PHYSICO-CHEMICAL CHANGES OCCURRING IN
FISH FLESH DURING FREEZING AND
THAWING AS MEASURED PILATOMETRICALLY
by
V. MAHADEVAN
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ARTS
IN THE DEPARTMENT
OF
ZOOLOGY
THE UNIVERSITY OF BRITISH COLUMBIA
APRIL, lolj-8
PHYSICO-CHEMICAL CHANGES •OCCURRING- IN
FISH FLESH DURING FREEZING AND
THAWING AS MEASURED DILATOMETRICALLY
ABSTRACT
The thesis deals with the use of a dilatometer
i n studying some of the physico-chemical phenomena occurr i n g i n f r e s h f i s h f l e s h when subjected to freezing at
o
o
temperatures ranging from 0 C to -30
C.
Two
different
kinds of f i s h f l e s h , marine lingcod (Ophlodon elongatus)
and fresh water rainbow trout (Salmo galrdnerll) were
used for comparison.
True freezing point determinations of samples
of f r e s h f l e s h cut from the above species of f i s h were
made and found to be the same, v i z , -1.5°C (29.1°F.).
The
percentage of water removed as ice at varying temperatures
below the I n i t i a l freezing point were calculated by
necessary adjustments of experimentally determined values.
A permanent net decrease i n volume accompanying
freezing and thawing of the samples of f l e s h was
and measured.
This change i n volume l s probably due to
the denaturation of the proteins, and was
0.075#.
observed
found to be
The c o e f f i c i e n t of cubical
expansion (oC ) of
anhydrous f i s h muscle was measured f o r the f i r s t time and
found to have the average value of 0.000118 over a temperature range from -30°C. to +20°C.
CONTENTS
INTRODUCTION . .
1 - 7
APPARATUS
8 - 9
FILLING THE APPARATUS
EXPERIMENTAL PART
9-12
13 - 1?
EVALUATION OF RESULTS
A*
Ad Justments of Experimental Data .......
18-25
B.
Results from Adjusted Data
25 - 28
DISCUSSION
SUMMARY
ACKNOWLEDGEMENT
LITERATURE CITED
29 - 3^
35
36 - 37
38
THE PHYSICO-CHEMICAL CHANGES IN FISH MUSCLE
DURING FREEZING AND
THAWING
AS MEASURED DILATOMETRICALLY
INTRODUCTION
I t Is a" common observation that f i s h a f t e r
landing at the shore do not' keep f r e s h f o r long and begin
to emit unpleasant
odours due to many biochemical
biophysical changes setting i n . During the past
and
few
years great strides have been made i n order to arrest
7
these changes, thus enabling the Industries successfully
to d i s t r i b u t e them to the' consumers without
app'reciable
loss i n the n u t r i t i v e value, taste and smell.
Before a r t i f i c i a l freezing was used as a
method of preserving f i s h , drying, s a l t i n g and p i c k l i n g
and l a t e r canning were adopted; hut f i s h preserved
the above methods are greatly changed.
a l l meet the demands for fresh f i s h .
by
They do not at
Freezing and re-
f r i g e r a t i o n are the only methods of preserving that keep
f i s h i n e s s e n t i a l l y i t s o r i g i n a l condition over long
periods.
The problem of freezing i s simply one of
2.
detecting the effect of lowering temperature upon the
rate at which these changes occur.
Unlike the f l e s h of t e r r e s t i a l animals, f i s h
f l e s h i s much more tender, for the simple reason that i t
l i v e s i n an entirely d i f f e r e n t environment (water) which
supports i t by hydrostatic pressure. Hence f i s h f l e s h
i s much more prone to mechanical 'injury and b a c t e r i a l
invasion.
I t may be merely coincidental that the proteins
of f i s h muscle a.re much more sensitive to chemical and
physical change, or i t may be i n some obscure \fay related
to the demands which environment makes upon i t . However,
It l s d e f i n i t e l y known that f i s h proteins undergo destructive alterations more quickly than do mammalian
proteins such as those present i n beef.
Most people consider that f i s h i s at i t s best
from the point of view of flavour, consistency and palat a b i l i t y Immediately a f t e r i t i s caught.
Storage i n
cracked melting ice immediately a f t e r catching w i l l keep
f i s h i n a marketable condition f o r some days.
Cod and
haddock c a r e f u l l y treated i n this manner may be kept from
i
eleven to fourteen days, and halibut up to almost twenty
days, depending upon the o r i g i n a l state of i n f e c t i o n ,
without s i g n i f i c a n t change.
But i t must be emphasized
that once this i n i t i a l latent period i s over, decomposition proceeds with r a p i d i t y and certainty as Beatty
3.
and Gibbons ( 1 9 3 5 ) have shown.
This period of time i s
i n s u f f i c i e n t f o r any hut the most.proximate markets and
freezing must be resorted to f o r the moi-e distant ones.
The effects of freezing upon a tissue are due
to two things, the removal of water as ice and the reduction i n temperature of the system.
Both w i l l cause
change i n the rate of physical, chemical, and microbiol o g i c a l reactions taking place i n the system, greatly
i n h i b i t i n g some and enhancing others.
The degree of
change vrt.ll depend upon the rate of freezing and the
extent or degree of severity of freezing.
A great deal of l i t e r a t u r e i s available r e l a t i v e to the freezing of b i o l o g i c tissue, but most of
them have concerned themselves with the effect which the
rate of freezing has upon v i s i b l e structure.
The "rapid
freezing" technique has been evolved mainly out of these.
The true implication of the phrase "rapid freezing" has
not been c o r r e c t l y understood since the demarcation line
between "slow" and rapid freezing had not been experimentally determined.
Much of the credit f o r accumulating experimental evidence i n this connection goes to Moran ( 1 9 3 2 ) ,
who by an ingenious method discovered that when the
temperature of beef muscle was lowered from ^ 1 ° to 2-3°F
( 5 ° to - 5 ° 0 ) within an i n t e r v a l of from forty to f i f t y
minutes, the rate of freezing was
i f any, physical damage was
such that very
done to the c e l l
little,
structure.
He also showed the formation of large ice c r y s t a l s with
resultant damage to the tissue on sloxv freezing.
He
therefore designated the former rate as the c r i t i c a l rate
of freezing.
The
success which attended the use of this
method i n the determination of the rate of freezing of
beef led to numerous investigations' on f i s h f l e s h .
