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The Acridine Orange Viability Test Applied to Bone
Marrow Cells I. Correlation with Trypan Blue
and Eosin Dye Exclusion and Tissue
Culture Transformation
By WILLIAM E. HATHAWAY, Loins A. NEWBY AND JOHN H. GITHENS
M ANY METHODS have been used to assay the viability of hematopoietic
cells during the course of procurement, preservation, and fransplanta-
tion of animal and human tissue. Tritiated thymidine is frequently used as a
cell marker in the study of hematopoietic proliferation.’ In vitro DNA
synthesis has been reported as an index of viability.2 Growth and transforma-
tion of living cells in tissue culture also indicate viability.3 Determination of
cell motility is used to detect living cells.4 The resistance of viable cells to
staining with trypan blue5 and eosin#{176} is probably the most frequently used
technic. The problems and limitations encountered in determining cell viabil-
ity by these and other in vitro methods have recently been reviewed.7 In the
field of bone marrow transplantation the most precise measurement of viabil-
ity is the ability of the tissue to transplant, proliferate, and protect a lethally
irradiated host. Because this in vivo method is not feasible prior to transplan-
tation with preserved human tissue, there is a need for a rapid and simple in
vitro estimation of cell viability. Following the suggestion of Pegg8 that the
acridine orange (A-O) staining reaction9’1#{176} may be correlated with the viability
of l)One marrow cells after preservation by freezing, the following studies were
done in order to establish an “A-O viability test of marrow cells.”
METHODS
Unless otherwise iIl(licated, the following conditions were carefully observed through-
out the experinlent.
Preparation of Marrow
The marrow was obtained froni the feniurs of freshly killed Balb/c mice �iiul suspended
in Puck’s tissue culture iriedia’ ‘ ( N-16 40 i)�r cent, saline F 30 per cent, fetal calf serum
30 per cent ) as single cells at an approximate concentration of 40-50 x 10� per ml.
fllis suspension was observed iillllle(liately after preparation and is called “fresh mar-
row. All other cell suspensions were at a similar concentration.
Frozen marrow samples were prepared by suspending marrow cells in Puck’s tissue
culture Illedia after passage through a 25 gauge needle to reduce particles to single cells
at a concentration of approxin�ately 50 x 1O� per ml. Glycerol or dililethylsulfoxide
( I)MSO ) was added prior to freezing in sealed 2 ml. glass ampules. The samples were
frozen in a C02-95 per cent ethanol I)ath l)Y slowly reducing the temperature 1 C. per
Illiflhlte to -40 C. and then rapidly to -79 C. The vials were tilen immersed in liquid
This work was �upported by American Cam�er Society Grants T138B and T255.From the Department of Pediatrics, University of Kentucky Medical Center, Lexingkrn,
Ky.
Submitted Sept. 4, 1963; accepted for publication Oct. 29, 1963.
517
BlooD, VOl.. 23, No. 4 (APRIL), 1964
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518 HATHAWAY, NEWBY AND G1THENS
nitrogen ( -196 C. ) for storage. Thawing was performed rapidly in a 37 C. water bath
and the specimen was examined immediately as described under “fresh marrow.”
Methods for Producing Injury to Ce&i
Aliquots of “fresh marrow” were treated in the following ways in order to note the
effects of various injurious agents on the staining characteristics with acridine orange.
a. pH-the pH of the cell suspensions were changed to 8.9 by addition of 0.1N sodium
hydroxide and to 6.0 by addition of 0.1N hydrochloric acid.’2 After incubation at 37 C. for
40 minutes, the cells were resuspended in fresh Puck’s media and observed.
b. Heat-Cell suspensions were heated to 60 C. for 30 minutes and observed.
c. Rapid freezing-Cell suspensions were frozen quickly by placing them in a freezer
at -20 C. without the use of a protective agent such as DMSO or glycerol. The cells
were then rapidly thawed and observed.
