PROTOPtaSMA

Protoplasma 102, 343--347 (1980)

9 by Springer-Verlag1980

A Method

for Localizing

Embryonal

Laticifers by Combined

Conventional and Fluorescence Microscopy ~

A. BI~UNI ': and BARBARA TOSI
Institute of Botany, University of Ferrara
Received October 23, 1979
Accepted November 4, 1979

Summary
The authors describe a simple method based on malachite green and acid fuchsin for the
detection of laticifers during the embryogenesis of some Euphorbiaceae plants by conventional and fluorescence microscopy. The strong sensitivity and specificity of the method make
it suitable for the ontogenetic studies of laticifers. The results obtained are discussed in the
context of the reactive mechanism of the staining and of the chemical composition of the
embryonal laticifers.
Keywords: Double staining; Euphorbiaceae; Fluorescent staining; Laticifers.

1. I n t r o d u c t i o n
Recently, great attention has been devoted to the study of the latex composition of Euphorbiaceae plants; first, for medical purposes due to the
vescicant, irritant, and co-carcinogenic properties in animal and h u m a n skin
and mucous membranes (WATT and BREYER-BRANDWIJK 1962, MORTON 1971,
KINGHOI~N and EVANS 1975, K~EI.ER et aI. 1978), and second, for purposes
pertaining to m a r k e t technology such as fish poison, insecticide and future
resources of gasoline (MORTON 1971, BIJCHANAN et al. 1976). Despite this,
the studies on the genesis and morphological organization of the latex system
represent a much neglected field of research (MAHImERC and SABHARWAI. 1968,
BRUNI et al. 1978). The genesis of non-articulated laticifers, the formation
of the latex system, and the correlation with the other tissues during growth
and differentiation show v e r y intricate problems, partially due to the difficulty of localizing the laticifers in the context of the embryonal tissues.
This paper was presented in part at the International Meeting on Botanical Microscopy,
organized by The Royal Microscopical Society in York, July 9-13, 1979.
':" Correspondence and Reprints: Institute of Botany, University of Ferrara, Corso Porta
Mare 2, 1-44100 Ferrara, Italy.

0033-183X/80/0102/0343/$ 01.00

344

A. BRt;NI and BAi~BARATosI

We have devised a simple method, based on malachite green-acid fuchsin dyes,

which has proved most efficient, both in conventional and fluorescence
microscopy.
2. Materials and M e t h o d s
The plant material used was mature embryos of Euphorbia rnarginata, E. lucida, E. amygdaIoides, and E. characias. The embryos, obtained by dissecting dry seeds, were fixed in a paraformaldehyde solution (10% w/v) in 0.2 M phosphate buffer at p H 7.4 for 1 hour, or in FAA
(ethanol 70-glacial acetic acid-formalin, 9 : 5 : 5 ) for 24 hours. In both cases, the material
was thoroughly washed after fixation, The embryos were dehydrated in graded ethanol and
embedded in a mixture consisting of butyl-methyl methacrylates 7 : 3 (Merck, Darmstad't).
After polymerization in a 50 ~ oven, the specimens were cut at 2-3 ~tm by an LKB Pyramitome with glass knives. Transversal and longitudinal sections were individually expanded
in a drop of water on a clean slide. The plastic was removed by benzene, and the sections
were immersed for 5 minutes each in absolute, 95%, 70~ and 50% alcohol.
The specimens were stained by the following procedure, modified from PINESE (1896):
a) staining with a solution composed of malachite green l~ aqueous-acid fuehsin 1~ aqueous-distilled water 5 : 1 : 9 for at least 2 hours; b) rinsing of specimens by pouring a few drops
of 95% alcohol over each slide, to wash away the excess dye; c) immersion for two minutes
in 950/o alcohol and then treatment with absolute alcohol and xilene to clean; d) mounting
in a rapid synthetic nonfluorescent medium (UV-inert, Serva, Heidelberg). As a control,
specimens were observed unstained, or after only malachite green or acid fuchsin staining.
All the preparations were examined and photographed with a Zeiss Photomicroscope II,
equipped with an incident fluorescence condenser and a 75 Watt Xenon arc source. The
band pass filter BP 546/10 was employed as a primary filter to give "green excitation", while
a chromatic beam splitter FT 580 and longwave pass filter LP 590 proved to be optimal for
these studies.

