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Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow
088 in Polyethylene Glycol-Glycerol

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DOI: 10.3109/10520299109110562 · Source: PubMed

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Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluorol Yellow
088 in Polyethylene Glycol-Glycerol
Mark C. Brundrett a; Bryce Kendrick b; Carol A. Peterson b
a
Soil Science and Plant Nutrition, University of Western Australia, Nedlands, WA, Australia b Department of
Biology, University of Waterloo, Waterloo, Ontario, Canada

Online Publication Date: 01 May 1991

To cite this Article Brundrett, Mark C., Kendrick, Bryce and Peterson, Carol A.(1991)'Efficient Lipid Staining in Plant Material with
Sudan Red 7B or Fluorol Yellow 088 in Polyethylene Glycol-Glycerol',Biotechnic and Histochemistry,66:3,111 — 116
To link to this Article: DOI: 10.3109/10520299109110562
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 Efficient Lipid Staining in Plant Material with
Sudan Red 7B or Fluoral Yellow 088 in
Polyethylene Glycol-G Iycerol
Mark C. Brundrett', Bryce Kendrick' and Carol A. Peterson2
'Soil Science and Plant Nutrition, University of Western Australia, Nedlands W. A. 6009, Australia, and
'Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3C 1 Canada
ABSTRACT. Polyethylene glycol (400)with 90%
glycerol (aqueous) is introduced as an efficient
solvent system for lipid stains. Various lipid-sol-
uble dyes were dissolved in this solvent system
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and tested for their intensity, contrast, and spec-
ificity of staining of suberin lamellae in plant
tissue. The stability (i.e., lack of precipitation) of
the various staining solutions in the presence of
fresh tissue was also tested. When dissolved in
polyethylene glycol-glycerol, Sudan red 7B (fat
red) was the best nonfluorescent stain and fluorol
yellow 088 (solvent green 4) was an excellent
fluorochrome. These two dyes formed stable
staining solutions which efficiently stained lipids
in fresh sections without forming precipitates.
Estimations of the solubilities of these dyes in the
solvent compared with their solubilities in lipids
of various chemical types indicated that they
should both be effective stains for lipids in gen-
eral.
onpolar, organic dyes commonly used
N as lipid stains include Sudan 111, SU-
dan IV, Sudan black B, and oil red 0
(Pearse 1968, Lillie 1977, Wigglesworth
1988).Many other dyes (referred to as sol-
vent dyes in industry) with properties
which suggest that they would be valuable
lipid stains (high lipid solubility, resist-
ance to fading, 'intense colors) are com-
mercially available (see Colour Index
1971). Lillie (1944) and King (1947) com-
pared the lipid-staining abilities of various
solvent dyes, but there have been few sub-
sequent comparisons of this type. The sol-
vent dyes oil red 0, Sudan black B, and
105L-OL9j/91/6h01-111/$3 OO/O
BlOTtCHNlC & HI5TOCtlEMISTKY
Copyright 0 1991 b y Willi,irns ti Wilkins
111
Sudan red 7B have been recommended for
use in routine microscopy (Pearse 1968,
Lillie 1977, Wigglesworth 1988). Green-
span et al. (1985) found that Nile red was
superior to other fluorescent dyes that
have been used to stain lipids. King (1947)
reported that a number of commercially
available oil soluble dyes, including oil
blue N , were superior to Sudan I1 and IV
as stains for suberin in plant tissues,
while Huisinga and Knijff (1974) recom-
mend Sudan red 7B (fat red) for this pur-
pose.
Solvent systems commonly used with
lipid stains include ethanol-water, ethanol-
acetone-water, supersaturated isopropa-
nol-water, propylene glycol and ethylene
glycol (Conn et al. 1960, Jensen 1962,
Pearse 1968). When these procedures are
used, problems may occur with dye precip-
itation, extraction of lipids from the sam-
ple, or weak staining (Gutierrez and Lillie
1965, Pearse 1968, Catalano and Lillie
1975, Stotz et al. 1986).The use of glycol-
ethanol-water (Gutikrrez and Lillie 1965)
or isopropanol with dextran (Catalano and
Lillie 1975) as solvents can partially over-
come these problems. Stain precipitation
can be eliminated by using a nonvolatile
solvent such as polyethylene glycol, but
weak staining of lipids results (O'Brien
and McCully 1981). Thus, lipid-stain sol-
vent systems must have hydrophobic
(nonpolar) properties or solvent dyes will
not remain in solution. However, this com-
patibility must not preclude efficient
 112 Biotechnic & Histochemistry