d. Aging of cells without tissue culture media or serum-Bone marrow cells were al-
lowed to stand in a balanced salt solution ( saline F ) for 6 hours at room temperature, andperiodic observations were made. The depletion of serum or tissue culture media from
the suspending fluid for long periods decreases the protective ability of bone marrow cells
being prepared for transplantation.’3
e. Poison.-0.25 ml. of a 700 mg. per cent solution of sodium cyanide was added to
1.5 ml. of marrow cell suspension for 1 hour at 37 C.14
Viability Test
a. Acridine orange: One drop of a murme marrow suspension was placed on a clean
microscope slide and mixed with a second drop of acridine orange suspension. A number
0 coverslip was then applied with firm pressure to produce a layer of single cell thickness.
The edges were sealed with petrolatum or Kronig’s cement. The acridine orange dye
( National Aniline Lot l4llp) was prepared fresh every 2 weeks as a 9 mg./100 ml.
suspension in Puck’s media buffered with tris(hydroxymethyl)-amino methane to pH
7.4. The slide-cover slip preparation was observed with fluorescent microscopy at 25-27 C.
in a partly darkened room and the necessary differential count or photograph made within
1 to 2 minutes of fluorescence per field.
The microscope was a Leitz Ortholux with the Osrarn HBO 200 mercury vapor lamp
as the light source. Two ( UGI ) exciter filters, one 4 mm., the other 2 mm., were used
with a Blau-absorption filter ( Leitz). Differential cell counts of the supravital preparations
were done with 430X and 970X magnification. Color transparencies were produced with
35 mm. Ektachrome, type B, color film with an exposure index of tungsten 100 and ex-
posure times of 30 to 60 seconds.
b. Eosin and trypan blue: Differential counts were done in the same manner ( one
drop of cell suspension plus one drop of dye ) under light microscopy with 1 per cent
and 0.1 per cent eosin Y and 0.05 per cent and 0.02 per cent trypan blue (in Puck’s
media at pH 7.4 ) . The percentage of unstained cells ( viable cells ) was determined. Thepreparations with eosin Y were examined within 5 minutes in order to avoid possible
photodynamic cytoxicity.’5C. Tissue culture transformation (TCT): A semiquantitative estimation of growth in
tissue culture was devised following the suggestion of Porterfield and Ashwood-Smith.’6
Aliquots of each specimen were plated on cover slips in Petri dishes at cell concentra-
tion of 12 x 10� per ml. When frozen specimens were used, the DMSO was reduoed
to 2 #{189}per cent. Puck’s culture media and a 5 per cent C02-95 per cent air incubator
( 37 C. ) were used. After 4 days, the cover slips were removed, air-dried, and stained with
Wright’s stain. The average number of “fibroblastic” or large “mononuclear” cells per
h.p.f. ( average of 25 random fields ) was recorded. The control was fresh murine marrow
in Puck’s media or 2’,� per cent DMSO in Puck’s media at the same cell concentration
(see figure 1).
For personal use only.on November 17, 2018. by guest www.bloodjournal.orgFrom
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ACRIDINE ORANGE TEST AND BONE MARROW CELLS 519
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Fig. 1.-Above, photograph of Wright’s stained cover slip demonstrating the
tissue culture transformation into “fibroblasts” and “mononuclear cells” of freshInurine bone marrow cultured for 4 days. Below, photograph demonstrating poor
tissue culture transformation of bone marrow cells treated with sodium cyanide
prior to 4-day culture.
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520 HATHAWAY, NEWBY AND GITHENS
RESULTS
With the factors of dye concentration, cell concentration, pH of the sus-
pending media, length of exposure to ultraviolet light, and thickness of the
preparation carfully controlled, a uniformly reproducible appearance of fresh
marine bone marrow cells was obtained. The fresh, “viable,” supravitally
stained nucleated cell displayed a bright, apple-green nucleus, and pale,
dull-green cytoplasm with varying amount of bright orange cytoplasmic
granulations present. With experience, a conventional differential count based
on the usual morphologic characteristics could be done even at 430X magnifi-
cation. In general, the supravital staining properties of mouse bone marrow17
cells are the same as for human marrow,’8 but decidedly different from fixed
preparations’9’2#{176} of either mouse or human marrow.
