367
A silver-aldehyde reaction for studies of chromosome
ultrastructure
By JOHN H. D. BRYAN and BILL R. BRINKLEY
(From the Department of Zoology and Entomology, Iowa State University, Ames,
Iowa, U.S.A.)
With 3 plates (figs. 1 to 3)
Summary
A method is presented for the visualization of structures containing deoxyribonucleic
acid at the electron microscope level. This is achieved by the substitution of an ammoniacal silver reagent for leucobasic fuchsin in a Feulgen-like procedure. Tissue aldehydes
reduce this reagent to metallic silver, which is deposited at the reactive sites in the
form of fine particles. The particles range in size from about 4 mfi downwards to
below the resolving power of the microscope used. 3 fixatives have been investigated
(buffered osmium tetroxide, formalin, glutaraldehyde) and the special problems
encountered with each fixative are discussed. Detailed considerations regarding the
preparation and handling of this somewhat unstable staining solution have been
described previously (Bryan, 1964).
Results obtained from an application of this procedure in an investigation of the
ultrastructure of polytene chromosomes are also reported in detail. T h e fibrillar
nature of these chromosomes is depicted more clearly following staining than in
preparations resulting from the employment of the more conventional methods in
current use. The fibrils are clearly delineated by silver particles and may be grouped
into 2 classes. Thus, the finest fibrils are of about 4 mju diameter and they are usually
associated in pairs to form units of about 10 m/x diameter. Although there is a predominant orientation of the fibrils composing individual bands, the nature of the
association of the 10 m/i units which results in this is not clear. Evidence for the
occurrence of similar fibrils in chromatin of interphase nuclei of mammalian origin is
also presented.
Taken together, the results of the various experiments suggest that this silver
procedure may be used to advantage in a wide variety of investigations concerning the
ultrastructure of chromosomes and chromatin.
Introduction
A T T E M P T S to elucidate the ultrastructure of nuclei and chromosomes with
the electron microscope have not met with the same success as has attended
investigations of other cell structures. The most significant progress has,
perhaps, been made in regard to polytene chromosomes from the salivary
glands of various species of diptera, lampbrush chromosomes of oocytes from
a few species of amphibia, and early meiotic prophase chromosomes of
certain plants (see Ris, 1957; Gall, 1958; Kaufmann and others, i960, for
reviews). Hay and Revel (1963) have presented evidence which strongly
supports the view that the dispersed chromosomes in interphase nuclei are
composed of microfibrils of similar dimensions to those reported in certain
[Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 367-374, 1964.]
368
Bryan and Brinkley—Silver
technique
of the studies reviewed above. By and large, however, less information of
structural significance has been obtained with respect to the more typical
chromosomes of somatic tissues.
It has long been evident that further progress in this area would require
the development of new techniques for the selective introduction of electron
contrast in chromosomal structures. The standard procedures, which
employ osmium tetroxide both as a fixative and electron stain, have several
disadvantages, among which is the fact that the osmium tends to obscure fine
structural details. Such images are difficult to interpret and are not very
satisfactory for studies at the high levels of resolution demanded in such work.
As a means of overcoming such difficulties much interest has been centred
around the development of methods specific for certain structural components
of chromosomes. Thus, uranyl acetate staining for structures containing
nucleic acid has been introduced by Huxley and Zubay (1961), and Watson
and Aldridge (1961) have advocated the use of indium chloride for the same
purpose. Albersheim and Killias (1963) have recently introduced a method
employing a mixture containing bismuth as a staining reagent for nucleic
acids.
The use of silver reagents in a Feulgen-like procedure has also attracted
attention, on the premise that such procedures should precisely localize
strands containing DNA. Staining routines have been described by Bretschneider (1949), van Winkle and others (1953), Bradfield (1954), and
Jurand, Deutsch, and Dunn (1959). However, these methods do not appear
to have borne out the theoretical expectations and have not received widespread attention or application. Furthermore, these procedures were applied,
in most cases, with little mention of possible side-reactions which tend to
detract from the specificity of the staining and, therefore, confuse the results.
