A New Method of Dissociating Cells.
By
Edwin S. Goodrich, F.R.S.
With 17 Test-figures.
IT is often desirable to ascertain and to demonstrate the
shape of isolated cells of animal tissues, not only in the course
of research, but also and particularly when teaching students.
For these purposes a simple method easy to apply and certain
in its results would be very useful.
More and more our modern text-books of histology seem to be
illustrated not by figures of complete cells as they occur in
nature, but either by photographs of sections showing only
portions of cells or by diagrammatic reconstructions of cells.
These often convey to the student a very inadequate idea of the
structure of tissues and the cells of which they are made up.
Even Schneider's excellent 'Lehrbuch' (1902), one of the best
books on the subject, contains but few figures of whole isolated
cells.
The method here described seems to fulfil the necessary
requirements; moreover, the ingredients used are easily obtained and are quite cheap. It consists in immersing small
pieces of tissue, or whole small animals such as H y d r a , in a
saturated solution of boric acid (H3B03) in normal salt solution,
to which a trace of Lugol's solution of iodine has been added.
Boric acid is not very soluble, and at ordinary temperature it
takes several days to obtain a saturated solution. A convenient
method is to have two bottles of 0-75 per cent, solution of NaCl
to which an excess of boric acid has been added; the bottle not
in use can be constantly refilled either with the acid or with the
solvent as required. Just before use sufficient of the iodine
should be added to give the mixture a pale yellow colour—about
2 drops of Lugol's solution to 25 c.c. The use of this reagent for
preserving delicate protoplasmic processes or membranes and
246
EDWIN S. GOODEICH
cilia with little or no distortion has already been described in
a previous work (Goodrich, 1919).
After the specimen has been thoroughly impregnated, the
iodized solution may be replaced by pure saturated solution of
boric acid in which it may remain for days or weeks. The best
results seem to be reached on the second or third day of immersion ; but the time varies somewhat according to the nature of
the tissue. After some days the cells may slowly deteriorate;
some, however, seem to undergo little change even after a month.
The dissociation may be combined with staining when desired.
Solutions of various appropriate stains which mix with the boric
acid without precipitation will be found useful. Such are
haemalum, carmalum, and aniline dyes such as toluidin blue,
methylene blue, &c. A drop of the stain may be introduced
under the cover-glass, or the stain may be added to the boric
acid solution with the specimen. A little dilute glycerine added
under the cover-glass will prevent the preparation from drying
up and enable it to be kept for some days. For marine Invertebrates the boric acid solution should be made up with sea-water.
The method, of course, has its limitations. It does not dissolve
certain membranes, such as the connective tissue membranes
which surround most organs in the Metazoa. Although the
intercellular substance may be readily dissolved the covering
membranes may prevent the cells from separating. The method
works best with epithelia having a free surface. In such cases
the cells may fall off, or be readily pipetted off and placed on a
slide; in other cases they may have to be separated by tearing
with needles on the slide. In this paper no attempt will be made
to give a detailed description of histological details, but a few
instances will be given of the general results obtained by treatment of the tissues of certain common animals, more especially
those used in teaching.
Hydra.—Both H y d r a fusca and H y d r a v i r i d i s have
been treated. The whole animal when moderately extended can
be plunged into the iodized solution. It usually dies without
excessive contraction. After one or two days the specimen may
be placed on a slide, if necessary teased with needles, covered,
the cover-glass tapped to help the cells to separate. The musculo-
DISSOCIATING CELLS
247
epithelial cells have been well described by Schneider (1890),
Gelei (1924), Eoskin (1922), and others.
Both ectodermal and endodermal muscnlo-epithelial cells are
somewhat elongated transversely to the long axis of the animal,
and their contractile fibrils are disposed in two layers, one on
each side of the intervening mesogloea. Thefibrilscross at right
angles, those of the ectoderm running longitudinally and those
TEXT-FIG. 1.
Two ectodermal cells of H y d r a v i r i d i s seen from inner surface,
showing muscular processes, pr; c, cell outline; n, nucleus.
of the endoderm running transversely (circularly). But it will
be noticed that whereas each endoderm cell has only one basal
fibril, each ectoderm cell may have several (from 1 to 5 or 6)
set at intervals along its base (Text-figs. 1 and 2). For these
ectodermal fibrils, set closely in parallel lines, are more numerous
than the cells (Text-fig. 2). They do not radiate from the base
of the cell, as figured by Schneider (1890), and the appearance
of occasional branching seems to be due merely to overlapping.
The endodermal musculo-epithelial cells, on the other hand,
have only one basal contractile fibril (Text-figs. 3 and 4).
The contractile fibres are covered with an irregular layer of
cytoplasm; delicate processes along each side and extending into
the mesogloea can generally be seen. The endodermal cilia,
though very slender, usually show clearly. Irregular amoeboid
processes project from the nutritive endodermal cells, which
248
EDWIN S. GOODBICH
pr
3OjJL
TBXT-FIG. 2.
Hydra viridis, inner view of ectoderm, showing longitudinal
muscular processes, pr, and cell outlines, c. Preparation made
with Bichromate of Potash.