(1933)
has
Finn
shown that the maximum zone of c r y s t a l f o r -
mation l i e s between 3l°F (-.5°C) and 23°F (-5°G), about
82/£ of water appearing as lce»
Young
(1935)
Choosing this i n t e r v a l ,
calculated that the time for the
critical
rate of freezing for f i s h would be about t h i r t y f i v e
minutes*
Although rapid freezing has been evolved i n
order to preserve the o r i g i n a l structure of the tissue.,
recent experimental work has indicated that the
condit-
ions of storage a f t e r rapid freezing are more important
to the ultimate condition of the f i s h *
Quite apart from
this v i s i b l e physical e f f e c t , the depth of freezing has
been shown to be responsible
the frozen
for most of the changes i n
state.
Proteins i n t h e i r natural state are
colloidal
suspensions of albuminous material i n aqueous s a l t s o l -
ution.
The relationship \tfhich water bears to the system
is very d e l i c a t e l y balanced, and among other things,
i s dependent for i t s equilibrium upon certain d e f i n i t e
concentrations of n a t u r a l l y occuring s a l t s , acids and
basic substances.
When water i s removed from the system
as i c e , s a l t and acid concentrations
where an i r r e v e r s i b l e disturbance
(called denaturation)
increase to a point
i n the equilibrium
takes place.
When s t i l l further
water i s removed as ice by lowering the temperature,
another zone of concentration i s reached which does not
have this effect, as shown by Finn
(1932).
In t h i s
zone the proteins may be stored f o r some months without
apparent harm.
It
i s necessary at this point to understand
c l e a r l y what freezing and freezing points are.
It is
frequently but erroneously assumed that the f i s h f l e s h
is
"frozen" by the time they become^ r i g i d at a tempera-
ture of about 25°F (-^°C).
Finn
(1933)
demonstrated
that 9% of the l i q u i d s of f i s h muscle juice could
be separated
i n the unfrozen.state
still
at 1^°F (-10°C), but
t h i s method could not well he applied at much lower
temperatures.
Carter (19^)
l a t e r demonstrated by a
d i f f e r e n t method that about 11% of the l i q u i d s of halibut
muscle juice remained unfrozen at -7°F (-21.5°C).
This
method, depending on the delicate measuring of the
expansion
is
i n volume c a u s e d by
applicable
at s t i l l
C a r t e r and
Complete
Freezing
lower
knowledge c o n c e r n i n g the
a t what t e m p e r a t u r e
is
actually
the
Flesh"
on
"The
summarize o u r p r e s e n t
subject,
freezing
i n an a r t i c l e
end
proceed
to
ascertain
o f the f l e s h
of
flounder
complete.
To
f u r t h e r the knowledge o f the p h y s i c o - c h e m i -
cal
effects
and
to undertake
resulting
f r o m the f r e e z i n g
f o r meaeuring
experiments
that
these e f f e c t s
I, I I , III and
As
of f i s h
a more c a r e f u l a n a l y s i s
tometric e x p e r i m e n t a l p r o c e d u r e
suitable
liquid,
temperatures.
(19^5)
Frost
of F i s h
the f r e e z i n g o f t h e
a preliminary
o f the
appears
t o be
IV below were
muscle,
dila-
to b e
most
examined,
conducted.
to t h e s e e x p e r i m e n t s ,
a some-
what n o n - r e l a t e d e x p e r i m e n t was
undertaken
f a m i l i a r w i t h the a p p a r a t u s and
method o f i t s o p e r a t i o n .
The
results
viding
fishing
I.
II.
of t h i s
e x p e r i m e n t were d e s i r e d a l s o
some d a t a r e q u e s t e d by t h e B r i t i s h
as p r o -
Columbia
industry.
The
true
freezing-point
and
fresh-water f i s h
The
proportion
The
magniture
range
of n o n - o i l y
marine
flesh.
o f aqueous l i q u i d
frozen at d i f f e r e n t
III.
t o become
i n such f l e s h
temperatures.
o f any n o n - r e v e r s i b l e ,
permanent
change i n a p h y s i c o - c h e m i c a l c o n d i t i o n
(e.g.
as
7.
v o l u m e ) of
the
flesh
after being
frozen
and
thawedo
' •
Incidental
ments, one
In
IV.
type
available
The
fish
to
of d a t a n o t
literature
coefficient
muscle
conducting
had
the
above
explicitly
t o be
of cubic expansion
tissue.
experirecorded
determined:
of
anhydrous
8.
APPARATUS
The
apparatus
used throughout
g a t i o n was t h e same a s , o r s i m i l a r
C a r t e r and F r o s t
(19^5)
this
investi-
t o , t h a t u s e d by-
i n an e a r l i e r
investigation of
some p h a s e s
o f the same p r o b l e m .
essentially
a d i l a t o m e t e r , o p e r a t i n g on t h e same
as a t h e r m o m e t e r , n a m e l y
an
enclosed substance
that
by
substance
flesh)
a s the t e m p e r a t u r e o f
The change i n volume i s i n d i c a t e d
the change i n p o s i t i o n o f a c o l u m n o f m e r c u r y
c a p i l l a r y bore
the substance
The
principle
to measure changes i n volume o f
(fish
alters.
The a p p a r a t u s i s
c o n n e c t e d w i t h the e n c l o s e d s p a c e
in a
i n which
i s contained.
d i l a t o m e t e r was c o n s t r u c t e d o f P y r e x
glass
as shown d i a g r a m m a t i c a l l y i n f i g u r e I .
A cylindrical
by
b a r r e l A about
a standard-taper ground-glass
c o n t a i n t h e sample o f f i s h
constricted
in
t h e shape
flesh.
15 x 2 cm.
sleeve joint
The b o t t o m
t o be c o n t i n u o u s w i t h a c a p i l l a r y
against
a millimeter
l o n g a t i o n was made h o r i z o n t a l
s c a l e S.
o f A was
stem
K bent
This
pro-
I n s t e a d o f v e r t i c a l to
a v o i d undue m e r c u r o s t a . t l c p r e s s u r e b u i l d i n g
up i n t h e
A.
During
cal
J s e r v e d to
shown t o a f f o r d a h o r i z o n t a l p r o l o n g a t i o n
of u n i f o r m b o r e
barrel
divided
portion
experiments,
o f the c a p i l l a r y
t h e b a r r e l A and t h e v e r t i tube K were immersed
in a
Fig.
1. DILATOMETER
(To face page 8.)
c y l i n d r i c a l Pyrex glass j a r (not shown i n the diagram)
containing about f i v e l i t e r s of a 28$ calcium chloride
brine.