It occurred to one of us that a new investigation of this class of reagents
might be rewarding and might lead to the development of a suitable electron
microscope technique for the localization of DNA, provided particular attention was paid to these largely unexplored variables. Our own studies were,
therefore, conducted along such lines and culminated in the development of
a method suitable for use at the light microscope level (Bryan, 1964). The
present report describes the adaptation of this procedure for use with the
electron microscope and presents the results obtained from its application in
a study of the ultrastructure of polytene chromosomes. Some of our preliminary findings have been reported previously (Bryan and Brinkley, 1963);
however the procedure reported here, which incorporates further refinements
in technique, is described in detail for the first time.
Materials and methods
To facilitate direct comparison of our results with those already in the
literature, our initial studies were carried out primarily on material fixed in
buffered osmium tetroxide. Additional fixatives have been used as described
below.
for chromosome ultrastructure
369
Salivary glands of Chironomus decorus Johannsen and Drosophila melanogaster Meigen were dissected out in insect Ringer and fixed for one h at room
temperature in either (1) 1% osmium tetroxide buffered at pH 7-4 with
veronal-acetate buffer after the method of Palade (1952) (optimum fixation
was obtained with an osmium/ buffer ratio of 3:1), (2) 10% formalin, or (3)
2-4% glutaraldehyde. The latter 2 fixatives were also buffered at pH 7-4
with veronal-acetate buffer. Following fixation, tissues were dehydrated in a
graded series of ethanol and embedded in a mixture of 3 parts of N-butyl to
2 parts ethyl methacrylate and containing 1 % w/v benzoyl peroxide as catalyst.
Polymerization was carried out for 24 h at 6o° C. After polymerization, the
blocks were trimmed to expose a tissue face of about 1 mm. square.
In an attempt to reduce the osmium content, some of the trimmed
blocks were immersed in either a 1:1 solution of 15% hydrogen peroxide and
o-6 N HC1 (after Marinozzi, 1961), or in un-acidified hydrogen peroxide
of the same final concentration. We deem this step to be necessary since
preliminary studies have indicated that bound osmium detracts from the
specificity of the silver reaction. Blocks were exposed to either reagent at
room temperature for periods of from 20 min to one h. After peroxide
treatment, blocks were washed with 1 % oxalic acid followed by distilled water.
Hydrolysis was carried out by incubating blocks (after the method of
Drum, 1963), or sections, in 1 N HC1 for 20 min at 6o° C. However,
hydrolysis of sections usually gave rise to pronounced tissue damage and is
not recommended. After hydrolysis, blocks were washed until free of
chloride.
The preparation of the silver reagent, together with necessary precautions
concerning its handling and use, has been described in detail in a previous
publication (Bryan, 1964). Briefly, it is prepared by titrating dilute silver
nitrate with dilute ammonium hydroxide until the precipitate which forms is
just redissolved. For electron microscopy, it is also advisable to filter the
solution through Whatman No. 1 paper. The reagent should be made up
immediately before use; it should be discarded promptly after use (see
Bryan (1964) for further details concerning these remarks). Staining was
accomplished by floating grids, tissue side down, on the surface of the
solution in a covered dish. After cooling to room temperature, grids were
washed in 3 changes of 2% ammonium hydroxide for a total time of 10 min
(if this step is omitted deposits of silver oxide are found to be scattered over
the sections). Finally, grids were rinsed in distilled water and blotted dry.
The sections were then overlayed with a methacrylate film (Roth, 1961) and
examined with an RCA-EMU3F (30 p objective aperture; 50 KV).
