Mitochondrial
Population in Mammalian
Cells
I. Description of a Counting Technic and Preliminary
Results on Rat Liver in Different Physiological
*
and Pathological Conditions
CLAUDE
ALLARD,
R. MATHIEU,
(Montreal Cancer institute,
G. DE LAMIRANDE,
Research Laboratories,
(17).However,
noaccuratemethodshavebeende
vised to count mitochondria.
briefly a method of counting
clophorase―
(6). This has been, to our knowledge,
to the number
of particles.
We have
dis
missed Harman's method a priori for two reasons.
First, it is too elaborate to be practical; and, see
ond, its accuracy is not indicated.
Variation
in the population
of mitochondria
in
the liver cell has been qualitatively
estimated (18).
Furthermore, Potter, Price, Miller, and Miller (10)
have proposed a hypothetical
mechanism of car
cinogenesis in terms of the deletion of cytoplasmic
particles or of an enzyme contained
therein.
S4±neider and Hogeboom
have stressed
the neces
sity of determining the yield in terms of the num
ber of mitochondnia (17).
We present a method for the estimation of the
number of mitochondnia
or large granules in
•
homogenate,
or when
intracellular
Montreal, Canada)
properties
characteristic
liver mitochondnia
of normal
(8).
The method consists essentially in counting
fresh mitochondria
dispersed at random in a
known volume of suspension,
to the study of the
normal rat liver cell.
with reported for the
cal and pathological
and it can be applied
number of mitochondria per
Preliminary results are here
liver cell in other physiologi
conditions.
METHODS
isolation of mitoc/zondrialparticulate.—Theanimals (Wistar
rats) were killed by a blow on the head and were immediately
Harman presented
decapitated. Exsanguinationwascompletedby holdingthe ani
the particulate “cy mal by the tail and massaging the chest and back. The whole
the only attempt to express enzymatic activities in
relation
Notre Dame Hospital,
and staining
INTRODUCTION
The evaluation of the total number of mito
chondria in cells has been the subject of a few in
vestigations. Attempts have been made in the past
to count them in cells (3, 18). These methods were
histological and are considered at the present time
as tentative. Since centrifugation has been applied
to isolate particulates from the cell, the yield in
grams of mitochondnial material has been inten
sively investigated
(10, 1@—15).Schneider and
Hogeboom have discussed this matter recently
AND A. CANTERO
isolated
in 0.@5 M or in
0.88 Msucrose from a known number of normal rat
liver cells. It has been shown that mitochondria,
isolated as such, seem to retain the morphological
liver was excised, placed on cracked ice, and chilled for 2 mm
utes, then rolled on filter paper to sponge off water and blood.
The tissue was immediately minced in a cold (5°C.) plexiglas
squeezer.
Collected
liver
pulp,
thus
prepared,
was
mixed
by
hand. An amount of 2.5 gm. was rapidly weighed in a 20-ml.
beaker. Five ml. of ice-cold 0.25 u salt-free sucrose (or other
medium) was immediately added to the weighed amount of
liver pulp and again mixed. The suspension was poured into an
ice-cold 15-mi. Potter homogenizer tube. Five more ml. of
medium was used to rinse the beaker. The time interval of
homogenization
with a pestle turning at a speed of 300 r.p.m.,
was 2@minutes, which gave a maximum cell rupture with the
homogenizer
used in this work. This was controlled
by de
terminations of enzymatic activity (1) and by phase and light
microscopic examination. The suspension was then poured into
a £5-mi. volumetric flask and diluted to the mark with the
medium.
Exactly 20 ml. of the resulting 10 per cent homogenate, pro
pared in 0.25 M or 0.88 u salt-free sucrose, was immediately
centrifuged to isolate a nuclear (N),' a mitochondrial (M) and a
supernatant (5) fraction. Thecentrifugal fractionation was car
ried out as described by Schneider and Hogeboom (16), except
that the (S) fraction obtained after isolation of (N) and (M)
was not further fractionated.