By comparing the acridine orange staining characteristics with the degree
of viability demonstrated by the various marrow preparations in tissue culture,
it was possible to recognize several staining patterns which indicated cell
death or injury. It became evident that the typical “injured” or “dead” cell
nucleus and cytoplasm stained diffusely bright red, and could be readily dis-
tinguished following injury by pH change, NaCN poisoning, and aging
without culture media. In addition, certain cells displayed an increase in red
granulation of the cytoplasm and an orange hue to the nucleus. These cells
were considered to be injured or damaged to a lesser degree than the “dead”
cells.
A second type of “dead” cell staining reaction was observed in the cells
killed by rapid freezing or heating. The nuclei of cells disrupted by quick
freezing stained a deep, dull-green. They could be distinguished from fresh
cells by the absence of intact cytoplasm and total loss of red granulations.
In a similar manner, the bare nuclei produced by mechanical trauma in
preparation of the suspensions could be distinguished by a dull-green stain and
absence of cytoplasmic granulations. Heated cells showed a diffuse, dull-green
staining reaction. Figure 2 illustrates the difference in the above designated
A-O staining properties of “live,” “injured,” and “dead” cells.
The results of the experiments designed to produce injured or “dead” cells
are summarized and compared with the eosin and trypan blue dye exclusion
test and tissue culture transformation in table 1.
a. pH-Change of pH to 8.9 revealed decreased numbers of viable cells
by the A-O method and TCT which was not indicated by eosin or trypan.
However, pH 6.0 effected the A-O test but not the TCT or dye exclusion tests.
b. Heat-All methods indicated death of most of the cells. The A-O
stained cells were diffusely dull-green.
3. Quick freezing toithout protective agent-The A-O cells were stained
a dull-green, easily differentiated from “live” cells. Most of the cells were
stained with eosin and trypan blue and showed little growth in tissue culture.
d. Aging “without” serum or tis.s’u� culture media-The TCT indicated de-
creased viability of cells suspended for 6 hours in saline F. This observation
correlated well with the A-O test but poorly with the dye exclusion tests.
e. Sodium cyanide-A-O cells were diffusely red and orange. The TCT also
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1
3
5
2
4
6
ACRIDINE ORANGE TEST AND BONE MARROW CELLS I 521
�. � 4t.4. �#{149}
, � #{149}5’� �I
4 1.
Fig. 2.-i. Fresh marrow suspension, X430. 2. Fresh marrow suspension, X900( oil immersion) . Note cluster of “dead” cells in center. 3. Fresh marrow, X900,
predominately granulocvtic elements. 4. Fresh marrow, X900. Note megakaryocyte.
5. Non-viable cells, X900. 6. “Injured” cells, mixed with two “live” cells, X900.
iIldiC�1tC(I decreased viability which was not apparent with eosin and trypan
blue.
f. Varying amounts of l)rotecticc agent ( DMSO )-Reduction of DMSO
concentration below 10 per cent gave evidence of decreased viability by all
the tests employed.
The results of the application of the A-O viability test to fresh and freshly
thawed specimens of murine marrow preserved by freezing in DMSO or
glycerol, and the comparison with eosin and trypan dyes is shown in table 2.
With A-O, the cells staining diffusely red or red-orange and those with bare
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522 HATHAWAY, NEWBY AND GITHEN5
Table 1.-Percentage of Viable Cells as Determined by Supravital_________ Staining and Tissue Culture_Transformation
Treatment of Marrow Staining Methods Tissue Culture
% Viable cells Average--�� numberof
1.0% 0.1% .05% .02% live cells perModality used A-O Rosin Rosin TB. TB. h.p.f.