It was necessary to depart from the above procedure in those experiments
involving the use of deoxyribonuclease (DNase). In these cases digestion with
the enzyme was carried out after fixation but before embedding. After
fixation, tissues were washed in several changes of buffer and then in 0-003 M
MgSO4 adjusted to the same pH. Following this they were incubated for
6 h at 370 C in a solution of DNase (5 mg/ml) in 0-003 M MgSO 4 adjusted to
37°
Bryan and Brinkley—Silver
technique
pH 7-0 with veronal-acetate buffer. This solution was changed 3 times during
the 6 h period. Controls were treated in the same manner and incubated in a
similar solution without enzyme. After washing in distilled water the tissues
were then processed as described above.
Results
Sections of polytene chromosomes of Chironomus decorus are shown in
fig. 1. Micrographs A and c are survey shots at low magnification, while
B and D represent enlargements of the regions indicated on A and c respectively.
In the unstained control preparation it may be seen that a fibrillar constitution
is suggested but that fine details are obscured by the high density imparted
by the bound osmium (fig. 1, A, B). The fibrillar nature of the chromosome is
much more clearly displayed after staining with silver (fig. 1, c, D). At high
magnification, 2 classes of fibrils are usually seen (fig. 1, D). Furthermore,
these fibrils are delineated by linear arrays of silver particles. The finest
fibrils showing silver particles are approximately 4 n\fM in diameter; frequently
they appear to occur in pairs. In these cases the component fibrils may be
loosely twisted about one another or, for short distances, they may be arrayed
in a parallel fashion. These paired fibrils constitute an apparent unit about
10 m/jL in diameter. The silver particles themselves range in size from about
4 m/t diameter to dimensions considered to be below the resolving power of
the microscope. Similar results are obtained with Drosophila chromosomes
(fig. 2, A, B) and thus these micrographs may also serve to illustrate the reproducibility of the technique.
Micrographs are also presented which illustrate various aspects bearing on
the specificity of the reaction for the demonstration of DNA (fig. 2, c, D).
A comparison of figs, i, D and 2, c will indicate the effect of hydrolysis.
Figure 2, c has been subjected to the same procedure except for the omission
of the hydrolysis step. As can be seen, the chromatin is free of silver particles
in the unhydrolysed control. For further evidence of specificity, sections
were stained with the silver reagent following prior incubation of the tissue in
solutions of DNase (fig. 2, D). Comparison of this micrograph with fig. 1, D
will indicate that staining of the fibrils is not evident following removal of
DNA.
In order to demonstrate the versatility of the staining procedure fixatives
other than osmium tetroxide were also investigated. Fixation in 10%
formalin gives rise to much coarser-looking fibrillar images (fig. 2, E, F).
FIG. 1 (plate). Portions of salivary gland chromosome of Chironomus decorus; buffered
osmium fixation.
A, an unstained preparation.
B, a portion of A at higher magnification showing the electron-dense nature of the bands
characteristic of osmium fixation.
c, a preparation stained with the silver reagent.
D, a portion of c at high magnification showing numerous fibrils delineated by particles of
silver.
FIG. I
J. H. D. BRYAN and B. R. BRINKLEY
FIG.
2
J. H. D. BRYAN and B. R. BRINKLEY
for chromosome ultrastructure
371
However, while it may not be clearly evident from the micrographs reproduced here, the association of fibrils is sometimes more apparent than in
the osmium-fixed material. In sections of formalin-fixed glands, the silver
particles appear to be more densely arrayed along the fibrils. The dimensions
of the fibrils are closely similar to those displayed in the osmium-fixed
material and the associated silver particles are also of the same order of size.
Again, unhydrolysed controls showed no staining of the fibrils. Studies
involving the use of formalin-containing fixatives are being continued and the
results will be reported separately.