The l e v e l of immersion i s shown by the l i n e LL
in the diagram.
This volume of brine allowed s a t i s -
factory temperature control.
Cooling of the brine bath
was accomplished by lowering to i t s bottom a small wire
ce.ge containing lumps of "dry i c e " whose generation of
C0
2
gas also provided e f f i c i e n t s t i r r i n g of the brine.
Other appurtenances to the apparatus are des-
cribed i n the following section.
FILLING- THE APPARATUS
To f i l l the dilatometer i n readiness f o r an
experiment, a known weight of clean mercury P was i n troduced into the b a r r e l A and through the stopcock F
attached to the f a r end of the c a p i l l a r y K suction was
applied u n t i l the mercury i n the c a p i l l a r y reached a
convenient reading near the left-hand end of the scale
S.
The stopcock F, lubricated with grease, was then
closed.
A sample of fresh f i s h f l e s h Q, x»/as cut with
scissors or a knife from the fleshy body part of the
f i s h so that, i t s dimensions (e.g. l x l x 17 cm.) would
allow i n s e r t i o n of the sample through J into the barrel
A.
The sample of f l e s h was c a r e f u l l y weighed
(25 to ^0
10,
gm.)
and a c o r r e s p o n d i n g
same f i s h
was p r e s e r v e d
preparation
symmetrical
f o r moisture
o f a n h y d r o u s muscle
phase IV o f t h e e x p e r i m e n t a l
The
into
sample f r o m t h e
sample o f f i s h
d e t e r m i n a t i o n and
tissue
(see l a t e r
part).
f l e s h Q, was t h e n
the b a r r e l A b y I n s e r t i n g
i t through
while p r o t e c t e d by a p i e c e of cellophane
ground-glass
moist
flesh
under
Introduced
the opening
to p r e v e n t the
s u r f a c e o f J from b e c o m i n g c o n t a m i n a t e d
samples.
a weight o f f i s h
I n some e x p e r i m e n t s ,
as c o u l d be accommodated
a p o r t i o n of the weighed
J
by
where as g r e a t
i n A was d e s i r e d ,
sample Q, was i n s e r t e d
into
that
p a r t o f A above the j o i n t J .
Before
hexane
fill
the
(from petroleum,
A from
joint
J , a recorded
a wire
around
a burette.
were r e l e a s e d .
J was t h e n
fitted
closed
to
The
together.
5 0 - m l . b u r e t t e B c o n t a i n i n g t h e hexane was now
the s h o r t g l a s s t u b e
stopcock
V.
By
t h e s i d e s o f the c o l u m n o f
any a d j e r i n g a i r b u b b l e s
joint
from
n e c t e d " b y meajis o f t h e s h o r t p i e c e o f r u b b e r
to
volume o f
Ea,stman Kodak) s u f f i c i e n t t o
introduced d i r e c t l y
specially-lubricated
The
joint
t h e s u r f a c e o f t h e m e r c u r y t o the b o t t o m "of
J xms
poking with
fish,
closing
con-
tubing R
above the s p e c i a l l y - l u b r i c a t e d
Before
opening
V, hexane was
allowed
d i s p l a c e a l l a i r a b o v e V and t h e r e a d i n g o f t h e
hexane on t h e b u r e t t e was r e c o r d e d .
applying gentle
intermittent
By o p e n i n g
V and
s u c t i o n to the t o p of the
11.
"burette, hexane f l o w e d down to f i l l
In
A*
the r e s t o f the space
Any a d h e r i n g a i r b u b b l e s i n the upper p a r t of A
were u s u a l l y e a s i l y removed by t a p p i n g , though
sometimes
s t r o n g s u c t i o n a t the top of the b u r e t t e had t o be a p p l i e d
to
expand the b u b b l e s to cause them to r i s e .
Any d e s i r e d
f i n a l adjustment o f the mercury column a g a i n s t the s c a l e
S was made a t t h i s s t a g e by c a r e f u l l y opening s t o p c o c k F
and l e t t i n g i n a l i t t l e
a i r or a p p l y i n g a l i t t l e
suction,
as r e q u i r e d , w h i l e the hexane i n A was s t i l l i n communic a t i o n w i t h the hexane i n the b u r e t t e .
F i n a l l y the s t o p -
cock V was t i g h t l y c l o s e d , the a d d i t i o n a l amount o f
hexane added to f i l l
the upper p a r t o f A was
recorded
from the b u r e t t e , and the b u r e t t e and rubber c o n n e c t i o n
tube were
removed.
Stopcock F was now opened i m m e d i a t e l y and
left
open t o o b s e r v e from any change i n t h e s c a l e r e a d i n g o f
the
mercury whether the system was p r e s s u r e - t i g h t , a l s o
to
p e r m i t volume changes i n the a p p a r a t u s a g a i n s t atmos-
pheric pressure.
Mercury was employed as a f i l l i n g l i q u i d f o r the
c a p i l l a r y because i t does not wet the g l a s s and would
not
to
e v a p o r a t e through F; i t was not p r a c t i c a l ,
use mercury to f i l l
all
however,
the space i n A not o c c u p i e d
by the f l e s h sample Q, s i n c e Q, would be compressed i n t o
the
top p a r t o f A, muscle j u i c e would be squeezed o u t ,
and r e m a i n i n g a i r b u b b l e s c o u l d not be seen,
Hexane was
therefore
it
chosen to f i l l
the r e m a i n i n g
h a s a low f r e e z i n g p o i n t , i s n o n - m i s c l b l e
aqueous l i q u i d s
peratures
action
used
i n the f i s h
i n these
The
solvent
proved
i s non-miscible
allowed
of A
volatility
difficult.
with
pressure
dissolving
f o r J and V.
hexane, a n d when u s e d s p a r i n g l y ,
surfaces
t o be g e n t l y
a lubricant
i n A to f o r c e
V.
out the g l y c e r o l ,
V and J were s e a l e d w i t h
lubricant
diffi-
that g l y c e r o l had s u i t a b l e q u a l i t i e s .
f o r c e out the p l u g o f stopcock
pelling
Petroleum
action hut i t s high
t o g e t h e r whereas too g r e a s y
bath
sample Q,.
of a suitable lubricant
the ground-glass
mercurostatic
Q, and a t t h e low tem-
u s e o f h e x a n e , however, i n t r o d u c e d
i n the c h o i c e
Experience
w i t h the
e x p e r i m e n t s h a s a low s o l v e n t
made q u a n t i t a t i v e f i l l i n g
culty
flesh
on any o i l i n the f l e s h
e t h e r has a l e s s
It
space i n A s i n c e
i n v i t e d the
Joint
To a v o i d
the o u t e r
J apart or
the brine
surfaces of
a r i n g of " s i l i c o n e "
or " p l a s t i c i n e " .