3. R e s u l t s

Results indicate that the proposed method offers several advantages, both for
the facility in execution and for the inexpensive cost of the materials
employed, when compared to the fluorescamine method (BRuNI et aI. 1977).
By conventional microscopy, the embryonal laticifers are easily localizable by
malachite green-acid fuchsin double stain. The cytoplasm matrix is coloured
in a pale purple-red, while the small protein bodies, typical of the embryonal
laticifers, and nuclei are dark green in colour (Fig. 1 a). The chromaticity of
the dyes is good; malachite green, however, permits an easier detection of the
laticifers since acid fuchsin only furnishes a weak purple contrast. The cells
surrounding the laticifers react very poorly to the double staining, and only
nuclei and some protein bodies appear weakly stained.
With fluorescence microscopy, embryonal laticifers are easily detectable
because of the strong red fluorescence emitted by the cytoplasmic matrix
(Fig. 1 b). The nuclei are weakly fluorescent and difficult to localize. The
protein bodies of laticifers, which are smaller (l/4-i/8) than those of other
embryonal cells, are quite evident, but less fluorescent than the cytoplasmic
matrix. Generally, in comparison with observations in "white light", the

A Method for Localizing Embryonal Laticifers

345

microscopical image formed under "green light" excitation has a higher

contrast and also permits the detection of very small latex vessels. In addition,
the brilliance of the image in fluorescence is reinforced because the remnant
tissues of the embryo appear weakly fluorescent.
The control reactions have shown that the unstained sections emit a pale
autofluorescence which does not disturb the staining. The sections, stained

Fig. 1 a-b. Longitudinal section of an embryonal radicle of Euphorbia marginata showing

the vascular iaticifers fixed in FAA and stained by the malachite green-acid fuchsin procedure.

a Photographed in "white light" by tungsten lamp. b Photographed under "green

light" excitation by Xenon lamp

only with malachite green and observed by fluorescence microscope, confirm

that this dye is not fluorescent with the excitation filter used in this work.
The embryonal laticifers, stained only with acid fuchsin, emit a red fluorescence similar to that engendered by the malachite green-acid fuchsin mixture,
but the efficency of fluorescence is weaker.
Concerning the fixative action which assumes great relevance in fluorescence
microscopy (PEARSE 1972), both coagulant and non-coagulant fixative mixtures may be employed, but the best results are obtained using the coagulant
ones.

346

A. BRUNI and BARBARATosI

4. Discussion

One advantage of the proposed histological method is that it is suitable for

observations both in conventional and fluorescence microscopy. The uses of
a combined techniques increases the probability of obtaining deducible information for each specimen. The images produced by xenon lamp are of
better quality when compared with those obtained by tungsten lamp: fluorescence microscopy furnishes highly contrasted images in which fine details are
also detectable. The red fluorescence of the specimens is due to the particular
chromatic characteristics of acid fuchsin. Commercial acid fuchsins are a
complex mixture of sulphonated derivatives of triphenylmethane compounds,
and they are well known for their capability of acting as a base towards
anionic or weakly cationic dyes (GuRR 1965). Whereas acid fuchsin acts as a
complementary stain of malachite green in conventional light, it is the primary
stain in fluorescence microscopy.
Control reactions have shown that red fluorescence is stronger when acid
fuchsin is used with malachite green dye. This evidence indicates that malachite
green interacts with acid fuchsin enhancing its emission intensity. The
mechanism of staining reaction may be explained as an interaction of the two
stains by which acid fuchsin also links with tissue elements that have already
been stained by malachite green. In this way, malachite green acts as a kind
of mordant for the acid fuchsin, adding new reactive sites. This interaction
is not evident in white light, but is strongly manifested under "green excitation".
Malachite green and acid fuchsin are not specific dyes for certain chemical
groups, but they are normally used in plant histology to differentiate primary
and secondary cell walls. With fluorescence microscopy, their combined use
may furnish a higher number of histological parameters particularly in tissues
which are usually difficult to stain.
In conclusion, red fluorescence emission of the embryonal laticifers under
"green light" excitation is not caused by the presence of particular substances
or chemical groups, but it results from the high amount of protein-carbohydrate complexes contained in the cytoplasm matrix of these internal
secretory structures (BiauNI et al. 1977). The proposed method offers advantages in procedure and outlay and, therefore, is very convenient to use in
routine work. Furthermore, the strong fluorescence and its resistence to
fading permit the use of low speed film, normally difficult to employ in
fluorescence microscopy.

Acknowledgements
The authors are grateful to the Italian National Research Council (CNR) for financial
support.

A Method for Localizing Embryonal Laticifers

347

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