staining of lipids which occurs by dye par-
titioning into hydrophobic domains of the
cells. These staining solutions may also
require some hydrophilic properties so
that water associated with fresh biological
specimens does not cause excessive stain
precipitation and extraction of lipids from
the tissue is minimized.
In this paper a new staining vehicle for
lipid stains, designed in accordance with
the principles described above, is intro-
duced. This solvent system uses polyeth-
ylene glycol (400 Daltons), a nonvolatile,
water-miscible liquid polymer which effi-
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(w/v) or 0.01% (in the case of fluorescent
stains) final solution was dissolved in pol-
yethylene glycol (average mw 400 Daltons)
by heating a t 90 C for 1 hr. (2) An equal
volume of 90% (v/v) glycerol (containing
10% distilled water) was added to the pol-
yethylene glycol plus stain. Synonyms and
sources of the solvent dyes used are pro-
vided in Table 1.
Staining procedure. Folded Parafilm
was used to immobilize plant material dur-
ing sectioning with a razor blade (Frohlich
1984). The numerous sections generated
in this way were examined under a dis-

ciently dissolves solvent dyes, but which
allows rapid and intense staining of lipids
when mixed with glycerol and water. This
solvent system was used in comparisons
of the staining intensity, specificity, and
solution stability (absenceof precipitation)
of various lipid stains, including some
dyes that have not previously been used
for this purpose. Staining of suberin la-
mellae (localized lipid and phenolic depo-
sitions in plant cell walls, Kolattukudy
1984)was used as a sensitive test of these
properties. The best stains for light and
fluorescence microscopy in this system
are recommended after testing their solu-
bilities in various lipids.
MATERIALS A N D M E T H O D S
Preparation of staining solutions. (1)A
sufficient amount of dye to make a 0.1%
secting microscope, and thin sections
were selected for staining. Sections of
fresh or 50%alcohol-preserved plant ma-
terial werk stained for 1 hr a t room tem-
perature, rinsed briefly in water and
mounted on slides in 75% (v/v) glycerol.
To process many sections simultaneously,
specially designed section holders were
used (Brundrett et al. 1988). In this case,
excess stain was blotted off, then the hold-
ers were rinsed several times in water.
Stain comparisons. The lipid-staining
properties of each of the solvent dyes listed
in Table 1 were compared. Suberin lamel-
lae in cross-sections of potato tuber peri-
derm and onion root were examined after
sectioning and staining as described
above. Staining intensity, color contrast,
and the stability of staining solutions (in-
versely related to their degree of precipi-
tation) were noted for each dye.

