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Staining - Materials and methods in Practical

microtechnique
Whilst observation in the natural state is important, it may be difficult for the student to
differentiate, for example, between unlignified and lignified tissues and it is for this reason
that we recommend staining sections.

Staining
Whilst observation in the natural state is important, it may be difficult for
the student to differentiate, for example, between unlignified and lignified
tissues and it is for this reason that we recommend staining sections.
There are several staining combinations that may be used to enhance
details with-in sections. Sections of freshly cut material should be washed
gently, to re-move cell debris which will obscure details once the section
has been stained.

Two main types of stain can be used: (i) those which are
temporary, whose colour fades, or which gradually damage the
section; and (ii) those which are regarded as permanent. Even
permanent stains may lose their colour if exposed to sunlight, so
be careful and store your collection in the dark.
With care, stains can be selected to give the maximum contrast between
the various cell and tissue types in the plant. They might be selected be-
cause they colour particular parts of the cell wall structure and indicate its
chemical composition. The stains and the protocol described below are in
daily use in many laboratories throughout the world, and not just those
as-sociated with the authors. Those who want comprehensive lists of
stains, procedures and protocols, should see the books by Gurr (1965),
Foster (1950) or Peacock revised by Bradbury (1973) to mention but
three of the many guides to micro technique.

We have included other techniques which will be useful to the student on

the CD-ROM. These can be accessed quite simply by following the
‘Tech-niques’ links associated with each of the exercises.

Temporary stains
 

1 .  1% aqueous methylene blue. All cell walls turn blue, except cutin
orcutinized walls which remain unstained; cell walls take up a degree of
in-tensity of blue depending upon their chemical composition and
physical structure; various wall layers frequently stain differently. The
stain may be mixed with 50% glycerine, about 10 ml of a 1% aqueous
stain, to 90 ml of 50% glycerine, and sections mounted directly into this
medium. This mixture is also useful for staining macerated tissues which
are difficult to handle. A drop of washed macerate in water is mixed with
a drop of the mixture on a slide, and the coverslip put on.

2   Chlorzinciodine solution (CZI, Schulte’s solution). This

solutionconsists of: 30 g zinc chloride, 5 g potassium iodide, 1 g iodine
and 140 ml distilled water. Cellulose walls turn blue, starch turns blue-
black, lignin and suberin turn yellow and moderately lignified walls turn
green-blue. Sections are placed on the slide, and a drop or two of CZI
added. This can be drawn off and replaced by 50% glycerine after 2–4
minutes, but satisfac-tory results can be obtained by adding 50%
glycerine directly, and mount-ing in the mixture. This stain swells the
walls and eventually dissolves them. Consequently, care must be taken
when describing wall thickness.

4   Chlorazol black saturated solution in 70% alcohol. Stains walls

black or grey and is particularly good for showing pitting.

5   Saturated carbolic acid solution. Sections are mounted directly inthe

solution (which should be kept off the hands). Silica bodies usually turn
pink; this helps to distinguish them from crystals which remain
colourless.

6   Phloroglucin/HCl. The phloroglucin is added to the section, and

thenthe dilute HCl. Lignin turns red. Caution: HCl is highly corrosive of
skin, clothes and the microscope!

 
7   Sudan IV. Sections can be mounted directly in the stain. Stains
fats,cuticles turn orange.

 
8 Ruthenium red.Mucilage and some gums turn pink. Sections can
bemounted directly in the stain.

A simple double stain

Fuchsin, aniline blue and iodine in lactophenol (FABIL) is a most useful

stain and mountant for all types of plant material. Although not available
commercially it is easily prepared from the following stock solutions: lac-
tophenol : phenol (crystals), glycerol, lactic acid and distilled water in
equal parts by weight.

Aniline blue, 0.5% in lactophenol.    A

Basic Fuchsin, 0.5% in lactophenol. B

 
Iodine, KI, 0.6 g : lactophenol, 1 litre.        C

The stain is made up by mixing the stock solutions in the proportions of

A,4 : B,6 : C,5. This is allowed to stand overnight and after filtering it is
ready for use and will keep indefinitely.