After the fractions were isolated,
they were immediately diluted with the medium to the follow
ing concentrations, as compared to the originalweight of tissue:
(N), 20 per cent; (M), 20 per cent; (S), 4 per cent, i.e., 1 ml. of
* This
work
Cancer Institute
was
supported
by
a grant
from
of Canada.
the
National
the (N) or (M) fraction contained 20 per cent of the particles
1 The
Received
for publicatio@i November
27, 1951.
nuclear
fraction
is
contaminated
with
blood
cells,
cell
debris, and mitochondria.
407
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1952 American Association for Cancer Research.
Cancer Research
408
from 1 gm. of tissue or 0.2 gram-equivalent
of nuclei or mite
chondria. Servall centrifuges 58-1 and SS-2 were employed
throughout.
The temperature
of the tissue or the particulate
suspension never exceeded 15°C.
Purity of the (M) fraction was established by total nitrogen
and pentosenucleic acid determinations,
by histological exami
nation of smears, and by phase and electron microscopy.
The mean total nitrogen content of eight normal rat liver
mitochondrial fractions isolated in 0.25 u sunrose was found to
be 6.4 ±1.4 mg. per gram of fresh liver and corresponded to
21.4 ±1.8 per cent of the total nitrogen content of liver. The
mean pentosenucleic acid phosphorus content, estimated on the
mitochondrial fraction from five normal livers, was 123 pg. and
corresponded to 15.6 per cent of the total liver PNA phos
phorus.
This confirmed
earlier results
obtained
on pure (M)
could be computed.
Dealing
with 0.2 per cent concentrations,
the value for the total number of particles present in the (M)
fraction
per gram of fresh tissue was obtained
by multiplying
the mean number of mitochondria counted in one prism by the
factor 10'°.
RESULTS
The number of granules found in the (M) frac
tion from nine normal rat livers, isolated in 0.@ M
or in 0.88 M sucrose, lies within the limits of @9.0to
30.@X 10'°,
with a probabilityof 0.975.The mite
chondria contaminating
the nuclear fraction were
counted from eight normal rat liver (N) fractions.
fractions (17). Electron microscopicstudies confirmedthe fact
Withthe sameprobability,the numberlies within
that only one type of particulate was present. Fig. 1 illustrates
the homogeneity of the suspension. Small debris seen in the
photograph are probably parts of broken mitochondria, since a
few disintegrating mitochondria are always present, as shown
in Figs. 2 and 3. Examination of the (5) fraction reveals this
fraction to be free of mitochondria.
Counling technic.—The apparatus
adopted for counting
the limits of @.84—4.18
X 10'°,which gives, per
gram of fresh rat liver tissue, a total ranging from
31.84 to 84.18 X 10w. Counts in the nuclear free
tion are made in the same manner as with the
mitochondrial fraction, but at a concentration of 1
per cent. Nuclei at this concentration are so scarce
(8 or 4 per 400 prisms) that they do not interfere
with mitochondrial counts. These results are pre
sented in detail in Table 1. Results of a standard
method of verification when working with particu
laths are given in Table
@.Summation of counts
made on three different (M) and (N) and (S) free
mitochondria was the Petroff-Hausser bacteria counter. The
chamber consisted essentially of 400 prisms,' each of 0.05 X
0.05 X 0.02 mm., thus having a volume of 5 X 10@ cm'. A
Zeiss-Winkle binocular microscope with phase contrast (Ph2)
attachment
was used. Oil immersion bOX and 1.3 numerical
aperture was employed in conjunction with 12.5 Hygens eye
pieces. KtShler illumination, with a Wratten filter No. 22, was
utilized. Four glass fibers, cemented at right angle with glass
ware cement, were fixed in one eyepiece. This reticule was do
signed tobeoptically
superimposed on asquare of the chamber.
Mitochondria suspended in 0.25 u or in 0.88 u sucrose could
not be counted in the chamberbecause of Brownianmovement
of the particles. All attempts to stop this movement or to sedi
ment mitochondria
chemically proved unsuccessful.
