Technical variations in Koelle's histochemical method for
demonstrating cholinesterase activity
By N. T. NAIK
(From the Department of Anatomy, King's College, University of Durham,
Newcastle-upon-Tyne)
With 4 plates (figs, i to 4)
Summary
Koelle's histochemical method for demonstrating cholinesterase activity and the
literature on its subsequent modifications have been reviewed. Experiments were
carried out on the effect on the cholinesterase reaction of formaldehyde fixation, cold
storage of tissues, pH of incubation solution, and progressive increase of incubation
time. A series of experiments was also carried out in testing the specificity of substrates and selective inhibitors used in the Koelle method. Enzyme reaction was visualized by the ammonium sulphide method. As a result of these experiments the
following technical desiderata have been established:
1. Fixation of tissues for 3 h in neutral formaldehyde solution at 40 C preserved the
morphology of the tissues without appreciably affecting the histochemical results.
Fixation for more than 6 h produced definitive inhibition of cholinesterases,
especially AChE, in most tissues.
2. Periods of up to 24 h of cold storage before fixation had no appreciable effect on the
cholinesterase reaction.
3. Incubation at pH values between 5 0 and 6-o produced neither significant diffusion
artifacts nor loss of enzyme activity. Below pH 5 the AChE reaction was affected to
a varying extent according to the tissues used.
4. B.W. 284 at a concentration of 5 X icr 5 M and ethopropazine hydrochloride at a
concentration of 1 X io~4 M were found to be suitable selective inhibitors for AChE
and ChE respectively.
5. Visualization of results by means of ammonium sulphide method was found to be
preferable to phase-contrast microscopy.
Introduction
T H E acetylcholine-cholinesterase system, which involves the enzymatic
hydrolysis of acetylcholine (ACh), forms an important part of the mechanism
of nervous activity. Dale (1914), studying the pharmacological properties of
ACh, suggested that an enzyme was responsible for its removal. Loewi (1921)
showed that stimulation of the vagus nerve caused the formation or liberation
of a substance which he called Vagusstoff. Later, Loewi and Navratil (1926 a,
b) identified the 'vagus substance' as a derivative of choline, possibly acetylcholine. They also showed that the vagus substance and ACh were destroyed
by an enzyme present in aqueous extracts of the frog's heart. Thus, not only
was the original suggestion of Dale (1914) confirmed, but the theory of neurohumoral transmission (Langley, 1901; Elliot, 1905) was significantly extended.
Final agreement is lacking on the details of the role of cholinesterases in
[Quart. J. micr. Sci., Vol. 104, pt. 1, pp. 89-100, 1963.]
90
Naik—Koelle's method for cholinesterase
nervous activity. Some authorities even suggest that this enzyme is essential
not only in all synaptic activities but also in nervous conduction (Nachmansohn, 1959). In general, it is believed that ACh is the active agent of nervous
transmission and that cholinesterase, especially acetylcholinesterase (AChE),
acts by removing the released agent to restore the nervous mechanism for
repeated activity (Eccles, 1957; Nachmansohn, 1959).
Before the introduction of a histochemical method the distribution of
cholinesterases was studied biochemically (Glick, 1937, 1938; Nachmansohn,
1937, 1938, 1940; Mendel and associates, 1943; Augustinsson, 1948).
Gomori (1948) proposed a histochemical method for the direct visualization
of cholinesterase activity in tissues with higher fatty acid esters of choline as
substrates. The method did not, however, give satisfactory results.