We have also carried out some preliminary studies with glutaraldehydefixed tissues, even though the use of this recently introduced fixative is
contra-indicated by its chemical composition. Glutaraldehyde is a dialdehyde and therefore introduces extra aldehyde groups which can react
with Schiff's reagent. However, since it has other desirable qualities as a
fixative for electron microscopy we have tried to use it in combination with
an aldehyde-blocking agent (dimedone). Although dimedone gave fairly
acceptable results at the light microscope level when used in conjunction with
Feulgen staining, it could not be used for electron microscope purposes
since it introduced serious tissue damage. Therefore, glutaraldehyde is not
recommended as a fixative for the silver-aldehyde procedure reported here.
Our studies are being extended to include tissue samples from a wider
variety of sources. As an example of the staining of interphase nuclei,
micrographs of portions of early spermatids of the mouse are shown in the
survey micrograph (fig. 3, A). The fibrillar nature of the chromatin is more
evident in the enlargements (fig. 3, B, c). Note that silver particles are arrayed
along these fibrils just as in the case of the polytene chromosomes (fig. 3, c).
However, owing to the superimposition of the individual elements, the
fibrillar nature may not be as sharply displayed.
Discussion
Successful development of a specific staining technique often depends
upon a knowledge both of the specificity of the reagent under a variety of
conditions and the nature of possible side-reactions which may detract from
this specificity. While such problems are of direct concern at the light microscope level, they become of even greater importance in investigations involving electron microscopy.
FIG. 2 (plate). A, salivary chromosome of Drosophila melanogaster stained with the silver
reagent; buffered osmium fixation.
B, a portion of A at high magnification showing 2 classes of fibrils. The uppermost arrow
indicates a 10 mj* fibril, and the pairs of arrows indicate fibrils of about 4 m^t diameter.
C to F, salivary chromosomes of Chironomus.
C, an unhydrolysed control preparation; buffered osmium fixation.
D, a preparation fixed in buffered osmium and stained with silver following incubation of
the gland in deoxyribonuclease (5 mg/ml). Note the lack of silver staining of the fibrils.
E, F, buffered formalin fixation and silver staining. Note the coarser appearance of the
fibrils.
372
Bryan and Brinkley—Silver
technique
The results of our experiments, as typified by figs. 2, c and 2, D, demonstrate that the silver procedure described here is specific for tissue aldehydes.
We should point out, however, that removal of bound osmium is necessary;
in sections stained without prior removal of osmium some staining is found in
the unhydrolysed controls. This result would appear to be due to the direct
interaction of osmium with the ammoniacal silver complex. Thus, many
of the membrane systems, which are principally lipoprotein in nature and
strongly bind the heavy metal, also have silver particles associated with them.
In order to remove as much osmium as possible from the tissues, blocks were
treated with hydrogen peroxide as advocated by Marinozzi (1961). This
method calls for treatment of the tissue with hydrogen peroxide acidified with
HCI (final concentration about 0-3 N) and it might be anticipated that this
could lead to incipient hydrolysis of DNA. Evidence in support of this has
been obtained. Thus, if after treatment with the Marinozzi reagent sections
are stained without hydrolysis (unhydrolysed controls) some silver particles
are found in chromatin-containing areas of the tissue. It is obvious that this
finding is of little consequence in routine applications of the full staining
procedure. However, in a rigorous examination of the questions relating to
specificity it may give rise to some concern. We have, therefore, attempted to
remove bound osmium by treatment with unacidified hydrogen peroxide. In
these cases we find a somewhat less satisfactory preservation of the cytoplasmic
components but no incipient staining of the unhydrolysed controls. Undoubtedly, the nature of this interaction between osmium-fixed tissue and
hydrogen peroxide is complex. As yet we cannot adequately account for the
fact that treatment with the acidified reagent yields a more satisfactory endproduct than does the employment of hydrogen peroxide alone.
