The use of clinostats and centrifuges to explore the hypogravity range between zero and 1 g is described. Different types of clinostat configurations and clinostat-centrifuge combinations are compared. Some examples selected from the literature and current research in gravitational physiology are presented to show plant responses in the simulated hypogravity region of the g-parameter (0 is greater than g is greater than 1). The validation of clinostat simulation is discussed. Examples in which flight data can be compared to clinostat data are presented. The data from 3 different laboratories using 3 different plant species indicate that clinostat simulation in some cases were qualitatively similar to flight data, but that in all cases were quantitatively different. The need to conduct additional tests in weightlessness is emphasized

Chapman, David K.

English

NASA Technical Reports Server

N90-13953
DO THE DESIGN CONCEPTS USED FOR THE SPACE FLIGHT HARDWARE
DIRECTLY AFFECT CELL STRUCTURE AND/OR CELL FUNCTION
GROUND BASED SIMULATIONS
David K. Chapman
University of Pennsylvania
Philadelphia, PA 19104-6018
ABSTRACT
The use of clinostats and centrifuges to explore the hypogravity range between
zero and 1 g is described. Different types of clinostat configurations and clinostat-
centrifuge combinations are compared. Some examples selected from the literature
and current research in gravitational physiology are presented to show plant
responses in the simulated hypogravity region of the g-parameter (0 < g < 1). The
validation of clinostat simulation is discussed. Examples in which flight data can be
compared to clinostat data are presented. The data from 3 different laboratories
using 3 different plant species indicate that clinostat simulation in some cases were
qualitatively similar to flight data, but that in all cases were quantitatively
different. The need to conduct additional tests in weightlessness is emphasized.
Introduction
Several methods to either simulate the weightlessness state or to produce short
periods of weightlessness have been used in ground based simulations. They
include bed rest studies, tail suspension tests, water immersion, clinostats and free
fall using parabolic flight manuevers and drop towers. Many biological studies
require that g be made an experimental variable. For many of these studies the use
of clinostats and centrifuges have been used to explore the hypogravity g-range
between zero and 1 g.
Most of these experiments fall into two major categories: (a) The phenomenon to
be studied is believed to be quanitatively dependent on a g- force and the
investigator wants to define the g-function of his test subject's response to
different g-levels in the hypogravity region: (b) The test system responds in a
similar way to gravity and some other factor of special interest and to measure the
latter it may seem necessary to decouple the response to gravity from the response
to the other factor.
Clinostat and Clinostat-Centrifuge Configurations
Ciinostats have been used to simulate the weightless condition for about a
century (Brown, 1979). They are rotating machines that rotate the test subject
slowly around some axis with respect to the coordinates of the subject (usually the
longitudinal axis). The simulation effect is achieved by rotating the subject in a
manner such the axis of rotation is normal to the earth's g-force vector. As the
clinostat rotates the earth's g-force moves around the axis once each revolution and
if summed the effect is assumed to be zero. The rotation rate should be fast enough
to achieve gravity compensation, but slow enough to prevent significant
centripetal forces. Acceptable levels of centripetal force vary, depending on the g-
167
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force required to elicit a response. For some systems a centripetal force of 10 -4 g
can be detected and in others a level of 10-2g can be tolerated. The rates most
frequently used are between 1 10 rpm.
The axis of rotation may be horizontal or less than 90 degrees from the plumb
line. The subjects longitudinal axis may be in the plane of its rotation or it may be
normal to that plane. The subject may be rotated on two or even on three (usually
orthogonal) axes simultaneously. Several of the many possible modes of clinostat
function which have been used in plant physiological experiments are shown in
Table I and Figure 1.
There are special applications that employ rapid rotation (ca. 50 - 200 rpm) and
are referred to as "fast clinostats" (Briegleb, 1967). They usually are used to suspend
cell particles within the cell. One of the requirements is to locate the cell in the
center of rotation. If the cell moves off center by a few millimeters it will
experience centripetal forces which may be significant. For example if the speed is
50 rpm the test subject will have to be retained within a radius of 3.6 mm to prevent
it from experiencing centripetal forces greater than 10 -2 g.