"wrung"
water-re-
EXPERIMENTAL PART
1.
Determination Of The True F r e e z i n g P o i n t Of F r e s h
F i s h Muscle.
(a)
L l n g c o d (Ophlodon elongatus) muscle.
The samples
o f f l e s h used were cut from
f r e s h l y - c a u g h t l i n g c o d obtained d i r e c t l y from l o c a l
handling p l a n t s .
fish
The columns of f l e s h were out from the
f l e s h y "back w e l l under the s k i n and were f r e e from bone.
After f i l l i n g
the
the d i l a t o m e t e r as d e s c r i b e d ,
b a r r e l was immersed i n the b r i n e bath a t 0°C.
After
a l l o w i n g some f i f t e e n minutes f o r the apparatus and
sample, to a t t a i n bath temperature, the tempera.ture of
the
bath was then s t e a d i l y and s l o w l y lowered w i t h d r y
ice
a t the r a t e of about 0.1 degree C. p e r minute.
slow and steady r e t r e a t of the mercury
the
( c o n t r a c t i o n of
system) a l o n g the s c a l e stopped almost immediately
i f the temperature was h e l d constant f o r a t r i a l
of
The
interval
a minute, i n d i c a t i n g that t h i s r a t e of c o o l i n g was
s u f f i c i e n t l y slow to a l l o w s u b s t a n t i a l l y continuous
e q u i l i b r i u m between the temperature of the contents of
the
b a r r e l and that of the bath as measured by a t e s t e d
mercury, thermometer graduated i n tenths of a degree
Centigrade.
At
some temperature a few degrees below -3°C
(varying
i n different
mercury h a l t e d . a n d
formation
e x p e r i m e n t s ) , the r e t r e a t
a sudden a d v a n c e
( e x p a n s i o n due t o
o f i c e i n the sample)' o c c u r r e d .
a t u r e was now h e l d
constant, this
due t o i c e f o r m a t i o n f r o m
supercooled)
aqueous l i q u i d s
had
of this
experiment,
I f t h e temper-
advance
many m i n u t e s
purpose
continued f o r
the ( s l i g h t l y
i n the f l e s h .
F o r the
however, once t h i s
commenced, i t was d e s i r e d
to h a l t
warming t h e b a t h a few d e g r e e s
of the
until
advance
i t immediately by
a retreat
o f the
m e r c u r y showed m e l t i n g h a d commenced, b u t t o a v o i d complete melting i n order that
remain
as s e e d i n g c r y s t a l s
cooling.
Furthermore,
as p o s s i b l e
erature
in
i t was d e s i r e d
o f the f l e s h
the r e t r e a t
taking
was a g a i n l o w e r e d
slowly.
a s much
t o s u b - f r e e z i n g temp-
unavoidable
freezing re-
o f the m e r c u r y
indicating
the thawing,
place,' the bath
This lowering
and the t e m p e r a t u r e
t r e a t o f the mercury h a l t e d
o f i c e would
still
temperature
eventually
a t which r e -
and ad.vanee a g a i n t o o k p l a c e
was t a k e n a s the t r u e f r e e z i n g
cooling.
to prevent
from s u p e r c o o l i n g .
t h a w i n g was s t i l l
crystals
super-
any c h e m l c o - p h y s i c a l changes
due t o t h e b r i e f
While
overtook
of i c e would
to a v o i d s u b s e q u e n t
I n o r d e r to m i n i m i z e
the f l e s h
sulting
exposure
some n u c l e i
point
of the f l e s h ,
be p r e s e n t t o a v o i d
since
super-
15 a
(b)
Rainbow Trout (Salmo g a l r d n e r l l ) muscle.
For the purpose of comparing the freezing point
of the muscle of a freshwater f i s h with that of a marine
f i s h , a sample of f l e s h from a freshly-caught rainbow,
trout was used i n an experiment duplicating (a) above.
In experiments under both (a) and (b) above,
the magnitude of the scale readings of the mercury as i t
advanced or retreated had no significance since only the
temperature i n r e l a t i o n to point of i n f l e c t i o n of the
movement was desired.
Hence no corrections f o r change i n
volume with temperature of the apparatus or i n d i c a t i n g
l i q u i d s were necessary as i n subsequent experiments of a
d i f f e r e n t nature,
II,
Freezing the Muscle Slowly In Different Stages to
-30°C(-20 F)
O
In doing this experiment, the bath was allowed
to come to equilibrium at various temperatures below the
freezing point of the f l e s h before reading the thermometer, and the dilatometer to aquire the bath temperature
before reading the scale.
stirred.
The bath was continuously
Both llngcod and rainbow trout f l e s h were used.
The results obtained i n these experiments are recorded
in "Results from Adjusted Data", part I I ,
16.
III.
Permanent change In Volume of F i s h Flesh Caused
by Freezing and Thawing
This experiment i s e s s e n t i a l l y the continuation
of experiment I I . The apparatus containing the frozen
f i s h f l e s h was brought to room temperature and allowed to
remain so overnight to ensure complete thawing out of the
frozen f l e s h .
I t was then immersed up to the l i n e LL
( f i g . I) i n the brine bath at exactly 0°C. u n t i l e q u i l i b rium was reached between the temperature of the bath and
the contents of the apparatus.
This was ascertained as
usual by awaiting constancy of the reading (N ) of the
2
mercury on the scale.
This scale reading was subtracted
from the already-observed reading (N-^) of the mercury at
exactly 0°C. before the f i s h f l e s h had been frozen.
The
difference (N^-^) i n scale readings corresponded to the
permanent change i n volume that occurred i n the sample of
f i s h f l e s h due to freezing and subsequent thawing.
The
results are shown i n "Results from Adjusted Data", part
III.
IV.
The C o e f f i c i e n t of Cubical Expansion of Anhydrous
F i s h Muscle
The c o e f f i c i e n t of cubical contraction of
anhydrous f i s h muscle was determined using the same apparatus and the same procedure.
This experiment was made
mainly with a view to providing data needed i n making
certain adjustments i n other experimental data.
