0038-9153/80/5501-0013E;0:!

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STAIN TECHNOLOGY Vol. 55, No. 1
Copyright 0 1980 by The Williams & Wilkins CO 2 S. A .
h n l e d in i

A VERSATILE STAIN FOR POLLEN FUNGI, YEAST AND BACTERIA'

Indian Institute
M.P. ALEXANDER~, of Horticultural Research, Bangalore-560006, India

ABSTRACT A versatile stain has been developed for demonstrating pollen, fungal hyphae and
spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20
ml; 1% malachite green in 95%ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1%
in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to
destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted
ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi,
bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture
plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts,
the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen,
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staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained
in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the
slides or by storing at 55+2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner
and then stained. The stock solution is durable, the staining mixture is very stable and the color of
the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not
necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the
coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting
and recording of aborted and nonaborted pollen is also discussed.

Estimation of pollen viability is necessary to plan any type of breeding program.

Usually, a count of the aborted and nonaborted pollen is taken since there is a
tendency for correlation between pollen stainability and pollen viability. Nona-
For personal use only.

borted pollen is stained by using nuclear dyes such as acetocarmine in glycerol

jelly or cotton-blue in lacto-phenol. These two stains have certain limitations since
they stain only nonaborted pollen while the aborted pollen is identified by the
unstained and empty pollen walls. The two dyes given above are not effective for
the pollen of most of the plants belonging to families such as Malvaceae and
Compositae, due to the thick and impervious nature of their pollen walls. They
also have prominent sculptures and a mucilagenous coating, due to which the
pollen grains cannot be separated from one another making staining difficult.
Pollen viability is estimated by using vital stains such as triphenyl tetrazolium
chloride (TTC) and tetrazolium red (Oberle and Watson 1953, Hauser and
Morrison 1964, Aslam et al. 1964). The procedure involved in vital staining is time-
consuming, complicated and not always reliable (Oberle and Watson 1953). Thus
the two methods reported for estimation of pollen sterility have certain limitations.
Alexander (1969) reported a differential stain in which aborted pollen took a
light green and nonaborted pollen a red color. The procedure for differentiating
the pollen is time-consuming, especially for pollen with thick walls. To overcome
these objections, the stain reported here was developed. This stain not only produces
better differentiation of aborted and nonaborted pollen but also stains fungi in
host tissue, fungal hyphae and spores, bacteria and yeasts. Thus, it is specific for
pollen and a general stain for fungi, yeast, and bacteria. This is the first report of
Contribution No. 789 of the Indian Institute of Horticultural Research, Bangalore-560006,India.
I
* Present address: Central Horticultural
Experiment Station, Ambavadi, Civil Lines, Godhra-389001
Gujarat, India
13
 14 S T A I N TECHNOLOGY

a versatile stain which could be used by plant breeders, cytologists, mycologists,

plant pathologists, microbiologists, and bacteriologists.
T h e staining procedure is very simple and is completed in 3-5 minutes. The
stock solution and the stain mixture are very stable. Since the stains are mixed in
glycerol the slides are semipermanent. The intensity of staining can be varied
according to one's liking by varying the amount of acid in the stain.

MATERIALS
A N D METHODS

T h e stain mixture is prepared by adding the various constituents in the order

qiven below, shaking after the addition of each item, and storing in colored reagent
bottles for 8-10 days before use. Stock solutions have been prepared from British
Drug House dyes. One gram of phenol is added to the acid fuchsin stock solution
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to prevent contamination.
Ethyl alcohol (95%) 20 ml
Malachite green 20 mg (2 ml of 1% solution in 95% alcohol)
Distilled water 50 ml
Glycerol 40 ml
Acid fuchsin 100 mg (10 ml of 1% aqueous solution)
Phenol 5g
Lactic acid as follows:
Pollen with very thin walls 0.5-1 ml
Pollen with moderately thick walls 1-2 ml
Pollen with thick walls 2-4 ml
For personal use only.

Pollen with very thick (sculptured) walls 4-6 ml

Fungi 2-3 ml
Yeasts 1-2 ml
Bacteria 6 ml
A solution to destain overstained specimens has also been prepared. T h e destain-
ing solution is compounded by adding the following constituents in the order
given, shaking after the addition of each and storing in reagent bottles.
Distilled water 50 ml
Glycerol 35 ml
Lactic acid 15 ml

