8 X 10-12
(1932)
Paramaecium caudatum Howland and Bern- 2.0 X 10-11
stein (1931)
Actinosphaerium eichornii Ibid 4.6 X 10-11
* Harvey cites the given
value, but indicates that the author was unknown
to him.

form that it is possible to determine the oxygen consumption per

yeast cell per hour. In Meyerhof's work, a given weighed sample
was used for the inoculum. No cell counts were made. The
oxygen consumption was reported in mols of oxygen per mg. dry
weight of yeast. From the data given, there is no way to deter-
mine the proportion of living and dead cells present. Stier (1933)
304 S. E. DONOVICK AND T. D. BECKWITH

when studying the effects of temperature upon oxygen consump-

tion of yeast, stated that standard suspensions were used through-
out the work, but failed to indicate how many cells were present.
Many other workers report the oxygen consumed per cell per
hour in terms of cubic millimeter with no reference to temperature
and pressure. Walker (1932), Meyerhof (1925), and others point

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out that the presence of sugar in the culture medium, and aera-
tion also, increase greatly the rate of oxygen consumption of
microorganisms.
To be able, then, to compare the rate of oxygen consumption of
yeast as determined by the present method with the rates as
measured by other methods available, it would be necessary to
use the same strain of organism, culture medium, etc., in all
series included. But, since the primary purpose of the present
work was to develop a respirometer which approached the opti-
mum more closely, the experiments were not designed specifically
for comparison with other work of similar nature. Nevertheless,
it may be of interest to compare the rate of oxygen consumption
of yeast as determined here with those of other organisms as
they have been observed by workers using various types of
respirometers. Table 3 affords such an opportunity. In the
cases in which the author has reported the amount of oxygen
consumed in cubic millimeter per cell per hour, it has been neces-
sary to assume definite conditions of temperature and pressure
in order to convert the results into mols of oxygen per cell per
hour.
SUMMARY
1. With the described aeration train it is possible to determine
the mean rate of oxygen consumption per cell per hour.
2. When Saccharomyces cerevisiae is growing in an aerated
culture medium rich in glucose, a 24-hour culture utilizes
0.18 X 10-12 mols of oxygen per cell per hour; a 36-hour culture,
0.28 X 10-12 mols.
3. The results for the rate of production of carbon dioxide by
yeast did not check in the present work as satisfactorily as did
those for oxygen consumption. This may be explained on the
basis that:
 GASEOUS METABOLISM OF MICROORGANISMS 305
(a) Only the gaseous carbon dioxide was determined.
(b) Some of the carbon dioxide produced will be found in the
"bound" form. To determine this would necessitate a careful
chemical analysis of the culture medium before and after the
respiratory tests are made.
(c) Much carbon dioxide is produced through enzymatic
activity which does not involve any consumption of oxygen.

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Therefore, in order to interpret respiratory quotients, it is neces-
sary to know what proportion of the sugar consumed during an
experiment is fermented and what is oxidized. This may be
determined by comparing at least three items: (1) total sugar
consumed; (2) total carbon dioxide produced (free and bound);
(3) total oxygen consumed.
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