Koelle and Friedenwald (1949) introduced a method with acetylthiocholine as a substrate. This is hydrolysed by acetylcholinesterase (AChE) and
non-specific cholinesterase (ChE) more rapidly than is ACh itself. The
method involved the incubation of fresh frozen sections or teased preparations
in a medium containing acetylthiocholine iodide, copper sulphate, and glycine
at pH 8-o. On hydrolysis the liberated thiocholine was presumed to act with
copper salt to give a rather insoluble product, copper thiocholine. To facilitate
precipitation and to prevent diffusion the incubating medium was saturated
with copper thiocholine before the sections were placed in it. After incubation the sections were treated with ammonium sulphide solution to convert the
white precipitate of copper thiocholine into a brown amorphous deposit of
copper sulphide. This produced massive diffusion of the precipitate because,
at pH 8-o, the products of hydrolysis diffused into adjacent regions until
sufficient acidity was built up locally (Koelle, 1950). To reduce the diffusion
artifacts, Koelle (1950) lowered the pH of the incubation solution to 6-4 with
phosphate buffer. He also introduced butyrylthiocholine as substrate for
ChE. Koelle (1951) further modified the method by incorporating sodium
sulphate in the storage and incubating solutions and by replacing the phosphate
buffer by the maleate buffer. By means of recovery tests he showed that
cholinesterases were completely precipitated at pH 6-o in the presence of
sodium sulphate. The phosphate buffer used in the original method was replaced by maleate buffer because of the greater buffering capacity of the latter
at pH 6-o and because it did not precipitate cupric or magnesium ions in the
concentrations employed. Gomori (1952), working on Koelle's thiocholine
method, used tissues fixed in neutral cold formaldehyde solution before incubation and omitted the storage solution.
Couteaux (1951), Taxi (1952), and Couteaux and Taxi (1952), trying
Koelle's method on fixed and unfixed tissues, varied the pH between 7-0 and
4-0 and found that pH between 5-3 and 5-6 gave the best results in tissues fixed
with formaldehyde. They also observed that the acetate buffer gave the best
results at pH 5-3 and 5-6.
The mechanism of the thiocholine method was re-examined by Zajicek,
Sylven, and Datta (1954) and by Malmgren and Sylven (1955). They observed
Naik—Koelk's
method for cholinesterase
91
that during reaction, minute rod-like crystals of copper thiocholine were
deposited at the sites of cholinesterase activity initially. If the medium was
saturated with copper thiocholine, as in the Koelle method, the crystals increased in size. Malmgren and Sylven, therefore, considered that only the
initial deposit could be regarded as a direct result of enzyme activity and that
several factors other than enzyme activity must influence the amount of deposit
at local sites. Similarly, they studied the effect of treatment of sections with
ammonium sulphide after incubation. When a purified sample of copper
thiocholine was placed on a coverslip and treated with dilute ammonium
sulphide, the crystals were dissolved and were replaced by an amorphous
deposit of copper sulphide. These authors considered that the process of
conversion might produce distortion of the true picture. On this suggestion,
Holmstedt (19576) introduced a modification of Koelle's method. He omitted
the saturation of the incubating medium with copper thiocholine and the conversion of copper thiocholine crystals into copper sulphide, and visualized
the results by phase-contrast microscopy. Pearse (i960) agreed that displacement of the original precipitate of copper thiocholine by the copper sulphide
might produce some inaccuracies, but suggested that this could be tolerated
since visualization by means of phase-contrast microscopy might produce
other difficulties.
In addition to the various modifications of Koelle's thiocholine method, now
in general use, other methods are sometimes employed for demonstration of
cholinesterase activity (Nachlas and Seligman, 1949; Crevier and Belanger,
1955). It should be emphasized that it is the products of enzyme activity and
not the enzymes themselves that are revealed by the present histochemical
methods, but the thiocholine method is more physiological than procedures
based on hydrolysis of other compounds employed in certain alternative
methods (Holmstedt and Sjoqvist, 1961).
Materials and methods
The main aim of this investigation has been to assess critically the following
variables in the technique: (a) the duration of fixation, (b) the pH of the incubating solution, (c) the specificity of substrates and inhibitors, (d) visualization methods of the results, (e) length of incubation, and (/) the effect of cold
storage of tissues before fixation.