FABIL is superior to other commonly used temporary reagents such as

aniline double stain, phloroglucinol or zinc chloride, the particular
advantage being that it incorporates a differential stain, a clearing agent
and a semi-permanent mountant. Sections cut from fresh or alcohol-
stored material are transferred directly to the stain, enclosed by a
coverslip and examined immediately. If desired, the stain may be
replaced after about 10 minutes by plain lactophenol or 25% aqueous
glycerol, but this is by no means essential. The solution is only slightly
volatile and mounts can be kept for several months without drying up,
although additions from time to time at the edge of the coverslip will
prevent air bubbles from being drawn in. However, if it is necessary to
store the mounts for a very long time the coverslip can be sealed with
melted beeswax or nail varnish. The cytoplasmic cell contents, including
nuclei and callose plugs of sieve tubes, are stained blue, Cellulose walls
stain a paler blue and lignified tissue becomes a bright yellow, orange or
pink, depending on the nature of the specimen. Staining is rapid but
improves with time and overstaining is not possible, even after immersion
for several weeks.

Much of the success of this reagent is due not simply to staining but also
to differential clearing, so that in practice the tissues are rendered more
dis-tinct than the above-mentioned colour reactions would suggest.
Moreover, because of the extremely good light-transmitting properties of
the solu-tion, cellular detail can be studied even in thick sections. FABIL
may also be used for mounting fungal or algal material, including
seaweeds, and causes very little distortion. Alternatively the aniline blue
solution A, or fuchsin solution B, may be used alone. With fungi, gentle
heating of the mount improves the absorption of the stain.

Safranin (1% in 50% alcohol) and Delafield’s haematoxylin is a very

useful combination. In cell walls cellulose turns dark blue; lignin turns
red and cellulose walls with some lignins turn purple.

Freshly mix the prepared safranin with matured Delafield’s haematoxy-

lin in the proportions of 1 : 4; filter. The stock mixture can be used for up
to about 1 week, but should be filtered before use each day.

Sections should be transferred from water (after washing all bleach away)
into a suitable dish containing the stain, and covered. Most sections take
up the stain in 24 hours, others will need less time. The sections should
then transferred to a Petri dish in which there is 50% acidified alcohol
(use a few drops of conc. HCI). This solution removes the stain, acting on
the safranin first. Whilst the object is to obtain a satisfactory colour
balance, only expe-rience will tell when this has been reached.

 
Sections should be removed when they still appear to be slightly dark or
overstained for best results – the colours look less intense under the
micro-scope. The decolorizing action is halted by placing the sections
into a Petri dish containing 95% alcohol. After about 5 minutes they can
be transferred to absolute alcohol in a covered Petri dish. Five minutes
later they can be transferred either to a 50–50 mixture of absolute alcohol
xylene in a covered dish, or this step may be eliminated and they may be
transferred directly into xylene. Xylene fumes should not be inhaled;
use a fume hood. After 10 minutes in the xylene, the sections can be
mounted in Canada balsam on the microscope slide. Any milkiness in the
section at this stage means that water is still present, and the section
should be taken back through xylene, then fresh absolute alcohol, fresh
absolute alcohol xylene and fresh xylene before remounting. Sections
which curl up or roll up should be straightened out in 50% alcohol. As
they progressively dehydrate in purer alcohol they become more brittle
and cannot be un-rolled without breaking. Curled wood sections can be
flattened by drawing them over the edge of a slide partly immersed in
50% alcohol (Fig. 10.5), a process needing three hands! Alternatively a
section lifter can be used. Once on the slide they can be ‘set’ using a few
drops of 95% alcohol.

If it is more convenient to stain overnight, the safranin-haematoxylin

mixture can be used in the proportions 94 : 6. Although fast green can be
used as a counter stain for safranin, we have found that haematoxylin pro-
duces a colour which photographs better on normal panchromatic film.
Al-cian blue may be used as an alternative to the haematoxylin; a 1%
aqueous solution is satisfactory. It is easier to use and gives blue colours
where the haematoxylin would have stained purple.

Fast Green can be used on its own as a stain for macerated material. The
macerate is dehydrated by decanting alcohols of 50, 70, 90, 95% and
absolute in turn from a tube containing the macerate. It is a help if a small
hand centrifuge can be used to settle the cells at each stage. Finally, the
cells are transferred to a slide bearing 23 drops of Euparal containing 23
drops of Fast Green per 10 ml. The coverslip is applied.