These
17.
data were obtained f o r the f i r s t time, since they were
not found i n any available l i t e r a t u r e .
The following procedure proved quite
satis-
factory i n obtaining the requisite quantity of anhydrous
fish flesh.
Ordinary methods of dehydrating, l i k e keeping
in hot oven or immersing i n absolute alcohol were avoided
since the former method denatures the f l e s h by cooking and
the l a t t e r also denatures the protein and possibly also
extracts some constituents*
About 500 grams of fresh llngcod f l e s h was
f i n e l y ground to a b r e i and then transferred to a 500-ml
narrow neck, round bottomed f l a s k f i t t e d with a one-hole
rubber stopper through which passes a glass tube.' The
f l a s k was immersed i n a water bath kept at 40°-50°C and
evacuated by connecting to a w.aterpump.
shaken by hand occassionally.
The f l a s k was
After four hours a con-
siderable amount of moisture was found to have been r e moved from the f l e s h .
The contents of the f l a s k were
transferred to a mortar and ground w e l l .
The r e s u l t i n g
s t i c k y powder was spread on a p e t r i dish and kept over
night i n a vacuum desiccator over P2°5«
Next day the
granular mass was ground and passed through a ^O-mesh
sieve and the resulting powder was kept f o r two days i n
a vacuum desiccator over ¥2^5'
18.
EVALUATION OF RESULTS
A*
Adjustments
of Experimental Data
Before interpreting the r e s u l t s the following
factors a f f e c t i n g the observed values were borne i n mind
and allowance was made f o r them.
During the lowering of the temperature of the
bath from 4°C down to the freezing point of the f i s h f l e s h
the apparatus and a l l i t s contents are contracting except
the muscle Juice, which commences to expand (since water
expands on cooling below ^°C.) and continues to do so
s l i g h t l y through any supercooled conditions, u n t i l i t
begins to freeze.
At and below the freezing point to a c e r t a i n
d e f i n i t e temperature, the f i s h muscle Juice progressively
freezes and hence expands greatly, but the rest of the
material, v i z . (a) hexane, glass, the solids of the f i s h
muscle and the ice already formed undergo contraction;
(b) the as yet unfrozen supercooled l i q u i d undergoes
expansion; and (c) the mercury i n the apparatus i s another
important f a c t o r to be taken into account.
During an
experiment, as the mercury moves to d i f f e r e n t readings
along the scale which i s at room temperature, there w i l l
be a varying amount of mercury subjected
temperature of the bath.
to the varying
Hence the net change i n volume
of the system recorded on the scale (due to changes i n
Fl&» „2
i*!o f a c o p a g e
19)
19.
bath temperature) l s the net result of the expansion due
to the freezing of the f i s h muscle Juice (and the supercooling of unfrozen l i q u i d ) , and the contraction due to
the
rest of the material.
The position of mercury on the scale reaches a
maximum at a p a r t i c u l a r , d e f i n i t e temperature (below 0°C)
and as the temperature i s lowered thereafter, contraction
due
i s noted on the scale because the expansion/to the
freezing of muscle Juice l s less than the contraction due
to the rest of the material.
1.
Adjustment of Readings f o r Change i n Volume with
Temperature of Organic F i l l i n g Liquid.
Fig.
2 gives the scale correction f o r volume
change of ^0 ml. of hexane (from petroleum Eastman Kodak)
as i t s temperature f a l l s below room temperature (20°C).
This graph was constructed from data on the density of
"hexane from petroleum", H e l l (1932).
To apply the cor-
responding correction f o r any other volume V of hexane of
o
measured at room temperature 20 C as i t s temperature f e l l
below this temperature, the result obtained from the
expression
V
x
Scale Correction, was added to the
observed scale reading.
2.
Adjustment of Readings For Change In Volume
with Temperature of Glass Apparatus
One cc. of pyrex glass contracts 0.0000108 cc,
for
each degree of temperature drop.
Therefore, 55 cc.
200
SCALE
250
DIVISION
300
READING
350
NOMOGRAM FOR READING THE SCALE CORRECTION
DUE TO TEMPERATURE DIFFERENCES
O F 80 GRAMS OF MERCURY,
(To face page 20)
20.
of volume enclosed by pyrex glass contracts 5*^ scale
d i v i s i o n s when the temperature of the glass f a l l s from
+20°C to - 3 0 ° C .
This correction i s so s l i g h t that f o r i n t e r mediate temperatures the proper correction to be applied
was readily estimated without a graph.
3.
Adjustment of Readings f o r Change i n Volume with
Temperature of Mercury Indicator Liquid
A nomogram was constructed to apply the proper
scale reading f o r change i n volume of the mercury from
one temperature to another.
( F i g . 3)
The following expression was derived f o r the
above purpose using the recognized densities of mercury
and certain ascertained c h a r a c t e r i s t i c s of the apparatus.
With the apparatus empty and with enough mercury
i n the c a p i l l a r y , the scale reading R at room temperature
T was taken from the point L ( = average l e v e l to which
the
l i q u i d i n the cooling bath comes).
Then the apparatus was t i l t e d gently so that
t h i s same volume of mercury lay, e n t i r e l y i n that portion of
the
c a p i l l a r y against the scale.
RQ_ and R
2
The two scale readings
opposite the ends of the mercury thread were
then recorded, the mercury was emptied out, and weighed.
Let
l a r y between ^
length of mercury column within the c a p i l and R
2
- (R - Ri) scale readings at
2
21.
temperature T°C.
Weight of mercury = W grama (kept constant
in several experiments)
Temp, of mercury
= T°C
I f density of mercury (from tables) at T°C = Dtp
then volume of mercury = W
Dip
And value of one scale d i v i s i o n i n the bore of
the c a p i l l a r y tube = W / (Rg-R-^ T
D
D
w
(R - Ri)
T
Q
2
C l a constant as long as the same c a p i l l a r y
tube i s i n use.
Also;
W
D
- CR = E = volume of the mercury from
T
point (L) to zero point on c a p i l l a r y scale.
This value E
always remains a constant during an experiment.
Then, i f R^ i s the reading on the scale at any
given temperature Tg at any stage of an experiment, the
scale reading correction to be added i s obtained from the
expression:
W -D E
m
- D R
T
3
X
D B - Dip
D D
B
T
where Dg s Density of mercury at the bath temperature Tg.