The material studied came from man and from a number of other mammalian species. Human material was collected from 35 individuals ranging
from late foetal stages to 81 years of age. Comparative material is represented
by the following species: 62 cats, 30 rats (hooded), 30 guinea-pigs (red and
black), 10 moles (Talpa europaea), and 5 hedgehogs (Erinaceus europaeus).
Human tissues were obtained during surgical operations and were fixed
immediately, or they were stored at 4° C from a few minutes to 6 h, or longer
if the effect of cold storage was studied on the tissues used for the cholinesterase reaction. Animals were killed by an overdose of chloroform or by
intra-peritoneal injection of sodium pentobarbitone (nembutal).
92
Naik—Koelle's method for cholinesterase
This paper reports only the results of the technical studies: other findings
based on the application of these methods will be reported elsewhere.
The tissues were fixed in 10% neutral formalin at 4° C adjusted to pH 7-0
with sodium acetate. Fixation time ranged from 30 min to 3 h for routine
studies; in a special study of the effect of the fixative it was extended up to 72 h.
Incubation was carried out with floating sections by the revised Koelle technique (Koelle, 1951, 1955) with certain simplifications and modifications. The
routine procedure was as follows:
(1) Fix small blocks of tissues or whole ganglia in 10% neutral formalin at
40 C for 30 min to 3 h.
(2) Cut frozen sections at 15 /u. or 20 (j. and collect them in distilled water.
(3) Wash in distilled water for 30 min. Place the sections intended for
eserine controls in 3 X icr 5 M eserine sulphate solution for 30 min before incubation (1-9 mg of eserine sulphate in 100 ml of distilled water).
(4) Incubate the sections for 15 min to 6 h (or longer) at 370 C and pH 5-3.
The basis of the incubation solution was prepared as follows (for the total
quantity of 10 ml of incubation solution):
1.
2.
3.
4.
2-5% copper sulphate (CuSO4, 5H2O)
3-7% glycine (amino-acetic acid)
o-i N sodium acetate
o-i N acetic acid
0-2
0-2
7-3
1-5
ml
ml
ml
ml
Items 3 and 4 are constituents of the acetate buffer solution adjusted to
pH 5-3. For eserine controls add 0-19 mg of eserine sulphate for every 10 ml of
incubation solution.
To the above basis add freshly prepared substrate. The substrate was prepared as follows: 15-0 mg of acetylthiocholine iodide or 16-5 mg of butyryl
thiocholine iodide was dissolved in 0-75 ml of distilled water in a centrifuge
tube. To this was added 0-3 ml of 2-5% copper sulphate solution; a brown
precipitate was formed. The contents of the tube were centrifuged for 10 to
15 min at 2,000 r.p.m., and the clear supernatant fluid was added to the basis,
of the incubation solution, bringing the total volume to io-o ml. The substrates were used with and without inhibitors. The different combinations of substrates and inhibitors and the enzymes visualized are shown in
table r.
After incubation, take samples at suitable intervals, wash in distilled water
for 1 min, transfer to approximately 5% ammonium sulphide solution for 30
sec to 1 min, wash again in distilled water, mount on slides, dry and mount
through xylene in neutral Canada balsam. If required, counterstain some
sections before dehydration.
The tissues intended for neurohistological studies were fixed in 10% commercial formalin solution for 3 to 10 days or longer. Nerve-fibres were
stained in frozen sections, 15 to 20 /n thick, by a simplified BielschowskyGros silver impregnation method (Cauna, 1959).
