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Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluorol Yellow088 in Polyethylene Glycol-GlycerolMark 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 ofBiology, 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 withSudan 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 I ycerol

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 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 (1 944) and King (1 947) 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 by Willi,irns ti Wilkins

Sudan red 7B have been recommended for use in routine microscopy (Pearse 1968, Lillie 1977, Wigglesworth 1988). Green- span et al. (1 985) found that Nile red was superior to other fluorescent dyes that have been used to stain lipids. King (1 947) 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 (1 974) 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

1 1 1

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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- 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 (absence of 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 METHODS Preparation of staining solutions. (1) A

sufficient amount of dye to make a 0.1 %

(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 at 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- 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, 11 920, 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 (61 525, 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.

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Lipid Staining in Plant Material 113

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 nm 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 (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, and the

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

Table 2. The Solubility of Sudan Red 7B and Fluorol Yellow 088 in Lipids and Staining Solutions

Lipids Classification Solvents Highest Soluble

Concentration (w/w)

Sudan Red 7B Fluorol

Oleic acid Lauric acid Stearic acid D-Lirnonene terpenoid Cholesterol sterol

18:l C fatty acid 1 2 : O C fatty acid 18:O C fatty acid

% 0.3 1 1 0.3 1

< 0.03

% 3 3 3

10 10 0.1 1 0.1

Glycerol Polyethylene glycol (400) 1 Polyethylene Rlvcol-glycerol 0.1

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Fig. 1 . Potato tuber cross-section with Sudan red 7B (fat red) staining of suberin lamellae (arrowheads) within periderm

Fig. 2. Onion root cross-section with Sudan red 7B staining of suberin lamellae (arrowheads) within exodermal walls.

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.

cell walls. Nomarski interference contrast. 280 X.

Nomarski interference contrast. 31 5 X.

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Lipid Staining in Plant Material 115

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- 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

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 an 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.

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116 Biotechnic & Histochemistry

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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