This l a s t expression was equated to successive
i n t e g r a l values 1 to 8 scale reading corrections. For
each of these corrections, a series of values of
was
22*
was found by substituting f o r Dg densities of mercury at
a selection of temperatures from 4,20° to -38°C.
these corrections f o r
Plotting
against Dg gave the family of
l i n e s shown i n the nomogram ( f i g . 3 ) which was then of
considerable value f o r ascertaining the proper scale reading correction f o r observed values of R^ and Tg.
In some experiments, a d i f f e r e n t weight W of
mercury was used i n f i l l i n g the apparatus.
In this case,
a d i f f e r e n t nomogram had to be constructed, but the p r i n ciple used i n correcting observed scale readings was
exactly the same as the above.
i+.
Adjustment of Readings f o r Change i n Volume with
Temperature of Anhydrous F i s h Muscle
When
25.00 gm (= 19.5 cc.) of anhydrous lingcod
muscle was used i n the dilatometer to determine the coe f f i c i e n t of expansion of the anhydrous f l e s h , i t was
necessary f o r experimental reasons to use
30.26 cc, of
hexane to f i l l the remaining space i n the b a r r e l .
This
r e l a t i v e l y large volume of hexane i n comparison with the
volume of the anhydrous f i s h muscle caused inherent d i f f i c u l t i e s i n adjusting observed values of the volume of
the system at d i f f e r e n t temperatures, owing to a s l i g h t
uncertainty i n the physical constants f o r the hexane.
There were no uncertainties i n the contraction constants
f o r the glass and mercury.
23.
A f t e r calculating
the values for contraction of
the hexane, and adding to these the contraction of the
glass and mercury at the various temperatures from +20°C
to -30°C, then using the totals as f i n a l adjustments f o r
the
observed values of the contraction of the system as a
whole, a s l i g h t l y i r r e g u l a r curve was obtained when p l o t t i n g volume of the anhydrous muscle against temperature.
The straight l i n e best representing this curve was drawn,
end from i t the appropriate values were read for evaluating the expression f o r c o e f f i c i e n t of expansion:
V
where
T
V ( l + oC(T-t))
=
t
=
volume of anhydrous f l e s h at temp.T = +20°C
=
volume of anhydrous f l e s h at temp.t =
°C =
-30°0
c o e f f i c i e n t of cubic expansion over the
temperature range -30°C to +20°
19.50
=
19.39
whence oC z
f l + {20-(-30)}|
oC
0,000118
The graph prepared i s not i l l u s t r a t e d , since
i t s nature and use were similar to that shown for the
change i n volume of hexane ( f i g , 2 ),
Corrections to ob-
served scale readings to compensate f o r changes In volume
of the s o l i d constituents of the f l e s h were read and computed d i r e c t l y from the graph.
small.
The corrections were quite
2k.
5.
Adjustment of Readings for Change In Volume with
Temperature of Preformed Ice In Fish Muscle.
Since any ice formed at a given temperature below
the
freezing point of the f l e s h would contract on further
cooling, i t was necessary to know t h i s contraction to be
able to adjust the observed scale readings at the lower
temperatures.
But before this could be done, a f i r s t
estimate or approximation of the amount of ice present at
each temperature had to be secured.
This was computed by
f i r s t assuming that the known weight of water i n the
sample of f l e s h a l l turned to i c e , and calculating the
expansion that would have occurred. Then, from the adjusted
observed expansion, the f i r s t approximation of the
percentage of i c e formed at each temperature was found.
F i n a l l y , from data i n the International C r i t i c a l Tables
on the c o e f f i c i e n t of cubic contraction of ice at d i f f e r e n t
temperatures below 0 ° C , the contraction of the i c e formed
at one temperature as i t cooled to a lower temperature
was read from a graph (not shown) and these values were
used i n obtaining f i n a l adjusted values,
6.
Adjustment of Readings f o r Change i n Volume with.
Temperature of Supercooled Aqueous L i q u i d i n
F i s h Muscle.
The procedure used here was similar to that
described above f o r i c e . Once the f i r s t approximation for
25.
the amount of i c e formed at each temperature
waa
obtained
as mentioned, a f i r s t approximation f o r the amount of
supercooled unfrozen l i q u i d was known, since:
% supercooled l i q u i d = 100
- % water already frozen to i c e .
Then, from t h i s f i r s t approximation
of the amount of super-
cooled l i q u i d present, the amount of i t s expansion as i t
became colder was obtainable.
Unfortunately there appeared
to be no data on the density or s p e c i f i c volume of water
supercooled below - 1 3 ° C .
supercooled to - 7 2 ° C ,
i n the l i t e r a t u r e , although water
i s known.
Dorsey, an expression was
However, i n a book hy
found that warranted extrapol-
ation to lower temperatures:
Vr
c
'
=
7
u
By allowing V^OQ
s
1,
at -30°C
a
_
_
_
_
*
_
temperatures
(e.g. s p e c i f i c volume of supercooled water
1,0171).
From the table of s p e c i f i c volumes,
the contraction of any f i r s t approximation
to the weight
of supercooled water as i t cooled s t i l l further could be
found, and then these values were used i n obtaining f i n a l
adjusted values,
B.
—
a table was prepared showing s p e c i f i c
volumes of 1 gram of supercooled water at
below - 1 3 ° C .
3
e
Results From Adjusted Data,
I (a) Freezing point of llngcod f l e s h .
The I n i t i a l freezing point of d i f f e r e n t
samples
of llngcod f l e s h due to unavoidable supercooling varied
26.
from - 3 . 6 ° C ( 2 5 ° F ) to -7.8°(18°F). ,But the true f r e e z i n g
point obtained
aa described i n the experimental part was
the aame f o r a l l the aamples, namely -1.5°C(29.1°F)•
On
thawing and re-cooling, the aupercooling was again observed
to a varying degree.
(b) Freezing point of Rainbow Trout
The
flesh.
I n i t i a l supercooled freezing point of one
sample of rainbow trout f l e s h was found to be -6.8°C, and
the true freezing point -1.5°C.
II.
Percentage of Water Frozen Out at Different
Temperatures.
Tables 1 and 2 below show the f i n a l calculated
values of the percentages of the t o t a l water present i n
f i s h f l e s h that i s frozen out at d i f f e r e n t low temperatures.
The i n i t i a l value f o r rainbow trout f l e s h i a at
the true freezing point of -1.5°C, while the i n i t i a l value
for lingcod f l e s h i s given at the supercooled i n i t i a l
f r e e z i n g point.