Naik—Koelle's method for cholinesterase
93
TABLE I
Demonstration of cholinesterase activity by the use of substrates and their
combinations with inhibitors
Substrates
Inhibitors
(a) acetylthiocholine
iodide
(6) butyrylthiocholine
iodide
(c) acetylthiocholine
iodide or butyrylthiocholine iodide
{d) acetylthiocholine
iodide
(e) acetylthiocholine
iodide
J) acetylthiocholine
iodide
(g) butyrylthiocholine
iodide
(h) butyrylthiocholine
iodide
Enzyme visualized
AChE
ChE
ChE
eserine sulphate 3 X icr5 M (1-9 mg
in 100 ml)
ethopropazine hydrochloride 1X
io"4 M (3'I2 mg in 100 ml)
B. W. 284 5 X icr5 M (28-0 mg in
1000
AChE
ChE
ml)
B. W. 284+ethopropazine hydrochloride
B. W. 284
ethopropazine hydrochloride
ChE
AChE (after prolonged incubation)
Observations and discussion
Most histochemical studies of cholinesterase activity reported in the literature are based on various modifications of Koelle's technique. The results
obtained by different workers are basically similar, but in certain instances
differences have been reported which have been partly attributed to variations
in the techniques employed. The importance of critical application of the
technique was emphasized at the symposium on histochemistry of cholinesterase held in Basel in i960 (Schwarzacher, 1961). With this view in mind it
was decided to study the effect of fixation, pH, cold storage, incubation time,
and specific inhibitors on the reaction, and also the visualization of results by
means of phase contrast and ammonium sulphide treatment.
The effect of formaldehyde fixation on the cholinesterase reaction
The effect of formaldehyde fixation was studied on a variety of tissues from
human and animal material. The tissues were fixed in 10% neutral formalin
buffered with sodium acetate at 40 C for 3, 6, 18, 24, 64, and 72 h by immersing the whole ganglia or thin slices of other tissues. After fixation the
tissues were cut on the freezing microtome, washed for 30 min in distilled
water, and then incubated and compared with unfixed controls under similar
conditions.
It was found that fixed specimens required longer incubation time to obtain
the same intensity of reaction as in unfixed specimens. The activity of ChE
was less affected by formaldehyde fixation than that of AChE. Fixation for
up to 6 h did not appreciably change the pattern of histochemical distribution
of the enzymes. After fixation for from 6 h to 18 h definite loss was
94
Naik—Koelle's method for cholinesterase
progressively observed in tissues of low enzyme activity. Further fixation
reduced the intensity of the reaction even in sites of high enzyme activity.
The main findings are illustrated in fig. i, which shows the stellate ganglion of
the cat incubated for AChE activity, A is a section of unfixed tissue. B is a
section incubated after 3 h of formalin fixation; it shows all the features of A.
In both A and B three types of nerve-cells can be distinguished: heavily
stained (a), moderately stained (b), and unstained or lightly stained (c). c is a
section incubated after 24 h of fixation. The cells of high enzyme activity
show no noticeable change, but all the remaining cells appear cholinesterasenegative. This means that the enzyme has been inactivated in these cells by
prolonged fixation.
These experiments show that fixation for 3 h preserves the structure of the
tissues adequately, the specimens can be cut easily on the freezing microtome,
and incubation can be carried out by the floating-section method. The
histochemical distribution of cholinesterase activity in such tissues is similar
to, and the cy tological localization is better than, that in sections incubated without fixation. In addition, fixed tissues are easier to handle, they are less
cytolysed by reagents, and their soluble proteins are prevented from diffusion.
For this reason fixed specimens give a better morphological picture and more
accurate localization of enzyme activity (Gomori, 1952; Pearse, i960). This
can be seen if A and B (fig. 1) are compared. These findings are in agreement
with a number of investigators who have studied the question by biochemical
as well as by histochemical methods (Taxi, 1952; Couteaux and Taxi, 1952;
Holmstedt, 1957a; Eranko, 1959, Fukuda and Koelle, 1959; Lehrer and Ornstein, 1959).