As pointed out in the results section, the fundamental structural component
would appear to be a fibril of about 10 m/x diameter which is composed of 2
finer fibrils each of about 4 m/x diameter. This interpretation is in agreement
with the conclusions of other workers (see, for example, Kaufmann and
others, i960; Ris, 1961; Jacob and Sirlin, 1963). Although it may not be
clearly evident in the reproduced photographs, close inspection of the
original micrographs has revealed another aspect of these fibrils which may be
of potential significance. It appears that the linear pattern of silver particles
may be identical in each of the pair of fibrils contained in a 10 mju. unit. If this
observation may be taken at its face value it would suggest that, at least over
short linear sequences, the distribution of DNA is identical in each member
of the pair. On a genetic basis such a conclusion is not altogether unexpected.
However, the exact significance of this observation must await the completion
of further detailed studies. There is also the possibility that these patterns
FIG. 3 (plate). Mouse testis; buffered osmium fixation and silver staining.
A, a low magnification micrograph showing portions of early spermatids.
B, c, portions of spermatid nuclei at higher magnifications showing the fibrillar nature of
interphase chromatin.
#
• I
*
•
•
•
•L-
>
i
FIG.
3
J. H. D. BRYAN and B. R. BRINKLEY
for chromosome ultrastructure
373
may be, in some way, related to the beaded appearance of the 4 myx fibrils
noted by Jacob and Sirlin (1963). We also have observed fibrils of similar
appearance in certain of our unstained preparations.
Unfortunately, the present studies have not provided much information
regarding the probable nature of chromosomal constitution at higher levels of
organization. However, the fact that fibrils can be traced for only relatively
short distances would suggest that they may be coiled in a complex manner;
similar conclusions have been reported by Goodman and Spiro (1962), Jacob
and Sirlin (1963), and others. At still higher levels of organization there is a
suggestion that there is a predominant orientation which prevails in each band
region (fig. 1, c).
Because of its pertinence to genetic and structural studies, much attention
has been focused on the question of whether or not interband regions contain
DNA. The results of recently reported studies dealing with this problem
tend to be mutually contradictory. Thus Steffensen (1963) has concluded
on the basis of his investigations involving tritiated thymidine that DNA is
probably located entirely within the bands. Swift (1962), on the other hand,
has reached the opposite conclusion as a result of his photometric studies
involving the use of the highly sensitive Azure A procedure for visualization of
DNA. Our own studies were not specifically directed to this question so our
observations of interband structure, while they may be suggestive, do not
afford unequivocal evidence regarding the presence or absence of DNA.
Nevertheless, they do have some bearing on this question and so are worthy of
mention. Fibrils were often seen traversing the less dense regions separating
successive bands. These fibrils appeared to be free of associated silver
particles except, perhaps, in areas immediately adjacent to the parent bands.
These observations suggest that fibrils in interband regions may differ from
those in the bands, at least on a quantitative basis. Whether or not they are
qualitatively different must await the outcome of further studies specifically
directed to this question.
As mentioned earlier, there is evidence to support the view that fibrils of
10 mju. diameter may also constitute the basic structural unit of chromosomes
other than those of diptera. On this basis it might be anticipated that similar
fibrils should also be evident in thin sections of interphase nuclei. Strong
support for this view is supplied by the studies of Yasuzumi (i960), Claude
(1961), Hay and Revel (1963), and others. The work reported in this paper
offers further support for the fibrillar nature of interphase chromatin of
mammalian cells (fig. 3, A, B, c). Again, association of silver particles with the
fibrils indicate that they contain DNA.
It should be evident from this discussion that the silver procedure here
described has strong potentialities for application in studies of chromosome
structure at the electron microscope level. Furthermore, the selective nature
of the staining procedure should allow an alternative approach to several
problems of fundamental importance which have so far proven rather refractory to the more conventional methods in current use.
374
Bryan and Brinkley—Silver
technique
These investigations have been supported by grants from the USPHS
(CA-05591, 02, 03). Assistance is acknowledged from R. A. Jenkins and Dr.
L. E. Roth (Department of Biochemistry and Biophysics). The electron
microscope used was that provided by a USPHS research grant (CA-5581) to
Dr. Roth.
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