It was
found possible, however, to diminish this movement greatly by
diluting the mitochondria
with 1.8 u si@rose. When kept in
this mediumat 0-5°C., they did not sediment, and they main
tamed their originalform.Theirnumber seemedto remain con
stant for a period of as long as S days. Fading was the only
visible change in the particles after such a storage period.
The technic adopted for counting was as follows: A suspen
sion was prepared by diluting 0.5 ml. of a 20 per cent mite
chondrial suspension to 50 ml. with 1.8 v sucrose, giving a final
concentration of 0.2per cent, as compared to the original tissue
homogenate.
After thorough
mixing, an aliquot
and heldbetween thehands untilitattained
Movement
of the particles
was pipetted
room temperature.
due to the expansion
of the liquid in
the chamber was thus avoided. The chamber was finally filled
by capillary
attraction.
Five minutes
after the introduction
of
the specimen in the chamber, movement had usually ceased;
the particles, dispersed at random, were ready to be counted.
Since the particles did not sediment in the chamber, they
were counted
veniently
done
from the base to the top of a prism. This was con
by focusing
the
objective
followed by a very slow ascending
on thebase
movement
ofa
prism,
of the objective
to the upper surface of the same prism. The reticule in the eye
piece served as the true limit of the surface of counting. With
such a slowmovementof the objective, mitochondriaappeared
tions,
isolated
from
a normal
rat,
and
of those
from two regenerating livers agrees with counts
made on each of the original liver homogenates.
Meticulous care is required to count mitochondria
directly in the homogenate, and the method in this
case is not so convenient for routine assay.
Preliminary results of the study of the variation
of mitochondria population in different pathologi
cal and physiological conditions are presented.
Table S shows results obtained with total liver
with hepatoma and with pure hepatoma induced
by feeding a semi-synthetic diet (9, diet 3) con
taming 0.06 per cent of 4-dimethylaminoazoben
zene. The number of mitochondria
per gram of
fresh tissue seems higher in tumor than in normal
rat liver. The difference is statistically
very sig
nificant. This seems confusing, since it is known
that the yield of mitochondria
expressed as dry
weight, protein or/and PNA content per gram of
fresh tissue is lower in tumor than in normal tissue
(10, 12—15,
17). These data and ourswouldimply
that mitochondria may be much smaller in tumor
tissue. In fact, it is apparent, when tumor (M)
fraction is e@mined under the phase microscope,
that a greater number of smaller mitochondria is
and disappeared progressively. They were counted when per
present.
fectly focused. Since the volume of a prism and the concentra
tion of the mitochondrial
suspension were known, the total
are broken down during homogenization,
because
of an increased fragility. This problem is now un
der investigation.
number of particles in the (M) fraction per gram of fresh liver
‘By“prism―
is meant the volume between the cover glass
ofthechamberand thesurface
ofonesquareengraved
on the
plate.
Another
explanation
would
be that
they
It is of interestto knowwhetherthe numberof
mitochondria
fluctuates
during normal
prolifera
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1952 American Association for Cancer Research.
ALLARD
@
et al.—Mitochondrial
Population
in
Mammalian
CelLi
409
The number of cells per gram of fresh pure liver
tumor calculated from DNA concentrations taken
from Price of at. (11, 1%) is approximately
tion, as it appears to do during cancerous growth.
Preliminary study of this phenomenon has been
undertaken during rat liver regeneration. Partial
hepatectomy is performed as described by Higgins
and Anderson (7). Results are given in Table 4 for
a period of %days of regeneration. The total num
her of mitochondria
per gram of fresh tissue lies
within the limits of
@.8—@8.6
X 10'°. The dif
554 X 10'. The numberof mitochondriaper neo
plasic cell would fall to approximately
700. From
the value of Price and Laird for the number of
cells after
days of liver regeneration, the number
of mitochondria per cell is @089.After 4 days it
TABLE
1
No. o@MITOCHONDRIA
PERGRAMOFFREsHRATLIVERIN THENUCLEAR
(N) AND
MITOCRONDRIAL
@
Sucaos.Rat
(M) FRACTIONS
IaoL•&TZDiN 0.55
fraction
[email protected]
no.SeaWeight
Ix i0'.