The effect of variation of pH on the cholinesterase reaction
It has been established biochemically that pH 8 is optimal for cholinesterase activity (Bernheim and Bernheim, 1936; Easson and Stedman, 1936;
Glick, 1937, 1938; Alles and Hawes, 1940). However, when this pH is
employed for histochemical demonstration of the enzyme, massive diffusion
results. (See, for example, the paper of Koelle and Friedenwald, 1949, which
introduced the thiocholine method.) To reduce diffusion, most investigators
have lowered the pH to 6 (Koelle, 1950, 1951; Gomori, 1952; Holmstedt,
1957&). Others have reduced it further, between the values of 5 and 6
(Couteaux and Taxi, 1952; Coupland and Holmes, 1957; Lewis, 1961). Exceptionally the very low pH of 4-2 has been used (Snell, 1958).
The effect of pH on the cholinesterase reaction was mainly studied on the
sympathetic ganglia of the cat and on human skin. The hydrogen-ion
F I G . I (plate). Photomicrographs of sections of the stellate ganglion of the cat incubated for
AChE activity, showing the effect of formalin fixation, a, heavily stained nerve-cells;
b, moderately stained nerve-cells; c, unstained nerve-cells. Frozen sections, 20 ft.. p H 5 3 .
A, incubated for 45 min without fixation.
B, incubated for 1 h 30 min after fixation in neutral formalin for 3 h.
C, incubated for 1 h 30 min after fixation in neutral formalin for 24 h.
...V
>
i
B
L
v.
, 100//'
FIG. I
N. T. NAIK
FIG.
2
N. T. NAIK
Naik—Koelle's method for cholinesterase
95
concentration was varied between pH 7-0 and 4-2. It was found that at pH y o
the enzyme reaction was fast, and reasonably sharp localization of the copper
sulphide precipitate was obtained after short periods of incubation. However,
as the incubation period was progressively extended the staim diffused from the
perikarya of the nerve-cells into the nuclei and surrounding tissues, and the
enzyme could not be localized cytologically (fig. 2, A). At pH 6-o the reaction
was slower but the stain remained better localized (fig. 2, B). Some diffusion
took place, however, especially into the nuclei of the satellite cells after longer
incubation. Further reduction of the pH to 5-0 or 5-3 did not affect the pattern
of enzyme distribution, and cytological localization was further improved
(fig. 1, B; also fig. 3, B). Under these conditions it was possible to carry out
incubation for a long period without much diffusion artifact (see fig. 4, A to c).
At pH 4-0 or 4-2 the increased acidity inhibited the reaction, especially that
of AChE, in many tissues. The ganglia of the sympathetic trunk of the cat
incubated at pH 4-2 showed a negative reaction in nerve-cells which did in
fact contain AChE, and showed a positive reaction only in tissues surrounding
the nerve-cells, where ChE was mainly present (compare fig. 2, c with figs.
1, B; 2, B). Sections incubated with butyryl substrate at pH 4-2 showed a
distribution of ChE similar to that in sections incubated at higher pH
(compare fig. 2, D with fig. 3, c). Sections incubated with acetyl substrate plus
ethopropazine hydrochloride as an inhibitor of ChE on the contrary remained
blank or showed very little reaction even after long periods of incubation.
These experiments demonstrate that in the sympathetic ganglia of the cat
almost complete inactivation of AChE and slight inactivation of ChE takes
place at pH 4-2. A similar finding was reported by Lewis (1961) in the
cholinesterase of rat brain.
Sections of human digital skin incubated at pH 5-3 or higher showed a
strong positive cholinesterase reaction in various sites: Meissner's corpuscles
and Pacinian corpuscles showed ChE, while the adventitia of the sweat-glands
and blood-vessels contained AChE. When the skin was incubated at pH 4-2
it failed to show any enzyme reaction in Meissner's corpuscles (arrows in fig.
2, F; compare with arrows in fig. 2, E) or in the adventitia of the sweat-glands
or blood-vessels. But Pacinian corpuscles still showed a positive ChE reaction.
FIG. 2 (plate). Photomicrographs of sections incubated for cholinesterase activity, showing
the effect of variations in pH. Frozen sections, 15 ji.
A, stellate ganglion of the cat, incubated with acetyl thiocholine iodide plus ethopropazine
hydrochloride for 1 h. pH 70.
