staining differences in cell nuclei · 2006. 5. 26. · lison—staining differences in cell nuclei...

227

Staining Differences in Cell Nuclei

By LUCIEN LISON(Front the Department of Histology and Embryology, Faculty of Medicine of Ribeirao Preto,

State 0/ Sao Paulo, Brazil)

With one plate (fig. i)

SUMMARY

If the parenchymal cells of the liver of the rat, or certain other cells, are stainedwith a modified Mallory stain or with iron haematoxylin, the nuclei are stained intwo different ways. This effect may be observed after various fixatives.

Direct microscopical observation shows that when part of a nucleus is cut away bythe microtome-knife, the part of it remaining in the section stains differently from anintact nucleus. The probability of a nucleus being wholly or only partly contained ina section of a particular thickness can be calculated.

Counts made in sections of various thicknesses show that the proportion of nucleistained in the different ways is consistent with the calculated probability.

It is suggested that the nuclear membrane in the fixed state presents a relatively lowpermeability.

SHEININ and Davenport (1931) observed in liver cells two types of nucleithat may be distinguished by differential staining. This result was

obtained after fixation of the tissue in a complex mixture containing potassiumdichromate, sodium sulphate, mercuric sulphate, sulphuric acid, and aceticacid, and staining with Mallory's triple connective tissue stain. Although noother difference could be found between the cells with nuclei differentiallystained, Sheinin and Davenport postulated the existence of varying physio-logical states of the cells.

Recently, Parr, Mossberg, Rosenzweig, Breslar, and Clark (1953), usinga technique derived from Mallory's stain, were able to confirm these results.Furthermore, they observed that in rats starved or on protein-free diet, thereis a shift in the relative proportion of the two types of nuclei. They suggestedunknown histochemical differences between the two types of nuclei; however,no definitive explanation was given for the modification caused by the changesin diet.

Long ago, I chanced to observe striking differences between nuclei intissues stained with Heidenhain's iron haematoxylin. Some nuclei, generallyfew in number, strongly resist the destaining action of the iron alum andremain heavily stained, while others are nearly destained. Although nothorough search in the literature was made, it appeared that such 'dark*nuclei in iron haematoxylin preparations have occasionally been observedby numerous authors. An attempt to trace an explanation for this peculiarphenomenon was made. However, no positive evidence concerning the[Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp. 227-237, 1955.]

228 Lison—Staining Differences in Cell Nuclei

mechanism of the production of 'dark' nuclei could be found, and for thisreason the investigation was discontinued. It appeared to the author thatthe 'light' nuclei described by Parr, Mossberg, Rosenzweig, Breslar, andClark in modified Mallory preparations might correspond to the 'dark'nuclei observed in iron haematoxylin preparations. The observations ofParr and others seemed further to indicate the possibility of increasingexperimentally the proportion of these nuclei. In the present paper an attemptis made to find an explanation for the appearance of differently stainednuclei in tissues.

MATERIAL AND METHODS

The animals used in these experiments were young adult male rats,weighing about 250 g. and fed on a standard complete diet.

For the study of the action of various fixatives on the staining propertiesof the nuclei, fragments of the same liver were fixed for 24 hours in thefollowing fluids: Bouin; Zenker; Susa; Helly; Carnoy; alcohol formalin,90:10; alcohol formalin acetic acid, 85:1015; 10 per cent, formalin in a 0-9 percent, solution of NaCl buffered with M/50 phosphate buffers at pH 3-5 andpH 7-0. All the pieces of tissue were dehydrated and embedded in paraffinin an automatic inclusion apparatus. Sections were cut at 8 /x, 10 /x, or 12 //..In order to eliminate differences due to uneven handling, the sections fromthe nine fragments treated with the different fixatives were mounted on thesame slide.

Two staining methods were used as a routine to differentiate the two typesof nuclei. The first was Mallory's stain as modified by Parr, Mossberg, Rosen-zweig, Breslar, and Clark. A variety of modifications of Mallory and Azanstains was tried, but none proved to be superior to the procedure recom-mended by Parr and his associates, and the latter was adopted. The secondwas Heidenhain's iron haematoxylin, used as follows: mordanting in 4 percent, iron alum for 24 hours at laboratory temperature (26° C ) ; staining inwell-ripened Regaud's haematoxylin; differentiation in 2 per cent, iron alum.For the special purpose of demonstrating the two types of nuclear staining,the time used for the differentiation is not critical. As was mentioned before,the 'dark' nuclei resist destaining so energetically that they appear intenselystained even when the other ones are nearly completely destained. Thecontrast obtained with the latter method is better than with the Mallorytechnique.

For studying the influence of the thickness of the section, sections of thesame block of liver fixed in alcohol formalin acetic acid were cut at 4, 6, 8,10, 12, 14, and 16 JU. and mounted together on the same slide. This was donein order to make the staining as uniform as possible.