Table 1. Staining Intensity, Contrast, and Stability of Solvent Dyes in Polyethylene Glycol-Glycerol
Solvent Name Staining , Color Precipitate
(Other Names, C.I., Source) Intensity Contrast Formation
Solvent yellow 14 (Sudan orange 220, BASF) ++ + ++
Solvent orange 1 (Sudan orange G, 11920, Sigma) + + -
Solvent red 19 (Sudan red 78, fat red, 26050, Sigma) +++ +++ -
Solvent red 23 (Sudan I l l , 26100, BDH) + + ++
Solvent red 24 (Sudan IV, 26105, Sigma) ++ ++ ++
Solvent red 26 (oil red EGN, 26120, Sigma) +++ ++ +++
Solvent red 27 (oil red 0, 261 25, Sigma) +++ +++ ++
Solvent blue 14 (oil blue N, 61555, Sigma) ++ ++ +++
Solvent green 3 (61525, Sigma) + + +
Solvent brown 1 (fat brown RR, 11285, Sigma) ++ + +
Solvent black 3 (Sudan black B, 26150, BDH) +++ ++ ++
* Solvent green 4 (fluorol yellow 088, 45550, BASF) ++++ ++++ -
-
* Nile red (Nile blue A oxazone, Sigma) ++++ +++
Note: subjective results are indicated by: -, none; +, weak; ++, moderate; +++, strong; ++++, intense
' Fluorochrome. observed with ultraviolet illumination.
 Lipid Staining in Plant Material
Photography. Sections were observed
and photographed with a Zeiss Photomi-
croscope I11 using bright field, Nomarski
interference-contrast, or epifluorescence
optics. Ultraviolet illumination (excitation
filter G 365, 365 n m peak emission; chro-
matic beam splitter Ft 395, X = 395 nm;
and barrier filter LP 420, X 2 420 nm) was
used for fluorescence microscopy. Images
were recorded on 100 ASA color slide film
and prints were made from black and
white internegatives.
Dye solubilities in lipids. Dye solubili-
ties were tested in samples of the lipids
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(Sigma) listed in Table 2, as well as glyc-
erol, polyethylene glycol and the polyeth-
ylene glycol-90% glycerol stain solvent.
Mixtures of Sudan red 7B or fluorol yellow
088 were made in each solvent at the fol-
lowing percentages 0.03, 0.1, 0.3, 1, 3,
and 10% (w/w). Lipids and stains were
mixed in ELISA plate wells and, if neces-
sary, were heated to the melting point of
the lipid. The highest concentration of
stain which dissolved completely was
noted in each case.
RESULTS AND DISCUSSION
Polyethylene glycol effectively dissolved
solvent dyes and, when mixed with 90%
glycerol (aqueous), allowed intense stain-
ing of lipids in plant tissues. Solvent dyes
which are routinely used as lipid stains,
and several other colored substances with
high lipid solubility (Colour Index 1971),
were used to stain suberin lamellae in po-
tato tuber periderm and onion roots. The
staining intensity, color contrast, a n d the
Table 2. The Solubility of Sudan Red 7B and Fluorol Yellow 088 in Lipids and Staining Solutions
Lipids Classification
Oleic acid 18:l C fatty acid
Lauric acid 1 2 : O C fatty acid
Stearic acid 18:O C fatty acid
D-Lirnonene terpenoid
Cholesterol sterol
Glycerol
Polyethylene glycol (400)
Polyethylene Rlvcol-glycerol
113
degree of precipitation of each dye mixture
are reported in Table 1. Several stains
used in light microscopy, including oil red
EGN, oil red 0 and Sudan red 7B, produced
high contrast staining. However, the two
oil red dyes rapidly precipitated during the
staining procedure, depositing crystals on
the specimen. Precipitation often occurred
when water introduced from samples of
fresh plant material came in contact with
dye solutions (Table 1).Only Sudan red 7B
and the fluorochromes formed solutions
that were stable after absorbing some
water and also produced high contrast
staining (Table 1). Solutions of these
stains were stable at room temperature
unless exposed to high humidity. Indeed,
Sudan red 7B could be used to stain sec-
tions by mounting them directly in the
staining solution. (Precipitation was some-
times caused by water absorption but
could be reversed by heating the slides.)
When polyethylene glycol alone is used
as a vehicle for a stain, it tends to retain
the stain, preventing it from partitioning
selectively into the lipid components. Add-
ing glycerol and water to polyethylene gly-
col increases the polarity of the solvent to
the point at which the stain will partition
into the tissue lipid. Dextran, another high
molecular weight, water-soluble polymer,
also improves lipid staining by solvent
dyes (Catalan0 and Lillie 1975).The solu-
bility trials presented in Table 2 demon-
strate that Sudan red 7B and fluorol yellow
088 dissolve in a range of lipids as well as
in polyethylene glycol, while their solubil-
ity in the staining solution (polyethylene
Highest Soluble
Solvents Concentration (w/w)
Sudan Red 7B Fluorol
% %
0.3 3
1 3
1 3
0.3 10
1 10
< 0.03 0.1
1 1
0.1 0.1
Downloaded By: [The University of Western Australia] At: 07:13 24 April 2009