B, stellate ganglion of the cat, incubated with acetyl thiocholine idodide plus ethopropazine
for 1 h 5 min. pH 6-o.
C, stellate ganglion of the cat, incubated with acetyl thiocholine iodide for zz h. pH 42.
D, stellate ganglion of the cat incubated with butyryl thiocholine iodide for 22 h. pH 42.
E, human digital skin incubated with acetyl thiocholine iodide for 1 h 20 min. pH 5-3.
Meissner's corpuscles (arrows) give a strong positive cholinesterase reaction. Male, 42 years.
Frozen sections, 20 ft.
F, human digital skin incubated with acetyl thiocholine iodide for 22 h. pH 42. Meissner's
corpuscles (arrows) give negative cholinesterase reaction. (Compare with fig. 2, E.) Male, 19
years. Frozen sections, 20 /i.
96
Naik—Koelle's method for cholinesterase
These experiments show that the hydrogen-ion concentration of the incubation medium is an important factor. Incubation at pH 6-o or higher produces
considerable diffusion artifact, whereas incubation at pH 4-2 or lower usually
fails to demonstrate AChE and, in certain situations, ChE as well. These
findings are in general agreement with the biochemical observations of
Augustinsson (1948), who stated that the AChE of horse erythrocytes was
destroyed at pH 4-5 and the ChE of horse serum at pH 2-0. The best cytological and histochemical results were obtained at between pH 5 and pH 6 in all
tissues examined. For this reason pH 5-3 was chosen in this research for
routine purposes, although a higher pH (6-o) was also used for comparison.
The specificity of substrates and inhibitors
The substrates, acetylthiocholine iodide and butyrylthiocholine iodide, used
in Koelle's histochemical method, are rather specific for cholinesterases.
Occasionally they may be hydrolysed by some simple esterases, but these can
be identified by the use of eserine controls (Easson and Stedman, 1937;
Richter and Croft, 1942; Mendel and Rudney, 1943 a, b). Acetyl substrate is
hydrolysed by both AChE and ChE (Koelle, 1950, 1951; Holmstedt, 1957a).
Therefore, the positive reaction obtained by this substrate shows the total
cholinesterase (fig. 3, A). In order to differentiate between the two enzymes
selective inhibitors are used.
Koelle (1950, 1951) recommended DFP as an inhibitor of ChE. However,
it is not possible to select a concentration of this compound that gives complete
inhibition of ChE without affecting AChE (Holmstedt, 1957a). Besides, DFP
is inconvenient to handle and highly toxic; the prepared solutions are unstable.
Instead of DFP, Holmstedt (1957 a, b) recommended Mipafox, which has a
high specificity and is easily handled (Holmstedt and Sjoqvist, 1961). The
inhibitor sold under the name of Mipafox in this country (Light & Co.) has a
slightly different composition (Holmstedt and Sjoqvist, personal communication), and in our hands did not produce complete inhibition of ChE. Similar
experience is reported by Pepler and Pearse (1957) in their trials of this compound on the rat-brain cholinesterase. Bayliss and Todrick (1956) have
recommended the use of ethopropazine methosulphate as inhibitor of ChE.
According to Pearse (i960) this compound may produce precipitation in the
substrate solution. In the present work ethopropazine hydrochloride was
used instead of ethopropazine methosulphate. It does not affect the solutions
and its inhibitory action is reliable. Fig. 3, B shows the stellate ganglion of the
FIG. 3 (plate). Photomicrographs of sections of the stellate ganglion of the cat, fixed in
neutral formalin at 40 C and incubated for cholinesterase activity at pH 53. The plate demonstrates the specificity of substrates and selective inhibitors. Frozen sections, 15^.
A, incubated with acetylthiocholine iodide for 1 h 45 min to show the total cholinesterase.