In each case, the i n i t i a l value i s shown
as zero to indicate the condition when freezing was Just
on the point of beginning.
A l l values have been subjected
to a l l the s i x adjustments described^
27
TABLE 1
TABLE 2
Freeh Rainbow Trout Flesh
Freah Lingcod Flesh
Temp. °C.
-
1.5
% Total Water
Frozen Out
Temp. °C.
% Total Water
Frozen Out
0
-
7.6
20.2
-
7.8
86»>
0
-
5.0
79.1
-
9.0
88.0
-
6.0
82.9
-12 i0
91.7
-
7.0
85.7
-llf.O
92^8
-
8.0
86.9
-16.0
9^.0
-10.0
90.2
-20.0
96.0
-13.0
92.8
-25.0
97.9
-15.0
9^.8
-30.0
99.7
-19.0
96.8
-31.6
99.7
-23.0
99.3
-25.0
99.5
-26.0
100.5
-28.0
100.1
-29. ^
100.7
III.
-
Permanent Change In Volume of Flah Flesh Caused
by Freezing and Thawing.
The number of scale d i v i s i o n s corresponding to
the permanent change i n volume was 5 . 5 . The weight of
28.
f l e a h taken (llngcod) waa ^0.98
gms.
Since the s p e c i f i c
gravity of f i s h f l e s h l s very nearly 1, the volume of the
f l e s h was taken to be tyO cc.
The volume occupied by 1 scale d i v i s i o n of
mercury was known to be
0.005^6 ccs.
Hence the volume occupied by 5*5
of mercury
s
5-5
x
scale d i v i s i o n s
0.005^6 ccs. = 0.03005 ccs.
This change i n volume i s due to *K) cc. of f i s h
f l e s h alone since the apparatus and the rest of the
material Inside It have so f a r not been shown to exhibit
such phenomena.
No adjustment of observed values were
necessary since a l l readings were at the same temperature.
Hence the permanent change i n volume that occurs In f i s h
f l e s h during freezing and thawing =
IV.
0.03005 x 100 = 0.075^
Coefficient of Expansion of Anhydrous F i s h Muscle.
This was one of the adjustments necessary i n
the determination of the percentages of water frozen out
at d i f f e r e n t temperatures, as previously explained.
The
manner i n which the c o e f f i c i e n t was calculated has been
described already i n part k under "Adjustment of Experimental Data".
Therefore, only the f i n a l result i s repeated
here:
<£- = 0.000118 over the temperature range +20°C to -30°C
29.
DISCUSSION
So f a r the methods used f o r freezing point
determinations of f i s h muscle have yielded i n s u f f i c i e n t l y
accurate r e s u l t s .
Whole f i s h were kept i n temperature-controlled
chambers.
The f a l l i n the temperature of f i s h muscle due
to progressive cooling was measured by inserting thermoe l e c t r i c couples or s i m i l a r temperature-measuring devices
inside the f i s h muscle.
The freezing point was taken as
that temperature at which the reading of the Instrument
was constant
f o r a considerable period due to g i v i n g up of
the latent heat of freezing.
The drawbacks of the above method were several,
one
i n p a r t i c u l a r being the lack of a d e f i n i t e l e v e l plateau
i n the curve showing the r e l a t i o n between time and temperature.
The method described i n this thesis involves the
use of the dllatometer by means of which the sudden expansion due to freezing can be easily observed, p r i n c i p a l l y
due
to the fact that water expands some ^% of i t s volume
on freezing.
This i s the f i r s t time that this apparatus
has been used with a l l possible refinements to record the
true freezing point of f i s h muscle and other data.
The
operation of the apparatus i s simple and the r e s u l t s can
be accurately reproduced.
30.
Attempts were made to f i n d out whether once
frozen and thawed, examples of f i s h f l e s h exhibited any
difference i n the further magnitude of the freezing point
range.
I t was found with certainty that the true freezing
point can never be obtained
a seeding c r y s t a l of i c e .
i n the i n i t i a l freezing without
In other words, there was a
state of supercooling of the f l e s h before i t a c t u a l l y
began to freeze.
Experimentally,
this was overcome as
described by f i r s t supercooling to the point of Incipient
freezing, then taking advantage of the ice c r y s t a l s formed,
to determine the true freezing point.
Another important result obtained revealed that
the true freezing point of^marine and fresh water f i s h
f l e s h used i n these experiments was substantially the same,
though they showed varying degrees of supercooling.
A non-oily type of f i s h l i k e llngcod was chosen
for
the experiments described since presence of a high per-
centage of o i l i n the f l e s h would Interfere with the
expansion due to water alone, since o i l has not the same
c o e f f i c i e n t of expansion as water, and the c o e f f i c i e n t of
expansion of o i l l s seriously disturbed by separation of
s o l i d fats (stearine) on cooling.
Although Reay (1933) and Finn (1933) have examined the e f f e c t of various degrees of cooling upon the
f l e s h of f i s h and found that the degree of freezing and
the temperature of storage have a pronounced effect on the
31.
texture of the proteins i n the muscle, l i t t l e quantitative
data on the magnitude of observable physical e f f e c t s
accompanying the denaturatlon of the proteins of the f i s h
flesh i s available.
In my investigation i t i s found that there i s
a d e f i n i t e permanent decrease i n volume a f t e r freezing and
thawing, Since the quantity of f i s h used i n this i n v e s t i gation had to be limited to small quantities (about kO gms.),
and the duration of storage a f t e r freezing was also small,
the magnitude of the change i n volume was consequently
small, nevertheless quite perceptible.
I t i s hoped to devise further experimental
methods whereby larger quantities of f i s h f l e s h can be
frozen and kept f o r a longer length of time, and the magnitude of the change i n volume determined under more varied
conditions.
In the experiments recorded herein dealing with
the percentages of water frozen out at d i f f e r e n t temperatures below the freezing point, previously determined data
such as those by Carter and Frost were confirmed, but the
present values have been calculated from the observed data
using refinements not taken into account by them i n their
preliminary work.
However, some of the refinements are known to
be s t i l l inadequate.
needed to f i l l
The r e l a t i v e l y large colume of hexane
space around the f l e s h sample, and the high
32.
c o e f f i c i e n t of contraction of hexane, both tended to i n t r o duce s l i g h t discrepancies that are rapidly magnified by
unavoidable d i f f i c u l t i e s i n manipulation.
These discrepan-
cies were kept to a minimum, but showed up p a r t i c u l a r l y
when determining the c o e f f i c i e n t of contraction of the
anhydrous f l e s h .