(gm.)N
A*M
IX i0'fraction
±0.3030.2±1.80215M1703.68
±0.2420.5±2.14216F1673.72
±0.1229.2±1.35218M16729.5±1.45223M1503.58
±0.1430.0±2.08224M2002.80
±0.6230.9±1.70800M1802.00
±0.2128.2±1.70
@
ISOLATSD
1-88M
3-88M
@
@
*
A =
S
M
M
195
170
Ix
0.88
Sucaosa
4.02
3.60
±0.85
±0.21
. Where
to.osi
istheStudent
“1@'
andS= \I.!@(X1—
29.6
29.2
±1.58
±2.30
.i@represents
theindividual
counts.
andI the @sn
value of 56 e@untaon the same sample.
ference with that of normal rat liver (31.8—34.3 X
lOlO) is statistically
very significant.
Preliminary results on the influence of diet on
the variation of the number of mitochondria in rat
liver tissue are given in Table 5. After 6 days of
fasting (rat No.
@1),the number of mitochondria
has increased to a value much higher than for nor
mal rat liver. On a semi-synthetic diet (9, diet 3),
the number decreases ; the same phenomenon oc
curs with a diet of cooked polished rice.
Since all results given above are expressed per
gram of fresh tissue, this may lead to an erroneous
interpretation.
The cell as a base of calculation
seems much more appropriate. In Table 6 the total
number of mitochondria per liver cell, in different
conditions,
celLs
from
mean
tory.
is given.
The values
for the number
1 gram of fresh normal
2
No. X 10@-'°
or MITOCHONDRIA
PERGRAMOFFRESH
RAT LIvER IN THE HOMOGENATE AND IN THE
FRACTIONS ISOLATED IN 0.25 M SUCROSE
RssINRP.&TiNGDaSCRIPTIONNO&MS&L1*
daysRat
days15
no.22625-R26-RSexmalemalemaleWeight
(gsa.)180220210Homogenate
1.52NFraetion(I)
A)35.50
±2.0630.70
± 1.0926.00
±
± 0.284.18
10
±1.83
±1.22
(A)
S Fraction30.20
none25.
none21.50
noneTotal34.9329.0225.68Becovery(percent)98.894.598.8
±
appears that the mitochondrial
the normal value.
is half
0.26M
(A)4.70
±0.303.92
Fraction
(I)
±1.00
of
per gram of fresh tissue were taken in part
the literature. In normal adult rat liver a
value of 133 X 10@is obtained in this labora
This confirms the results of Price and Laird
(11). Since
TABLE
rat liver con
taiiis about 33 X 1O―@
mitochondria,
a rat liver
cell would thus contain approximately
@,5®
mito
chondria.
In total rat liver with tumor induced by DAB
feeding the number of cells seems to decrease.
From seven assays, a mean value of 9@ X 10@is
obtained m this laboratory. Thus, the mean num
ber of mitochondria
per cell would be approxi
mately 4,500.
population
DISCUSSION
Method.—The technic described above was sub
jected to an elaborate verification. The results
were compiled independently by three individuals,
and their counts agree with a probability of 0.95.
Table 7 includes values obtained with replicate
counts done by three different operators (a, b, c) on
the same specimen. Each operator did three counts
on the same prism. This was repeated with three
different
prisms
(Nos.
1,
tween the values obtained
@,3). The difference
by each operator
be
is not
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1952 American Association for Cancer Research.
TABLE 8
No. OFMITOCHONDRIA
PERGRAMOFFRESHTUMORTISSUEIN THENUCLEAR
(N) ANDMIT0CHONDRIAL (M) FRACTIONS ISOLATED IN 0.25 M SUCROSE
Rat
N fraction
1X10'°
A
Weight
no.