AND RESULTS
PROCEDURE
Procedure I (For immedzate use). This procedure is recommended for all types of
pollen of angiosperms except for those which have a tendency to adhere to each
other either due to mucilagenous substances on them or due to interlocking of
spines on the pollen walls. This procedure is also recommended for fungi, bacteria
and yeasts
Pollen is dusted onto 1-2 drops of stain on a slide, stirred well with a needle,
warmed lightly over a flame 4-5 times, coverslip mounted and examined after
letting the slide stand for 5 minutes. With a staining mixture having the optimum
acid level aborted pollen grain? are stained green and nonaborted ones crimson
red. Fungus in host tissues is also stained using this method; after storing the slides
 POLLEN STAIN 15

for 24 hrs the host tissue stains green and fungus a bluish purple. If fungal hyphae
and spores are placed on a slide, 2-3 drops of stain are added and warmed over a
flame till the stain begins to boil, the preparation is cooled and coverslipped and
the hyphae and spores assume a red color. If yeasts and bacteria are smeared on a
slide, these are warmed over a flame till the water evaporates completely, and then
a drop of stain is added to the fixed material, the slide is warmed over a flame till
the stain begins to boil, the slide is cooled and coverslip mounted, and excess stain
is removed with blotting paper; bacteria (both Gram positive and negative) stain
purple and yeasts red.
Procedure ZZ (For later use). This procedure is recommended for pollen of conifers
and of Malvaceae and Compositae where the pollen grains are sticky and have
reticulate walls. If the preparations are to be kept for many months then this
procedure can be used for other types of pollen also.
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Pour the stain mixture into a 5 ml culture tube or into any small glass vial. Add
pollen of fully dehisced anthers to the stain. Keep the ratio of pollen to stain at
1:2. Close the vial tightly, and store at 55+2 C for 24 hr. Afterwards, remove the
TABLE1 AMOUNT
OF LACTIC
ACIDTO BE ADDEDTO STAIN FOR IDEAL STAINING
FORMULA OF DIFFERENT
MATERIALS
Amount of lactic acid
Material
Procedure I Procedure I1
ml ml
impatiens balsamina 0.5 0.5
For personal use only.

Lycopersicon esculentum 0.5 0.5

Oryra sativa 0.5 0.5
Dolichos lab-lab 0.5 0.5
Pisum satmum 0.5 0.5
Crotolaria juncea 0.5 0.5
Solanum melongcna 1.o 1.o
Solanum 1.0 1 .o
Cymbopogon sp. 1.0 2.0
Citrus sp. 1.o 2.0
Carica papaya 1.o 2.0
Capsicum annum 1.o 2.0
Watermelon hybrid 2.0 3.0
Bottle gourd hybrid 2.0 3.0
Datura stramonium 2.0 3.0
Dianthus hybrid 3.0 4.0
Cocos nucifra 3.0 4.0
Amaryllis hybrid 3.0 4.0
Nelumbo hybrid 3.0 4.0
Jasminum species 3.0 4.0
Rosa hybrids 3.0 4.0
Mangifera indica 3.0 4.0
Chrysanthemum hybrids 4.0 5.0
Zinnia hybrids 4.0 5.0
Hibiscus hybrids 6.0 6.0
Abelmoschus hybrids 6.0 6.0
Fungus in host tissue 3.0 -
Fungi 3.0 -
Bacteria 6.0 -
Yeast I .o -
 16 STAIN TECHNOLOGY

anther and other debris. Place one drop of pollen-stain suspension on a slide.
coverslip and examine. When the acid content is ideal, aborted pollen grains stain
green and nonaborted ones crinison red. If deep green staining predominates, add
2--4 drops of destaining fluid, store the vial at 5 5 k 2 C for ten minutes and re-
examine. When the green of the aborted pollen has become very faint, then add 5-
6 drops of a stain mixture containing more acid than the one used earlier, store the
vial at 55+2 C for 10 minutes and reexamine. In this procedure, it is advisable to
select a stain mixture which produces a light green color in the aborted pollen and
a deep red in the nonaborted pollen since, on prolonged storage, the green will
deepen and give the ideal contrast.
T h e amount of acid used for different materials tested during development of
this stain is given in Table 1. Differential staining of pollen, fungus, yeast and
bacteria are shown in Figs. 1-8.
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DISCGSSION
Differential staining of aborted and nonaborted pollen was produced by the two
dyes malachite green and acid fuchsin. Cellulose in the pollen walls reacts with
malachite green producing a green color. Protoplasm reacts with acid fuchsin
producing a purple color. Since aborted pollen grains have no protoplasm, they
appear green. In nonaborted pollen the purple of the protoplasm can be seen
through the pollen wall if the acid content of the stain mixture is ideal. T h e cell
walls of fungi, yeasts and bacteria do not take the green stain, but their protoplasm
For personal use only.

stains purple. Hence, differential staining is not produced in these materials.

