644 MEDICAL MICROBIOLOGY
The slide bearing the paraffin section is placed in a jar of
In the case of fluid material, e.g. broth cultures, urine, sputum, pus, etc.,
one loopful (or more) is taken up with the inoculating wire (p. 791) and spread
thinly on the slide. A little experience will soon determine the amount
required, and in spreading the films it will be found that there are both thick
and thin portions, which is not disadvantageous. The slide is then held in the
palm of the hand high over a Bunsen flame and dried. The film is fixed by
passing the dried slide, film downwards, three times slowly through the flame,
or by heating through the glass slide. In the latter method the slide is held,
film upwards, in the top of the Bunsen flame fora few seconds so that the slide
becomes hot. Care must be taken not to char the film, and when the slide is
just too hot to be borne on the back of the hand, fixation is complete.
In making films on coverslips and staining them, Cornet's forceps are used
to hold the slip in a horizontal position, the forceps resting on the bench.
With solid material, such as cultures on agar, etc., it is necessary to place a
loopful of clean water on the slide. The loop is then sterilised and a minute
quantity of material, obtained by just touching the growth, is transferred to
the drop, thoroughly emulsified, and the mixture is spread evenly on the slide.
The resulting film is fixed and dried as above. Beginners are apt to take more
material than necessary from the culture and thus make too thick a film.
STAINING OF FILMS
The stains are poured directly or filtered on to the slide. When staining is
completed, the dye is washed off with water, and the slide is allowed to dry in
the vertical position or is placed between two sheets of white fluifiess blotting
paper or filter paper. The drying of the film is completed over the Bunsen
flame. Such stained films may be mounted in Canada balsam under a cover-
slip, or may be examined unmounted with the oil-immersion lens, a small drop
of cedar-wood oil being placed directly on the film. If it is desired to mount
the preparation later, the oil can be removed with xylol or benzol (i.e. benzene).
STAINING OF TISSUE SECTIONS
The sections being embedded in paraffin (p. 678), it is necessary to remove
the paraffin so that a - watery stain may penetrate. The paraffin is first
removed with xylol (xylene) or benzol (benzene), the xylol or benzol is then
removed with alcohol (95 per cent. ethanol), and the alcohol is replaced with
water. The staining is then done. After staining, the section must be
dehydrated with absolute alcohol, cleared in xylol and mounted in Canada
balsam under a coverslip. The Canada balsam (which is a resin) is dissolved
in xylol in order to render it suitable in consistency.
Alcohol (Ethanol) Solutions.The reagents most commonly employed in
preparation of sections are "absolute alcohol", which is 100 per cent. ethanol,
and "95 per cent. alcohol", which is a 95 per cent. solution of ethanol in water
by volume (i.e. 95 ml. absolute alcohol plus water to give 100 ml. solution).
Industrial methylated spirit (not mineralised) may be used for making up
stains, decolourising stained preparations, dehydrating tissues and treating
sections. The type known as "Toilet spirit, acetone free (66 O.P.)" is quite
satisfactory for use instead of 95 per cent. alcohol. Similarly, industrial
methylated spirit, absolute (74 O.P.), can be used instead of absolute alcohol.
Not only are these industrial spirits much cheaper than rectified spirit (90 per
cent. alcohol) and absolute alcohol, but permits for obtaining them duty-free
are more readily granted by the Customs Authorities.
S IM P LE S TAI N S 645
Technique.
xylol for some minutes to remove the paraffin. The section is then treated
with a few drops of absolute alcohol (ethanol), when it immediately becomes
opaque. A few drops of 50 per cent. alcohol are poured on, and the slide is
finally washed gently in water. If the tissue has been fixed in any mercuric
chloride preparation, such as Zenker's fluid, the section should be treated with
Gram's iodine solution for a few minutes (p. 650), then with 95 per cent.
alcohol and finally with water. The sections are now ready to be stained by
the appropriate method. After staining and washing with water, the slide is
wiped all round the section with a clean cloth to remove excess of water. The
bulk of the water in the section may be removed by pressing between fluffless
blotting paper. The section is treated immediately with a few drops of 95 per
cent. alcohol and then with absolute alcohol. The slide is again wiped all
round the section, a few more drops of absolute alcohol are poured on and the
slide is then immersed in xylol. When cleared, the slide is removed, and
excess of xylol round the section is wiped away, a drop of Canada balsam is
applied and the section mounted under a No. 1 coverslip. It is essential that
the section should not be allowed to dry at any period of the process, and that
dehydration with absolute alcohol should be complete in order that the section
may be thoroughly cleared.
When the bacteria are readily decolourised with alcohol, aniline-xylol
(aniline, 2 parts; xylol, 1 part) should be used for dehydration. After
washing, when the slide has been wiped round the section, the preparation is
blotted and then treated with the aniline-xylol mixture, which clears as well as
dehydrates. The aniline-xylol is then replaced with xylol. This can be done
conveniently by holding the slide almost vertically and dropping xylol from a
drop bottle on to the slide just above the section. The xylol flows over the
section and quickly removes the aniline. The preparation is mounted
immediately in Canada balsam.
DPX Mounting Medium
A mounting medium that replaces Canada balsam has been devised by
Kirkpatrick and Lendrum (1939, 1941). It consists of polystyrene (a synthetic
resin) dissolved in xylol, with a plasticiserdibutyl phthalateto ensure
flexibility. There is, however, much shrinkage and the mounting fluid should
be applied generously. The mountant termed DPX is made up as follows:
Mix dibutyl phthalate (B.D.H.) . . 5 ml.
with pure xylol . . . . 35 ml.
and dissolve "Distrene 80" 1 . . 10 g.
DPX medium is water-clear, inert and does not become acid or cause fading
of stained preparations. It is used in the same way as Canada balsam.
If polystyrene of a low molecular weight (about 3000) is used, much less
xylol is required and no plasticiser need be added. Moreover, there is
practically no shrinkage, which is a great advantage over DPX.
SIMPLE STAINS
These show not only the presence of organisms but also the nature of the
cellular content in exudates.
Normally obtainable from Messrs. Honeywill & Stein, Ltd., 21 St. James's
Square, London, S.W.1.