B, incubated with acetyl thiocholine iodide plus ethopropazine hydrochloride for 1 h 20 min
to show AChE.
c, incubated with butyrylthiocholine for 1 h 50 min to show ChE.
D, incubated with acetyl thiocholine iodide plus B.W. 284 for 1 h 20 min to show ChE.
Fie. 3
N. T. NAIK
Naik—Koelle's method for cholinesterase
97
cat incubated with acetyl substrate plus ethopropazine hydrochloride as an inhibitor of ChE. AChE is localized mainly in certain nerve-cells and to a small
degree in other tissue elements (cf. fig. 3, A).
As an inhibitor of AChE, B.W. 284 has been found effective (Denz, 1953;
Koelle, 1955; Bayliss and Todrick, 1956; Holmstedt, 1957a). This was found
reliable in the present work. Fig. 3, D shows the stellate ganglion of the cat
incubated with acetyl substrate plus B.W. 284 as an inhibitor of AChE. As a
result, the distribution of ChE is shown: all nerve-cells in A and B (fig. 3) give
a negative reaction, but the tissue elements surrounding the nerve-cells
are strongly positive (compare with fig. 3, c). When A, B, and c (fig. 3) are
compared, the effect of selective inhibitors is evident, B and c show each
enzyme separately whereas A shows the combined picture, namely the total
cholinesterase.
In comparison with acetyl substrate, butyryl substrate may be considered as
specific for ChE for all practical purposes and is being used as such without
inhibitors (Koelle, 1950, 1951; Couteaux and Taxi, 1952; Holmstedt, 1957^)The section in fig. 3, c has been incubated with butyryl substrate without
inhibitor. When the incubation is prolonged, however, some traces of stain
corresponding to the distribution of AChE can be detected.
As a result of these experiments, it can be seen that AChE can be demonstrated by means of incubation of sections with acetyl substrate plus ethopropazine hydrochloride (fig. 3, B). If ChE is not present in the tissues
concerned, acetyl substrate will show AChE without the use of the inhibitor. ChE can be demonstrated either by acetyl substrate plus B.W. 284 or by
butyryl substrate with or without B.W. 284 (fig. 3, c, D). It will be noted,
however, that butyryl substrate shows ChE in a shorter period of incubation
and therefore should be preferred.
The specificity of ethopropazine hydrochloride and B.W. 284 as selective
inhibitors was further confirmed by incubating tissues containing both
enzymes with acetyl substrate to which both inhibitors were added. Such
sections remained blank, and this demonstrates that both enzymes present
were completely inhibited by B.W. 284 and ethopropazine hydrochloride.
Visualization methods of the results
Most histochemists visualize the results of the histochemical cholinesterase
reaction by treatment of the sections with diluted ammonium sulphide
solution after incubation. In this way, the deposit of the colourless copper
thiocholine crystals is replaced by the brown copper sulphide crystals.
Holmstedt (1957&), however, recommends the study of the results directly by
the use of phase-contrast microscopy without treating with dilute ammonium
sulphide solution.
A series of experiments was carried out to assess the advantages and disadvantages of the two methods. Sections incubated for cholinesterase activity
were mounted in an aqueous mounting medium without treatment with
ammonium sulphide solution, and the localization of copper thiocholine
98
Naik—Koelle's method for cholinesterase
crystals was studied by phase-contrast microscopy. Subsequently, some of
these sections were removed from the slides and immersed in ammonium
sulphide solution; after mounting they were re-examined by ordinary transmitted illumination. On comparing the results obtained with and without the
use of ammonium sulphide, it was found that the localization of the crystals
was the same in both cases. It was, however, difficult and sometimes impossible to identify the deposit of copper thiocholine in sites of low concentration.
On the contrary, the stained sections showed clear localization even at sites of
very low concentration without difficulty. It was decided, therefore, not to use
phase-contrast microscopy in this work.