The most common use of a clinostat has been to achieve gravity compensation
with the axis of the clinostat rotation in the horizontal position. However if the
experimenter wishes to explore the entire hypogravity range between zero and 1 g
the axial g-force component must be altered. This can be accomplished by either
placing the clinostat on an angle from the horizontal or by applying an axially
directed centripetal g-force. If the clinostat is placed on an angle, the axial
component of earth's g-force depends on the cosine of the angle of inclination that
departs from the plumb line (Brown and Chapman, 1977). The use of a single axis
clinostat and a centrifuge can be employed to create a two axis clinostat with one of
the axes providing gravity compensation with a horizontal clinostat and the other
applying a centripetal acceleration in the horizontal direction. Both
configurations are depicted in Figure 2,
Hypocoytl Nutation in Simulated Hypogravity
A number of investigators have employed clinostats to simulate hypogravity,
but only a few have explored the entire range between simulated zero and 1 g. The
first reported use of a centrifuge and horizontal clinostat to investigate levels
above zero g was in 1961 by Finn and Brown (1961). A more recent study to
characterize hypocoytl nutation of sunflower seedlings in the hypogravity region
(0 < g < 1) rotated them on orthogonal axes using a horizontal clinostat to provide
gravity compensation and a centrifuge to apply centripetal acceleration along the
plants longitudinal axis (Chapman et al, 1980). The configuration used is depicted
in Figure 2B.
The results shown in Figure 3 indicate that below 1 g both the period and
amplitude changed markedly. There was a 35% reduction in the period of nutation
and a 80% reduction of the amplitude at simulated 0g. Neither the period or
amplitude extrapolated to the origin.
These ground based simulation tests which were conducted to characterize
circumnutation of sunflower hypocoytls provided useful background information
for an experiment that was conducted during the Spacelab-1 mission in 1983 to
determine the gravity requirement for circumnutation. These tests indicated that
168
gravity did influence circumnutation and that its mechanism could not be entirely
endogenous, but on the other hand, the fact that at simulated zero g it did not
completely damp out did not support the role of gravity as the exclusive driving
force for the oscillations.
Validation of Clinostat Simulations
The least g-force condition, attainable only in space, is microgravity, essentially
"zero g" or weightlessness. Gravity compensation, achieved by use of horizontal
clinostats is assumed to mimic zero g. In order to test this theory the effects of
weightlessness and of clinostats must be compared in adequately controlled
experiments of statistically competent design.
For higher plants, tissue cultures, microorganisms, and small animals the
horizontal clinostat has been employed with the usually tacit assumption that its
simulation of hypogravity (most often zero g) is at least an excellent approximation
of the true environmental condition it putatively imitates. Obviously that
assumption ought to be tested for, if it cannot be validated, an unfortunately large
number of experimental findings based on tests with clinostatted biological
material necessarily must be reevaluated (Brown et al, 1976).
Direct tests of the validity of clinostat simulations of course were impossible
until scientists could attain experimental access to a (nearly) weightless
environment achievable only in space. In recognition of the importance of
knowing the validity of hypogravity simulations NASA's first orbital mission
designed exclusively for its effort in space related gravitational biology (Saunders,
1971) included two major experiments with a large number of functional objectives
that would become biologists' first direct test of clinostat simulation valitity.
The experiments, flown on Biosatellites I and II, were designed to acquire
quantitative data of known precision and, for each of the biological processes
tested, results obtained from space flight were compared with appropriate ground
controls. One growth process that had been studied extensively on earth and on
earth bound clinostats was the epinastic response (altered position of lateral plant
organs such as leaves and secondary roots). It provided the best data for the desired
tests of agreement between results from the clinostat environment and from true
microgravity.
It is in principle nearly impossible to "prove a negative" and if, for one or a few
phenomena, results from space flight and results from clinostatting are in
statistical agreement, we can only conclude tentatively that there may be no "real"
difference-- a conclusion strongly encouraged by our wishful thinking. But if the
differences are large (unquestionably significant), that would be a serious blow to
our tentative conclusion of equivalency and would demonstrate that clinostat
simulation would not always be dependable without verification by space flight
tests for each new phenomenon to be studied.