TEXT-FIG. 3.
Hydra fusca. Endodermal musculo-epithelial cells.
contain various food vacuoles and other granules. In H y d r a
v i r i d i s the zoochlorellae occupy chiefly the more basal region,
while in the more apical region are many colourless spherical
bodies of uniform granular appearance—possibly stored food
material (Text-fig. 4). The glandular cells are usually conspicuous. Delicate branching cells are probably nervous.
DISSOCIATING CELLS
249
Lumbricus.—If a piece of the wall of the intestine of
L u m b r i c u s h e r e u l e u s be placed in the dissociating fluid
the chloragogen cells of the external coelomic epithelium and
the cells of the internal endodermal epithelium will begin to drop
off even in 24 hours, and more completely on the second or third
day. With a fine pipette they can then be placed on a slide and
TEXT-FIG. 4.
H y d r a v i r i d i s : endodermal musculo-epithelial cells; still connected by their basal process in c. ch, zoochlorella; ci, cilium;
gb, granular bodies; pr, basal contractile fibril.
examined. The chloragogen cells, filled with the characteristic
granules, vary considerably in size and shape, but are usually
club- or pear-shaped with a slender base provided with tapering
branching processes (Text-fig. 5 E-H). The endoderm cells are
of several kinds. Especially in the more anterior region are
numerous ciliated cells with broad ciliated apical end, and
tapering basal end often more or less branched (Text-fig. 6).
The details of the structure of the apical end, well described by
Schneider (1902), can be made out with the help of toluidin blue.
Glandular cells with central nucleus also occur; secretory
granules are seen in the basal tapering region, and larger masses
of secretion on the apical side of the nucleus. But, towards the
250
EDWIN S. GOODKICH
TEXT-FIG. 5.
Lumbricus herculeus. A-D, epidermal cells, E-H, chloragogen
cells from wall of intestine, bp, basal process; n, nucleus.
50H
CI
n
TEXT-FIG. 6.
Lumbricus herculeus. Ciliated endodermal cell of intestine.
posterior end of the intestine the ciliated cells become fewer and
fewer, being replaced by broader cells with smooth exposed
surface preserving, however, the superficial refringent layer
which encloses the basal granules in the ciliated variety (Textfig. 7). The basal end usually has several branched processes.
In the more extreme form the cell becomes broadened and the
basal processes more pronounced (Text-fig. 8).
It will be realized that these tissues depart very greatly from
DISSOCIATING CELLS
251
the usual conception of a 'columnar epithelium' as composed of
solid cells set like bricks on a flat surface.
The cuticle can easily be stripped off the surface of the bodywall, and the epidermal cells then dislodged. The ordinary
TEXT-FIG. 7.
Lumbricus herculeus: endodermal cells of intestine.
Lumbricus hereuleus: endodermal cells of posterior intestine.
A, side view, B, surface view, bp, basal process; c, outline of
cell-body; r, apical refringent layer.
epithelial cells are then seen to have a flattened refringent apical
layer and many short basal processes (Text-fig. 5 B, D). Similar
but less developed processes occur on the glandular cells of the
epidermis (Text-fig. 5 A, C).
Of some interest is the structure of the muscle cells of the
middle layer of the intestine. The contractile fibre often has
pronounced spine-like processes, sometimes forked (Text-fig.
9 A). Similar processes may be much developed in the muscle
252
EDWIN S. GOODRICH
cells of the larger blood-vessels and especially those of the hearts.
Here they tend to form on either side a spreading extension with
fringed edge and outstanding rounded lobes (Text-fig. 9 B).
These extensions seem to belong to a siieath of refringent
material enclosing the true muscle fibre, and it frequently shows
fine closely set folds transverse to the main axis of the fibre, and
TEXT-FIG. 9.
Lumbricus herculeus. Portions of muscular fibres of wall of
intestine, A, and of heart, B and C; showing processes and bilateral
fringes of sheath of contractile fibril.
giving it the deceptive appearance of a striated muscle (Textfig. 9 c, B).
Unfortunately, the convoluted canal of the nephridium affords
an instance of the failure of the boric-acid method owing to the
presence of closely investing insoluble membranes. The freely
exposed cells of the open nephridiostome can, however, be dealt
with. Text-fig. 10 A is an outline sketch of the upper lip which
may be made to separate off entire by tapping the cover-glass.
It shows the shape of the large central cell and the surrounding
marginal cells. The way in which the marginals fit into the
notched margin of the central cell is seen in Text-fig. 10 B.
E a n a.—From B a n a t e m p o r a r i a a few examples may be
taken of easily dissociated cells. Such are the ciliated and the
glandular cells lining the roof of the buccal cavity (Text-fig. 11).
While the basal end of these cells is usually simple, some of them
often have divergent processes (Text-fig, l i e ) .
Typical cells of the endodermal lining of the gut are shown
258
DISSOCIATING CELLS
in Text-fig. 12, where A and B represent a columnar and a
glandular cell of the large intestine; c, two cells from the small
intestine; and D, two glandular goblet cells of which the lining
of the cavity of the stomach is almost entirely composed. In A,
B, D well marked basal processes can be seen.