A further experiment was carried out with ammonium sulphide visualization to determine the effect of the time factor in its application. A number of
sections was simultaneously placed in the ammonium sulphide solution and
taken out one by one at intervals from 15 sec to 5 min. No difference in
results was obtained. Similarly, no difference was observed when the concentration of ammonium sulphide solution was varied. The stock ammonium
sulphide solution has a clear yellow appearance and it can be stored in the
refrigerator for several months. When, however, the solution becomes
opaque it should not be used. If incubated sections were placed in such
a solution the copper thiocholine was replaced by copper sulphide, but the
latter was then dissolved, giving false negative results.
The effect of progressive increase of incubation time on cholinesterase reaction
When a standard method is used the optimum time required for demonstration of cholinesterase activity depends upon the concentration of the
enzyme and also upon intrinsic factors including species differences. For that
reason no standard incubation time can be laid down, but it has to be determined empirically.
The effect of progressive increase of incubation time was studied in the
stellate ganglion of the rat. The nerve-cells of this ganglion contain enzymes
in different concentration. In the early stages of incubation only some nervecells give a positive reaction (fig. 4, A) ; later all cells show a positive reaction
(fig. 4, B, c). If A and c (fig. 4) are compared it becomes clear that A shows an
incomplete picture and c shows an excessive reaction, masking the true distribution of cholinesterase. In both cases misinterpretation of the findings
may result. Hence the optimum incubation time to demonstrate AChE in this
case is in the region of 1 to 2 h (fig. 4, B). Similarly, in the tissues supplied by
the autonomic nerves, variations in incubation time produce equally marked
differences. For instance, in the parotid gland of the guinea-pig, after a short
FIG. 4 (plate). Photomicrographs of the sections of the stellate ganglion of the rat, fixed in
neutral formalin for 3 h at 4° C and incubated for AChE activity at pH 5-3 to show the effect of
variation in incubation time. Frozen sections, 25 /x.
A, incubated for 25 min.
B, incubated for 1 h 10 min.
C, incubated for 4 h.
FIG.
4
N. T. NAIK
Naik—Koelle's method for cholinesterase
99
period of incubation, only the nerve-bundles give strong positive cholinesterase reaction. Later the finer branches and also the adventitia surrounding
the acini of the gland show a definite positive reaction as well.
The experiments show that it is not advisable to prejudge the incubation
time. For each newly studied tissue a range of incubation time should be used.
This is more easily done when incubation is carried out by the floating section
method.
The effect of cold storage of the tissues on cholinesterase reaction
The routine procedure for cholinesterase reaction requires continuous attention for a period of approximately 5 to 10 h, and cold storage of the tissues
enables one to time the beginning of the process. This is especially important
in the case of human material obtained during operations, sometimes late in
the day.
The effect of cold storage on cholinesterase reaction was studied on a
variety of tissues of various species. The tissues were closely wrapped in polythene sheets to avoid evaporation and stored in the refrigerator at 4° C for
periods up to 70 h. The tissues were then processed in a uniform manner and
compared with tissues not subjected to cold storage.
It was found that short periods of cold storage had no appreciable effect on
the cholinesterase reaction. Even after storage for 70 h the pattern of distribution and localization of the stain was not substantially affected as compared
with specimens incubated without cold storage. However, the reaction was
slower and required longer incubation time. These findings are in harmony
with those of Glick (1938), Augustinsson (1948), Koelle (1950, 1951), Gomori
(1952), and Gerebtzoff(i959) and it was considered that tissues obtained in the
afternoon could safely be stored overnight at 40 C if the need arose.
The author wishes to thank Dr. N. Cauna for his guidance and Professor
R. J. Scothorne, Head of the Department of Anatomy, for his criticism of the
manuscript and for the facilities offered. The author also wishes to thank Mr.
C. J. Duncan and the staff of the Department of Photography for their help in
photomicrography and Mrs. M. Young for typing the manuscript. This
publication is part of the work done in partial fulfilment of requirements for
the degree of Ph.D. in the University of Durham.
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