The i r r e g u l a r i t i e s mentioned as having
occurred i n the curve plotted to show the volume
variation
with temperature were such as to indicate the adjustments
of the observed readings f o r contractions of the hexane
were inexkct.
Discordant data i n the l i t e r a t u r e f o r the coeff i c i e n t of contraction of i c e , and lack of d e f i n i t e values
f o r the c o e f f i c i e n t of expansion or r e l a t i v e volumes of
water supercooled below -13°C, a l l contributed to the uncertainties
below -13°C.
surrounding the adjustments for these effects
Fortunately, the percentage of supercooled
water present was least at the low temperatures where the
uncertainty regarding the correction was greatest.
The
f i r s t approximation method used for the percentages of i c e
formed and supercooled water remaining was essential; hut
i t was considered unwarrantable to perform a second approximation to these values before computing
given i n tables 1 and 2.
the f i n a l values
The values exceeding 100$
in
table 1 are of course Impossible t h e o r e t i c a l l y ; but the
s l i g h t excesses over 100$
are considered inconsequential i n
33.
view of the numerous adjustments
o r i g i n a l observed readings.
that had to be made to the
These s l i g h t excesses i n no
way detract from the value of the r e s u l t s , and were not
present i n the results i n table Z.
The chief i n t e r e s t i n tables 1 and 2 l i e s i n the
percentages of water frozen out at around -20°C (-^°F)
because modern recommendations for commercial storage and
shipment of frozen f i s h point to a temperature
being the highest temperature
compatible with maintenance
of prime quality of the frozen product.
that above this temperature
of 6°F as
Tables 1 and 2 show
there i s more than 5$ of unfro-
zen aqueous l i q u i d present i n the unfrozen product.
It i s
probably this l i q u i d phase that allows such deleterious
e f f e c t s as r a n c i d i t y , denaturation, and the l i k e , to progress
more rapidly above 0°F than below 0°F,
F i n a l l y , i t should be confessed that i t i s
r e a l i z e d there may be other s l i g h t uncertainties i n the
accuracy of the results i n tables 1 and 2,
gation could not be extended
This i n v e s t i -
to explore f u l l y the probabil-
i t y that what has been c a l l e d "ice' at low temperatures i s
1
not r e a l l y pure water i c e ; l i k e l y i t i s a complex mixture
of various eutectlcs of s a l t s occurring i n the f l e s h tissue
Juices.
Also, what has been called "supercooled water" i s
probably a d i l u t e solution of s a l t s .
These would not have
the same physical properties as ice and water, but a complete investigation of the properties would be very complex,
and beyond the scope of t h i s work.
35.
SUMMARY
1*
The true freezing points of llngcod (Ophlodon elongatus)
and Rainbow Trout (Salmo galrdenerll) f l e s h were
determined and found to be ~1.5°C (29.1°F).
2.
The percentages of water frozen out from the f l e s h at
temperatures below the freezing point were found to
Increase rapidly f o r the f i r s t few degrees below the
freezing point, then more slowly, reaching 9 5 $ at
about -18.8°C (0°F), then approaching 100$ asymptotic a l l y at s t i l l lower temperatures down to - 3 0 ° C .
3.
The permanent decrease i n volume of f i s h f l e s h at 0°C.
a f t e r freezing and thawing was observed to be of the
magnitude of 0 . 0 7 5 $ based on the o r i g i n a l volume of
f l e s h used.
This change i n volume i s most probably
due to denaturatlon of the proteins of f i s h f l e s h .
The C o e f f i c i e n t of Cubical Contraction of anhydrous
f i s h muscle has been determined f o r the f i r s t
time and
found to be 0.000118 over the range *20°C. to - 3 0 ° C .
36.
ACKNOWLEDGEMENT
I hereby wish to express my deep gratitude to
the chairman and members of the s e l e c t i o n Board,
Education
Department, Government of India for the award of one of
the Foreign Scholarships to pursue higher studies i n
f i s h e r i e s technology with special reference to f i s h o i l s
and
processing..
My thanks are due to the University of B r i t i s h
Columbia, Vancouver, B.C., for enabling me to r e g i s t e r
myself for the M.A. degree and the P a c i f i c F i s h e r i e s
Experimental Station, of the Fisheries Research Board of
Canada, f o r laboratory f a c i l i t i e s afforded i n conducting
this investigation.
I wish to express my sincere thanks to Dr. N.M.
Carter, D i r e c t o r , P a c i f i c Fisheries Experimental Station
f o r his sixggesting the problem, and his kind help i n
d e t a i l s of the experimental work and his valuable
and
advice
c r i t i c i s m during the course of my stay.
The assistance of Mr. N.E. Cook, Chemist at the
F i s h e r i e s Experimental Station, i n modifying
the apparatus
used i s g r a t e f u l l y acknowledged.
My thanks are also due to Dr. W.A. Clemens,
Head of the Department of Zoology, and Dr. W.S. Hoar,
Professor of Zoology and F i s h e r i e s , University of B r i t i s h
37.
Columbia, f o r t h e i r keen Interest i n the above investigation
and
supervision of my academic work.
Acknowledgment i s made to B r i t i s h Columbia
P r o v i n c i a l Game Department f o r kindly providing us with
a sample of f r e s h l y caught Rainbow Trout.
38.
LITERATURE CITED
Beatty, S.A. and Gibbons, N.E.
(1935), A t l a n t i c Prog.
Rep.. B i o l . Bd. Canada, No.15, 1935.
Carter, N.M.
(19^4). Unpublished.
Carter, N.M. and Frost, P.J.
(19^5), Prog. Rep. P a c i f i c
Coast Stations, Fisheries Res. Bd., Canada, No.62,
21, 19^5.
Dorsey, M.E.
(19^)»
"Properties of Ordinary Water Sub-
stance", Rheinhold Publishing Co. 19^-0.
(1933).
Finn, D.B. (1932).
Finn, D.B.
He'll, L.M.
Moran, T.
Reay, G.A.
8, No.25, 1933.
Proc. Roy. S o c , B . 3 , 356, 1932.
Cont. Can. B i o l . N.S.
(1932). Phys. Rev. 39, 670, 1932.
(1932).. J.S.C.I., 51, 16, 1932.
(1933).
Young, 0,C.
J.S.C.I., _52,
265, 1933.
(1935).. P a c i f i c Prog. Rep. B i o l . Bd. Canada,
No.26, 1935.