Sex
219
220
222
(gm.)
Description
150
120
110
120
160
Hepatoma@parenchyma*
Pure hepatoma
Pure hepatoma
F
F
M
PoolM
F
225
F
228
P
* Total
1M
°°M
liver with
M fraction
1X1o-1o
A
36.8
±1.50
35.6
±2.17
88.1
±1.27
158
Cholangioma-hepatoma
5.10
±0.86
88.2
±4.60
160
150
Hepatoma@parenchyma*
5.08
±0.84
85.5
±1.90
tumor.
TABLE
4
No. OFMITOCHONDRIA
PER GRAMOFFRESHREGENERATING
RATLIVERIN THE NU
CLEAR (N) AND MITOCHONDRIAL (M) FRACTIONS ISOLATED IN 0.25 M SUCROSE
Rat
Weight
no.
Sex
221-A
Days
130
145
120
140
150
145
2
2-R
pcolF
M
Pool@'
F
M
@°°‘M
S@It
pooiM
185
F
125
1-It
M fraction
N fraction
(gm.)
Ix
i0'.
A
1X1o-1o
A
22.0
±1.75
2
4.69
±0.26
20.9
±1.80
2
4.15
±0.25
21.2
±1.14
2
5.30
±0.26
19.8
±1.20
TABLE S
No. OPMITOCHONDRIA
PERGRAMOFFRESHRATLwERIN THENUCLEAR
(N) ANDMITOCHONDRIAL
(M) FRAC
TIONS ISOLATED
IN 0.25 M SUCROSE.
RAT
Sax
Before
After
(gm.)
150
150
@A
Fasting
150
140
Synthetic
154
150
150
150
110
120
Normal
Total liver
withtumor
Pure tumor
Regeneration
2days
@
@
Cooked rice
90 days
N
M TRACTICS
TRACTION
@x1o-1o
A
6
88.OX1O'°
138X10'
2,480
41.9X10'°
89.4X10'°
92X10'
554X10'
4,550
711
£5.7X10'° 128X10't
2,089
26.7X10'°
1,940
137X10'
* Calculated fmm DNA concentrations giwn by Prira .1 at. (11, 15).
±1.17
2.82
±0.16
19.7
±1.20
8.45
±0.28
20.2
±1.10
3.69
±0.31
23.0
±1.10
90
TABLE
No. of mitochondria/cell
A
34.4
120
6
No. of cells/gm
of fresh liver
1X10'°
120
Cooked rice
TOTAL No. OF MITOCHONDRIA PER RAT LIVER Cmi.
IN DirnRENT
PIrYSI0wGICAL AND
PATHOLOGICAL CONDITIONS
No. of mitochon.
dna/gin of fresh
liver
OF THE DIET
DIET
ThaT
110
115
TABLE
INFLUENCE
DAYS ON
WIIGBT
7
CoMPARISoNOFREPLICATECOUNTSON THREEDIP
FERENT PRISMS BY THREE OPERATORS
Operators
Prisms
I
2
S
a
b
a
28 28 27
35 34 80
26 25 28
27 29 27
82 41 29
24 29 25
80 28 26
82 19 26
39 41 87
Operators
t
Prisms
a versus b: 0.3645
a
“ c:O.9000
b
“ c:0.5748
valuesbetweeno
prisms
for operator
bility of 0.95
ratorsforprisms
a, rand
5.150.
1 versus 2: 1.7064
1
“ 3:1.3884
2
“ 8:0.1471
1, 2 andS andtvaluesbetween
c,
he
S
@luebr si@ificance with a
t TakenfromPriceandLaird(11).
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1952 American Association for Cancer Research.
@ba
et al.—Mitochondrial Population
significant, as indicated in detail in Table 7. It
shows also that there is no significant difference
between prisms (Nos. 1, @,3).