Fig. 1 . Potato tuber cross-section with Sudan red 7B (fat red) staining of suberin lamellae (arrowheads) within periderm
cell walls. Nomarski interference contrast. 280 X.
Fig. 2. Onion root cross-section with Sudan red 7B staining of suberin lamellae (arrowheads) within exodermal walls.
Nomarski interference contrast. 31 5 X.
Fig. 3. Material as Fig. 1, with intense fluorol fluorescence of suberin lamellae. Ultraviolet excitation. 330 X.
Fig. 4. Suberin lamellae in the endodermis (En) and exodermis (Ex) of an onion root cross-section stained with fluorol.
Autofluorescence of epidermal (Ep) and xylem (X) cell walls can be distinguished by its blue color in color images.
Ultraviolet excitation. 190 X.
Fig. 5. White ash (Fraxinus americana) tree root cross-section with fluorol fluorescence of suberin lamellae in the
endodermis (En) and exodermis (Ex). Note passage cell gaps (P) and storage lipids within mycorrhizal fungus hyphae
(arrowheads) in the cortex. Ultraviolet excitation. 190 X.
Fig. 6. Abies balsamea (balsam fir) long root with ultraviolet-induced autofluorescence of secondary xylem (X) and
fluorol staining of terpenoid lipids (arrowheads) in cells adjacent to the central resin duct. 500 x.
 Lipid Staining in Plant Material
glycol-90% glycerol) is lower, which facil-
itates staining. They are practically insol-
uble in the mountant (glycerol),which has
the beneficial effect of effectively prevent-
ing destaining during subsequent storage.
Dye solutions must not generate precip-
itates if they are to be of practical value.
Sudan red 7B forms precipitates less read-
ily than other oil stains (Pearse 1968).and
we have found that fluorol yellow 088 is
even more resistant to precipitation. Dyes
such as oil red 0, which are good lipid
stains in other solvents (Pearse 1968, Lil-
lie 1977), form unstable solutions in pol-
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yethylene glycol-glycerol and may be too
hydrophobic to remain in solution in the
presence of even small amounts of water.
The observed solubility of lipid stains in
semipolar solvents (such as described in
the present manuscript) may be because
most dyes on the market are mixtures of
dyes or contain impurities; highly purified
dye samples tend to be much less soluble
(Catalan0 and Lillie 1975, Stotz et al.
1986).
Sudan red 7B produced intense red
staining of lamellar suberin in potato
tuber and onion root tissue (Figs. 1 and 2)
although some of this contrast was not
recorded by black and white film. At
higher magnifications it was possible to
see the location of a suberin lamella within
a wall, especially if sections were observed
with Nomarski interference contrast op-
tics (see Fig. 1). This observation agrees
with ultrastructural studies of suberized
wall layers in potato periderm (Schmidt
and Schonherr 1982) and onion roots (Pe-
terson et al. 1978).Sudan red 7B staining
of suberin lamellae within the walls of
onion exodermal’ cells (Fig. 2) also corre-
sponds with results obtained with other
staining procedures (Brundrett et al.
1988).
The fluorochromes, Nile red and fluorol
yellow 088, both formed stable solutions
in polyethylene glycol-glycerol and in-
duced intense fluorescence of lipids in
plant tissues (Table 1). However, some
nonspecific staining of plant cell walls
115
containing phenolic substances occurred
with Nile red. Fluorol yellow 088 specifi-
cally stained lipids bright yellow and pro-
duced very high contrast images of sub-
erin lamellae and other hydrophobic
structures (Figs. 3-6). Autofluorescence of
structures such as xylem and nonlamellar
suberin, e.g., in epidermal walls (Fig. 4),
also occurred but could be easily distin-
guished in color images. Fluorol yellow
088 staining is especially suited for low
magnification surveys of features that
would be hard to visualize using conven-
tional lipid stains. At high magnifications,
some fading of fluorol yellow 088 occurs
and the very high intensity of its fluores-
cence can partially illuminate adjacent
unstained structures. Fluorol yellow 088
staining was found to be a n efficient
method for identifying suberin lamellae in
exodermal, endodermal and phellem cells
during a survey of tree root anatomical
features (Brundrett et al. 1990).
The procedures presented here are es-
pecially valuable for observing suberin la-
mellae in the exodermal and endodermal
cells of roots (Figs. 2 , 4 and 5).but they do
not allow observation of Casparian bands,
which are nonlamellar suberin. However,
Casparian bands are revealed by another
procedure in which they are stained by the
fluorochrome, berberine sulfate, while the
fluorescence of suberin lamellae is par-
tially quenched by aniline blue counter-
staining (Brundrett et al. 1988). With Su-
dan red 7B and fluorol yellow 088 we have
also observed efficient staining of lipids
(terpenes) within secretory cells (Fig. 6)
and storage lipids within fungal hyphae
(Fig. 5). Small lipid droplets within plant
cells are also highlighted by these stains
and even the plasma membrane was rec-
ognizable in some preparations. The pol-
yethylene glycol-glycerol staining proce-
dure with Sudan red 7B and fluorol yellow
088 could potentially be used for many
classes of lipid. With appropriate modifi-
cations to the procedure, this technique
could be applied to other kinds of samples
and other methods of tissue preparation.
 116
ACKNOWLEDGMENTS
This research was supported by operating
grants from the Natural Sciences and En-
gineering Research Council of Canada to
B. Kendrick and C. A. Peterson. Ulricke
Schafer provided excellent technical as-
sistance. We thank Daryl Enstone for
bringing Sudan red 7B to our attention,
BASF Canada, Inc., for providing samples
of solvent dyes and technical information,
and Susan Kamula for supplying Fig. 2.
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