B
cc
TEXT-FIG. 10.
Lumbricus herculeus. A, detached upper lip of nephridiostome, cilia omitted, B, portion of same showing notches of central
cell, cc, into whichfitmarginal cells, m. me, nucleus of marginal;
nc, nucleus of central cell.
The epidermal cells form successive layers of epithelium progressively flattened towards the surface. These layers easily
come apart, but the cells separate with some difficulty, since
they are joined together by numerous intercellular bridges
(Text-fig. 13). Except in the most external layer holes between
or even through the cells can be seen to pierce the layer (Textfig. 13, h).
NO. 330
S
254
EDWIN S. GOODRICH
50fl
TEXT-FIG. 11.
Rana t e m p o r a r i a . A, B, glandular, andc, D, ciliated cells from
posterior roof of buccal cavity, bp, basal process; sc, secretion
cavity.
"SC
h
20 H*
TBXT-FIG. 12.
Rana t e m p o r a r i a . Cells from endodermal lining of large intestine, A, B; small intestine, c; and stomach, D. bp, basal process;
c, apical cup emptied of its secretion; n, nucleus; r, apical refringent layer; sc, secretion cavity.
DISSOCIATING CELLS
255
Lepus.—From the rabbit (Lep us (Or y c y t o l a g u s ) c u n i c u 1 u s) we may select the following tissues. The epithelial lining
of the trachea is composed of ciliated cells (Text-fig. 14 A, B)
usually provided with well developed and branching basal processes, and a refringent apical layer containing the basal granules
TEXT-FIG. 13.
Rana t e m p o r a r i a . Cells of epidermis: A, dissociated; B, still
connected by bridges, h, holes pierced through or between cells;
n, nucleus.
of the cilia. Cells lining the lumen of the colon are shown in
Text-fig. 14 c, D, and are of the more typical columnar form.
Very different are those of the inner lining of the urinary
bladder (Text-fig. 15). These vary much in size and shape, and
often have two or three nuclei. They form a kind of stratified
epithelium, and as the cells enlarge in the more superficial layers
they become pressed together, of irregular shapes with deep
impressions of neighbouring cells. The figures show how faithfully the shape of the cells is preserved after dissociation by the
256
EDWIN S. GOODRICH
30 [x
TEXT-FIG. 14.
Lepus cuniculus. A, B, ciliated cells from inner lining of trachea.
c, D, cells of inner lining of colon, ci, cilia; bp, basal processes.
TEXT-FIG. 15.
Lepus cuniculus. Cells of inner lining of urinary bladder.
boric-acid method. The same results are obtained with cells of
the liver (Text-fig. 16 B, F, G). Typical cells of the spleen are
drawn in Text-fig. 16 A-D.
The wall of the ventricle of the heart is composed of characteristic striated muscular bands, flattened and often branched
(Text-fig. 17). There has been considerable controversy as to
whether the nucleated pieces into which these bands readily
break up are really cells or merely fragments. Certainly the
limits of the pieces are generally visible in the unbroken bands
DISSOCIATING CELLS
257
TEXT-ITG. 16.
Lepus cuniculus. A-D, cells of spleen, E, F, O, cells of liver.
TEXT-HG. 17.
Lepus cuniculus. Muscle cells of ventricle of heart.
A, B, in outline; c, with contractile fibrils.
though even after prolonged treatment two or more may adhere
owing probably to the minute fibrils being continuous from
segment to segment (Text-fig. 17 B, C).
No mention has so far been made of the nervous system, a
tissue peculiarly ill adapted to dissociation owing to the tangle
258
EDWIN S. GOODRICH
of nerve fibres holding the cells together. Nevertheless, good
results have been obtained by treating pieces of the spinal cord
or brain with the fluid, and then teasing them on a slide. But
the results are possibly no better than those reached by other
well-known methods usually recommended.
In conclusion it may be said that the boric-acid method here
described has the merit of being very easy to apply to suitable
tissues. It is possible that, if combined with other reagents and
appropriate stains, it may help to reveal fine histological details
and thus help in the study of histological problems.
SUMMAEY.
A method for the dissociation of the cells of Metazoan tissues
is described consisting in their treatment with a saturated
solution of boric acid in normal saline to which a trace of Lugol's
solution of iodine has been added. Examples of the results
obtained are described and figured.
LIST OF REFERENCES.
Gelei, J . v., 1924.—"Cytologieder Hydragrisea", 'Z. f. Zellen u. Gewebel',
!•
Goodrich, E. S., 1919.—"Pseudopodia of Leucocytes", 'Quart. Journ.
Micr. Sci.', 64.
Roskin, Gr. v., 1922.—"Epithel-Muskelzellen v. Hydra", 'Anat. Anz.', 56.
Schneider, K. C, 1890.—"Histol. v. Hydrafusea", 'Arch. mikr. Anat.', 35.
1902.—'Lehrbuch d. vergl. Histol. der Tiere.' Jena.