Malachite green is a weakly basic dye and acid fuchsin stains only in an acid
niediuni. Hence, they tend to stain inversely. The intensity of the green staining is
also affected by the thickness o f the pollen walls. These factors,have to be taken
into account i n working with the stain; ideal differentiation can be obtained b y
varying the acid content of the stain. If pollen walls are very thin and membranous,
as in Inipatiens DaL~aminae,the acid content of the formula should be as low as 0.5
ml. O n the other hand Hibixus ro.sa-sinensi.c, with very thick and impervious pollen
walls requires 6 nil of acid for proper differentiation.
In practice it has been found useful to prepare seven bottles of the stain mixture
containing 0.5, 1, 2, 3 , 4, 5 and 6 ml of lactic acid respectively. When one is
doubtful about the staining properties of the pollen wall, then the stain containing
3 nil of acid should be tried first. If the results are not satisfactory the procedure
should be repeated with a mixture of greater or lesser acidity depending on the
actual color balance sought. With three to four trial runs it is possible to select the
stain mixture containing the right amount of acid and to obtain ideal differentia-
tion. From the data given in Table l , one may obtain a general idea of the amount
o f acid required for different materials.
T h e question of differential staining does not arise in case of fungi, yeast and
bacteria since their cell walls do not take up the malachite green. Therefore, only
the intensity of staining of their protoplasm has to be controlled. It has been
observed that for a perfect staining, the stain mixture should have 1 ml of acid for
yeast, 3 nil for fungi and 6 nil for bacteria.
Biotech Histochem Downloaded from informahealthcare.com by Nyu Medical Center on 11/25/13
For personal use only.

FIG. 1. Aborted (green; lighter shade) and nonaborted (red; darker shade) pollen of Nelembo hybrid
300 X.
FIG. 2. Aborted and nonaborted pollen of &oslegia uenusta. 300 X .
FIG. 3. Aborted and nonaborted pollen of Amaryllis hybrid. 1200 X.
Fie. 4. Aborted and nonaborted pollen of Achrus zapota. 1200 X.
FIG. 5. Aborted and nonaborted microsporangia of Aurucarza cnokiz. 300 X .
FIG. 6. Uredospores of rust on Crotilaria jeniculata. 1200 X.
FIG. 7. Yeast ‘Montraschk’ strain grown on PDA.
FIG. 8. Bacteria, Bascillus sp.
 18 STAIN TECHNOLOGY

T h e p H of the stain mixture regardless of the acid level does not change even
after prolonged storage. Alcohol is used to dissolve the malachite green and to
prevent its precipitation when the other ingredients are added. It also helps in
removing mucilaginous substances present in certain types of pollen and in aiding
their dispersal in the stain. Water is the carrier of the two dyes and due to the
glycerol the stain does not evaporate as do water-based stains. T h e phenol acts as
a preservative. Inorganic acids interfere with the malachite green staining. Among
the organic acids tried, lactic acid produced the best staining and differentiation.
Alexander (1969) reported a triple stain for differentiating aborted and non-
aborted pollen. He used 10 mg of malachite green, 50 mg of acid fuchsin and 5 mg
of orange G. This stain, though i t differentiated aborted and nonaborted pollen,
was not effective in differentiating fungus in host tissue, or for staining fungi,
bacteria and yeasts. T h e stain reported here has 20 mg of malachite green, 100 mg
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of acid fuchsin and orange G is omitted. Since lactic acid, which is a stronger acid
than acetic acid is used in this stain, the intensity of the acid fuchsin staining can
be varied, depending upon the material, with minor changes in the concentration
of acid.
Since aborted pollen is stained green and nonaborted pollen red, this technique
offers possibilities for the development of electronic pollen counters. The author
visualizes the development of a n electronic pollen counter which will eliminate
time-consuming manual counting by combining this versatile stain and advances
in image analysis.
For personal use only.

ACKNOWLEDGMENTS
T h e author is thankful to Dr. G.'S. Randhawa, Director of the Institute for his
keen interest and for providing facilities. He is also thankful to the Heads of the
Divisions of Plant Pathology, Plant Physiology and Microbiology, Floriculture and
Landscape Gardening, Fruit Crops and Olericulture and their scientific staff for
providing the required specimens to test the efficiency of this stain and also for
numerous suggestions during the course of its development.
KEFEKENCES
Alexander, M. P. I969 Differential staining of aborted and nonahorted pollen. Stain Technol. 44: 1 1 7-
122.
Aslarn, I,. M . , Brown, M. S. and Kohal, K. ,J. 1964. Evaluation of seven tetrazoliurn salts as vital pollen
stain in (;r,i.!ypiurn hinuturn. Crop Sci. 4: 508-5 10.
llauser, J . P. and Morrison, J. f-I. 1964. Cyrocheniical reduction of nitro blue tetrazoliurn as a n index
of pollen viability. Arner. J. Bot. 51: 748-753.
Oberle, G. D. and Watson, R. 1953. T h e use of 2, 3, 5-triphenyl tetrazolium chloride in viability of fruit
pollen. Amer. Soc. Hort. Proc. 61. 299-309.