Control ( fresh marrow) 97 90 93 98 94 16-18
Heat (60 C.-30 mm.) 0 0-i 0 0-1 0-i 0
Rapid freezing 5 3 0 6 8 0-1
pH 6.0 (40 mm.) 62 98 92 97 96 17
pH 8.9 (40 mm.) 10 93 97 99.5 98 8-9
NaCN (1 hr.) 11 54 82 80 92 2
Aging without serum
(6 hr.) 30 92 99 99 98 10
Freezing-2�/� % DMSO
(14 days) 48 5 83 30 70 1-2
Freezing-5% DMSO
( 14 days ) 74 65 90 73 81 6
Free7ing-10% DMSO
(14 days) 80 82 97 80 91 8
Freezing-12#{189} % DMSO
(14 days) 82 65 97 80 90 8-10
Freezing-15% DMSO
(14 days) � - 68 � � � 79 92 8
- #{176}T.B. = Trypan blue.
green nuclei were considered non-viable in the differential count. In general,
the A-O stain gave a consistent result from specimen to specimen and cor-
related well with the dye exclusion tests. One per cent ( 1 per cent ) eosin
occasionally gave markedly different values in comparison to the other stains.
DisCuSsioN
Our findings regarding the A-O staining reaction of healthy and damaged
marrow cells are in accord with the studies of Wolf and Aronson2’ for other
tissues. They have described the fluorescence and metachromasy of fibroblasts
from chick embryo hearts, rabbit lens epithelium and chorioidal melanocytes
cultured in the presence of acridine orange. They have demonstrated that
relatively healthy cells stained orthochromatically ( deoxyribonucleic acid
hues of green; ribonucleic acid hues of red ) ; more diffuse metachromatic
staining ( red and orange ) accompanied cell injury; and complete cytoplasmic
metachromasy accompanied irreversible injury ( red ) . In accord also, is the
finding of diffuse green staining ( loss of metachromasy ) at an early stage of
degeneration. This finding was demonstrated in our study by the staining
reaction of the cells damaged by heat. Wolf and Aronson have emphasized
that the “A-O viability test” must be interpreted along with a study of
morphology and history of the cells being tested.
Emphasis should be placed on factors which need to be carefully controlled
in order to have reproducible results when employing the A-O viability test.
( 1 ) The cell concentration:stain concentration ratio should be kept constant.
The number of “dead” cells at the usual concentration of 40-50 x i0� cells per
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ACRIDINE ORANGE TEST AND BONE MARROW CELLS I 523
Table 2.-Comparison of A-O Staining and Dye Exclusion to Indicate Viability of
Murine Marrow Preserved_by Freezing for Varying Periods of Time
Per Cent Viable Cells
Length of Eosin Trypan blueStorage
Protective Agent (daye) A-O 1% 0.1% 0.05% 0.02%
Fresh ( average 5samples) 93 94 97 98 97
10% DMSO 7 76 42 85 80 73
10% DMSO 14 83 74 84 - 73
10% DMSO 22 81 76 98 88 9310% DMSO 31 90 84 98 85 96
10% DMSO 48 77 79 88 91 8210% DMSO 78 83 77 98 88 91
15% DMSO 180 89 44 90 - 88
15e/( Glycerol 90 51 48 42 55 77
ml. and 9 mg. per cent A-O stain is appreciably fewer than would be noted
when the cell concentration is 10-20 x i0� cells per ml. (2) The pH of the
suspending media is critical and easily controlled by use of the tris buffer.
( :; ) The cell preparations should be promptly examined under the ultraviolet
microscope with as little U-V radiation per field as possible in order to avoid
photodynamic effect on cell viability. Khodas22 has emphasized the importance
of these factors in studies of acridine orange staining of peripheral blood cells
after X-irradiation.
The results of the present study indicate that the A-O test correlated well
with the TCT for all types of marrow cell injury, although the A-O staining
results suggested greater injury with pH change than was apparent from the
tissue culture growth. The A-O test compared favorably with the 0.1 per cent
(‘OSill and 0.02 per cent trypan blue dye exclusion tests in determining the
viability of fresh hemic cells and those damaged by heat, and by rapid or
slow freezing. The 1 per cent eosin and 0.05 per cent trypan blue occasionally
gave results that correlated much less well with the other methods than the
0. 1 per cent eosin and 0.02 per cent trypan blue.