Epinastic responses of wheat roots and of pepper plant leaves were not the same
on clinostats and in microgravity (Brown et al, 1976; Brown et al, 1974; Lyon, 1968;
Johnson and Tibbitts, 1968). The data in Table II indicate the differences between
space flight and clinostat data for the pepper plant. The initial angles at launch
were not significantly different. The initial rate of change of the petiole angles
169
(degrees/hr)was significantly different, P < 0.001. The final angles that were
attained were also significantly different at the 1% level or beyond.
The data in Table III indicate that while the epinastic responsesfor wheat roots
were qualitatively similar they were quantitatively different. The difference
between the clinostat and microgravity data were significantly different at the 1%
level. In both cases the experimenterschose to discount the importanceof
statistical analysesof Biosatellite II results--presumablybecausethe data showed
effects of clinostatting were less extreme than those of space flight; a possible
consequenceof only small deficiency in the clinostat's ability to simulate true
weightlessness(Lyon, 1968; Johnsonand Tibbitts, 1968).
Sixteen years after the flight of Biosatellite II NASA's Spacelab-l mission
provided data that permitted definitive quanitative comparisons for parameters of
sunflower circumnutation on earth based clinostats and in microgravity (Brown
and Chapman, 1984). The data in Table IV (adapted from Brown and Chapman, 1984)
show that when compared with plant behavior at 1 g circumnutation was less
vigorous on clinostats than during space flight. The changes were large but
especially significant was the difference between the effect of space flight and the
effect of clinostatting; the clinostat environment suppressed circumnutation much
more than did microgravity, a result that reasonably could not be attributed to
clinostat imperfection. One could not criticize the microgravity condition as a poor
simulation of the clinostat environment!
It seems evident that validation of clinostat simulations, especially for research
in plant biology, has warranted a high scientific priority for about twenty years.
However, the number of phenomena studied and the number of flights on which
such test were possible have been discouragingly few. It appears that this
important topic remains in the category of NASA science's unfinished business.
_N_
Briegleb, W. 1967. Ein Modell zur Schwerelosigkeits-Simulation an
Mikroorganismen. Naturwissenschaften 54:167.
Brown, A.H., D.K. Chapman and S.W.W. Liu. 1974. A comparison of leaf epinasty
induced by weightlessness or by clinostat rotation. Bioscience 24:518-520.
Brown, A.H., A.O. Dahl and D.K. Chapman. 1976. Limitation on the use of the
horizontal clinostat as a gravity compensator. Plant Physiol. 58:127-130.
Brown, A.H. and D.K. Chapman. 1977. Nutations of sunflower seedlings on tilted
clinostats. COSPAR Life Sciences and Space Research 15: 279:283.
Brown, A.H. 1979. Research opportunities and limitations of protracted hypogravity
simulation for plant gravitational physiology. The Physiologist 22: (No. 6)
Supplement 15-18.
Brown, A.H. and D.K. Chapman. 1984. Circumnutation observed without a
significant gravitational force in space flight. Science 225:230-232.
170
Chapman, D.K., A.L. Venditti and A.H. Brown. 1980. Gravity functions of
circumnutation by hypocotyls of Helianthus annuus in simulated hypogravity.
Plant Physiol. 65:533-536.
Finn, J.C. Jr., and O.D.R. Brown. 1961. Plagiotropism in petioles of Xanthium . Plant
Physiol. 36: (supp.) xxi.
Johnson, S.P. and T.W. Tibbitts. 1968. The liminal angle of a plagiotropic organ
under weightlessness. Bioscience 18:655-661.
Lyon, C.J. 1968. Wheat seedling growth in the absence of gravitational force. Plant
Physiol. 43:1002-1007.
Saunders, J.F. (Ed.) 1971. The Experiments of Biosatellite II. NASA SP-204, Sci. Tech.
Info. Office, Washington, DC.