Series of counts on replicate dilutions of the
same mitochondrial
fraction are statistically
the
@ same. Whether
or 50 prisms are counted, the
same accuracy is obtained. The mean values are,
@ respectively,
@8.9and 30.0 for
counts and 50
counts. The number of prisms counted has been set
at 26 for convenience in the statistical analysis.
Retults.—As reported in Table 1, the number of
mitochondria
found per gram of fresh tissue,
whether the particulates are isolated in 0.@5 M or
0.88 M sucrose, is the same. Furthermore,
we feel
that agreement between the summation
of the
iiumber of particles in the (N) and (M) and (5)
fractions and that of the homogenate confirms the
accuracy of the results.
The mean percentage of mitochondria present
in the nuclear fraction isolated in sucrose has been
approximated
at 10—18per cent by enzymatic
studies on particulates (17). A mean of 11 per cent
is obtained
by direct counting
on eleven
different
(N ) fractions isolated from liver. Accurate correc
tions of results in biochemical investigations on
particulates seem possible if counts are made after
each separation.
A correlationof the variationof the mitochon
dna population with the number of cells has been
presented in Table 6. The number of mitochondria
per cell seems much more interesting than the
number per gram of fresh tissue, though both
methods of expression are useful. Comparison of
the number of mitochondria with the yield in
grams may indicate whether the mitochondria are
normal or abnormal, and a comparison of the
number of mitochondria with the number of cells
per gram may show whether the cell. are normal or
abnormal. In fact, by comparing the number of
mitochondria
per gram
of fresh
tissue
with
pathological
mitochondria
in tumor
tissue.
The fact that the number of mitochondria per
neoplasic liver cell seems less than in the normal or
regenerating
liver
cell is of great
importance.
It
remains to be shown if the phenomenon is char
acteristic of neoplasia. Further investigation
on
@
these
topics
is necessary
may be accurately
Since the number
before
these
variations
of mitochondria
SUMMARY
A method has been devisedfor countingmito
chondria obtained by centrifugation
of rat liver
homogenate in sucrose. Values for normal rat liver
are given. Preliminary results are furnished for
tumor and regenerating liver and for fasting rats.
The influence of diet is also considered.
The number of mitochondria per normal rat
liver cell is approximately
@,500. The mito
chondrial population of the rat liver cell seems to
vary in different
conditions.
varies in dif
ferent physiological and pathological conditions, it
would appear more appropriate to express the con
physiological
and pathological
Boivin and the Vendrelys' suggestion to express
biochemical phenomena per cell is extended. When
dealing with mitochondrial fractions it is proposed
to express results per mitochondrion.
ACKNOWLEDGMENTS
We wish to thank Dr. A. Frappier, director of 1'Institut de
Microbiologic de l'Université de Montréal,for the use of the
electron microscope. Our thanks are extended to Mr. A. Nantel
of the same Institute for his guidance in the work with the
apparatus. We thank Dr. Genest, director of the Veterinary
Research Laboratories,
St-Hyacinthe,
Quebec, for the loan of
the phase microscope.
We express our gratitude to Mr. R. Daoust of our labora
tory, for cell counts and also to Mr. Y. Grandchamp, for the
statistical
analysis.
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FIG. 1.—Spherical
@
0.25
sucrose.
mitochonthma
Electron
from liver tissue
micrograph
isolated
gold shadowed
in
without
fixation. Meg. X 19,000.
Fia.
2.—Spherical
mitochondria
disintegrating.
Electron
micrograph gold shadowed without fixation. kIng. X26,000.
Pio. 3.—A spherical mitochondrion
graph gold shadowed without fixation.
ghost. Electron
Mag. X36,000.
micro
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Mitochondrial Population in Mammalian Cells: I. Description of a
Counting Technic and Preliminary Results on Rat Liver in
Different Physiological and Pathological Conditions
Claude Allard, R. Mathieu, G. de Lamirande, et al.
Cancer Res 1952;12:407-412.
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