Furthermore, cell damage due to injury of metabolic processes associated
with change of pH, depletion of serum, and treatment with sodium cyanide
was indicated by the acridine orange test and confirmed by tissue culture, but
was not indicated by the eosin or trypan blue dye exclusion tests. Eaton et al.�
have shown that the tumor-producing capacity of Ehrlich ascites cells injured
with NaCN or viruses tends to diminish at a somewhat greater rate than
cellular respiration or the ability to exclude eosin or trypan blue dyes. Cells
which have been frozen and preserved at low temperatures for long periods
of time probably undergo biochemical denaturation.24
Although in the present study eosin and trypan blue results correlated well
with the A-O and TCT in revealing cell death of marrow frozen up to 180
days, it has been shown that the eosin exclusion test does not correlate with
the ability of long-term frozen cells to transplant.25 Tullis has shown that the
eosin exclusion test is not sensitive in determining viability when compared
to amoeboid or phagoc�tic activity of preserved leukocytes�26 Whether the
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524 HATHAWAY, NEWBY AND GITHENS
A-O test is any more sensitive in indicating damaged or dead cells after long-
term freezing, or in indicating marrow which will not transplant, remains to
be shown.
An additional advantage of the acridine orange test is that the viability of
specific types of cells can be noted by performing cell type differential
counts simultaneously with the “live-dead” determination. Cell differentia-
tion is not possible with the eosin and trypan blue dyes.
SUMMARY
Suspensions of murine bone marrow cells were stained with acridine orange
( A-O ) and observed under fluorescent microscopy after treatment with
various injurious agents in order to establish the staining characteristics of
“live” and “dead” cells. The percentage of viable cells demonstrated by the
“A-O viability test” were correlated with eosin and trypan blue dye exclusion
and tissue culture transformation viability tests. In general, the A-O test
demonstrated the viability of cells preserved by freezing as effectively as the
other in vitro tests. In addition, the A-O test may be more sensitive in
determining the viability of cells where metabolic processes have been injured
by poisons or change in pH.
SUMMARIO IN INTERLINGUA
Suspensiones de murin cellulas de medulla ossee esseva tincturate con
orange acridinic ( 0-A ) e observate per microscopia fluorescente post tracta-
mento con vane agentes nocive, con le objectivo de establir le characteristicas
tincturatori de cellulas “vive” e “morte.” Le procentage de cellulas viabile
demonstrate per le “test de viabilitate a 0-A” esseva correlationate con tests
de viabilitate a exclusion de eosina e trypan blau e a transformation de cultura
de tissu. A generalmente parlar, le test a 0-A demonstrava le viabilitate de
cellulas preservate per congelation tanto efficacemente como le altere tests in
vitro. In plus, le test a 0-A es possibilemente plus sensibile in determinar le
viabilitate de cellulas in casos in que le processos metabolic es lesionate per
venenos o per alterationes del pH.
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\Villiam E. Hathaway, M.D., As�tistant Professor of Pediatrics,
University of Kentucky Medical Center, Lexington, Ky.
Louis A. Newby, B.S., Research Assistant, Department of Pedi-
atrics, University of Kentucky Medical Center, Lexington, Ky.
John H. Githens, M.D., formerly Professor arid Chairman,
Department of Pediatrics, University of Kentucky Medical
Center, Lexington, Ky.; presently Professor of Pediafrics,University of Colorado Medical Center, Denver, Cob.
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1964 23: 517-525
WILLIAM E. HATHAWAY, LOUIS A. NEWBY and JOHN H. GITHENS TransformationCorrelation with Trypan Blue and Eosin Dye Exclusion and Tissue Culture The Acridine Orange Viability Test Applied to Bone Marrow Cells I.
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