171
TYPE NUMBER
OFAXES
Table I. CLINOSTAT CONFIGURATIONS
COINCIDENCE OF
A ROTATIONAL
AXIS WITH A
PLANT AXIS
VF_L-q_R DIRECTION AXIAL EXAMPLE
OF FORCE g-FORCE
RELATIVE TO THE ON PLANT
PLANT AXIS
A-1
A-2
A-3
B-1
C-1
D-1 2
COINCIDENT VARIABLE 0 < g < 1
COINCIDENT
COINCIDENT
NOT COINCIDENT
BUT PARALLEL
NOT COINCIDENT
COINCIDENT
NON-ORTHOGONAL
TRANSVERSE g = 0
PARALLEL g= 1
TRANSVERSE g = 0
CHANGES g = 0
CONTINOUSLY
VARIABLE g _> 0
VARIABLE
ANGLE
CLINOSTAT
CONVENTIONAL 90°
(HORIZONTAL)
CLINOSTAT
VERTICAL
ROTATION
PERIPHERAL
ARRAY
TUMBLING
CLINOSTAT AND
CENTRIFUGE
172
TABLE II. Epinastic responseto altered gravity
S P JohnsonandT W Tibbitts (1968)
Initial petiole angles (Degrees) a
Flight Data 153.8 __+3.6
Clinostat Data 158.7 + 3.7
Difference 4.9 + 5.2
Probability of difference occurring merely by chance, P = 0.36 b
Initial rate of change of petiole angles (Degrees/Hr) a
Flight Data 3.04 + 0.10
Clinostat Data 4.24 + 0.13
Difference 1.2 + 0.16
Probability of difference occurring merely be chance, P < 0.001
Final petiole angles (Degrees curvature after 20 hr in orbit) a
Flight Data 103.6 +0.7
Clinostat Data 113.0 + 0.6
Difference 9.4 + 0.9
Probability of difference occurring merely by chance, P < 0.001
aData are expressed as mean + Standard Error
bNot a significant difference
173
TABLE III. Epinastic responseof wheat lateral roots to
alteredgravity. (Data of C J Lyon, 1968)
Condition
Microgravity
Clinostat
1 g Controls
a... Liminal Angle (Degrees)Percent change from
96 99.6 + 1.4
97 94.2 + 1.5
127 62.4 + 0.8
1 g controls
59.6 + 1.61%
51.0 + 1.70%
Conclusion: Probability that plants on clinostat and those in
microgravity were different only by chance, P _<0.009
TABLE IV. First quantitative measurements of parameters of sunflower
hypocotyl circumnutation on clinostats and microgravity,
(Data from Spacelab-1 experiment A H Brown)
Number of cycles
observed in 13 plants
Amplitude of
circumnutation
oscillation (ram)
Period of
circumnutational
oscillation (rain)
On Clinostat a In Microgravity a
50 121
1.66 + 0.16 b
78.47 + 2.55 c
2.77 + 0.13 b
87.60 + 2.58 c
aData are expressed as mean + Standard Error
bprobablility of difference occurring merely by chance, P _<0.00006
Cprobability of difference occurring merely by chance, P _<0.012
174
A-1 A-2
/z
/
---
/
/
A-3
B-1 C-1 D-1
,,,,
Figure 1 Clinostat configurations that have been used for research in
gravitational plant physiology.
I
_-_
_ --...... -_-- --.---_-- • _
\ 1 \
\ \
'., \
\ \
(_) \ .- 9
CENTRIFUGE AXIS
Figure 2 Force diagram for equivalent conditions of clinostatting.(Modified from
Brown, A.H. 1979. The Physiologist 22 (No. 6) Supplement 15-18).
A. (Above) Type A-I; Axial component of earth's 1 g also imposes 0.3 g in
axial direction. Force magnitude depends on cosine of angle of
inclination.
B. (Below) Type D-l; Centripetal force of 0.3 g imposed in axial direction.
Force magnitude depends on rotation rate and radius.
175
120
W
_.1
110
100
I.-
Z
-- 90
8O
7O
5
O3
LLI
¢4
i,-
"_3
.J
2
(__ER IOD
+
PLITUDE I_
0 J I I i
0 .5 1.0 1.5
CENTRIPETAL G-FORCE
Figure 3 Amplitude and period of circumnutation over a range of axial forces
between 0 and 1.5 g achieved by rotation on 2 axes. Earth's gravity was
compensated as in Figure 2B.
176


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