Counts of the nuclei were made in the following way: A reticule dividingthe field into twelve parts was placed in the ocular of the microscope.Since, according to Parr and others, nests of 'light' nuclei were more

Lison—Staining Differences in Cell Nuclei 229

numerous near the central veins, precautions were taken to ensure a randomsampling in the liver. Keeping this in view, the nuclei were counted in suc-cessive fields, always in the same direction, beginning at one surface of theliver and continuing vertically to the other surface. Usually 600 to 750 nucleiwere counted. The counts were reduced to percentages of the two types ofnuclei. Upper and lower limits of the confidence-interval for a probability of95 per cent, were calculated from the tables of the confidence-intervals givenby Snedecor (1946) for the binomial distribution, by interpolating the valuesof the table for the number of items in the count. The significance of differ-ences between the proportion of both types has been tested by the x2 test basedon the null hypothesis, using the tables of distribution of the probability of xz

established by Fisher (1950).

RESULTS

Identity of the 'yellow' nuclei in modified Mallory preparations and the 'dark'nuclei in iron haematoxylin preparations

A first series of observations was made to ascertain whether the nucleidifferentially stained by the methods of Sheinin and Davenport and of Parrand others are the same that are differentiated by Heidenhain's iron haema-toxylin.

In the preparations treated with the method of Parr and others, most of thenuclei are stained blue, while others are golden yellow. The inspection of thepreparations gave the impression that these 'yellow' nuclei may be identicalwith the nuclei that remain heavily stained in black in strongly destainediron haematoxylin preparations.

However, since no method was found that would make it possible to stainthe same cells by the two staining methods, one after the other, some doubtremained as to the identity of the nuclei selectively stained. In order to givea valid proof of this identity, a statistical analysis of the results of comparativecounts was made.

It will be shown later that it is possible to modify at will the ratios of bothtypes of nuclei. If the Mallory technique and the iron haematoxylin techniquereveal selectively the same types of nuclei, the ratios of the two types mustbe identical in preparations treated in the same way, but stained by the twomethods. Hence comparative counts must differ only to an extent controlledby the degree of accuracy of the sampling technique. In order to verify thishypothesis, material was selected to cover a wide variation of the relativeproportion of the two types of nuclei (from o per cent, to 43 per cent, 'yellow'nuclei); counts were made on parallel preparations stained, one by the Malloryand the other by the iron haematoxylin technique.

The statistical analysis was made by using the x2 test applied to a test ofindependence in a fourfold table. Table 1 summarizes the results of a seriesof determinations. The nuclei counted in modified Mallory preparations areclassified as 'yellow' (y) or 'blue' (b), those in iron haematoxylin preparations


as 'dark' (d) and 'light' (/). The values of x2 have been calculated from theabreviated formula:

The individual values found for x2 must be compared with the distributionof probability of xz for 1 degree of freedom. From the table of Fisher (1950),the limit of probability P = 5 per cent, for 1 degree of freedom correspondsto the value of x2 = 3 "84- From the inspection of the results it is obviousthat no value found for xz x% superior to this limit. It may be concludedthat the distribution of the cell types is identical with the two types of staining.

This result is reinforced by the combination of all the x2 tests.It is known that the sum of a number of value of x2 follows the distribution

of x2, with the appropriate number of degrees of freedom. Entering the tableof the distribution of x2 with 5 degrees of freedom, the value x2 = 4"Oo8,obtained in pooling the individual values of x2, corresponds to a probabilityP = 0-55, far above the critical limit of P =0-05.

TABLE I

Nuclear counts in preparations stained by Mallory's stain and iron haematoxylin

Prep.no.

ABCDEF

Numberyellow

01 0 0

2362 1 0

233166

Numberblue

5 1 93 2 2

3473 0 0

385

Numberdark

01 0 02 2 92 0 12 9 2

177

Numberlight

6713 1 1

39S3544 0 1

Per cent.yellow

0

16-242-23774373 0 1

Per cent.dark

01 3 0

4 2 433'745'230-6

TOTAL

X2

1-7330-00141-9800-2610-033

4-008

The mere fact that there is such good correspondence between the resultsobtained with the two staining methods demonstrates that the factors in-volved in the staining (concentration of the solution, duration of the stainingand of destaining) are unimportant in determining the relative numbers ofthe two types of nuclei, and that the results are conditioned by factors inherentin the nuclei themselves.

Influence of fixation

The fixative recommended by Sheinin and Davenport is of the chromicacid mercuric type. Parr and others used perfusion with 10 per cent, formalingum acacia, followed by the completion of fixation in 10 per cent, formalin.It appeared interesting to find whether the possibility of differentiating thetwo types of nuclei is related to a particular type of fixation. With the ninefixatives used it was possible to differentiate the nuclei either by the modifiedMallory technique or by the iron haematoxylin technique. However, thecontrast between the two types of nuclei was found to be very different


according to the fixative used, so that for practical purposes some of themappear unsuitable.

With both staining techniques, good contrast was observed in liver blocksfixed in Carnoy's and Bouin's fluids. The best results, however, were obtainedafter fixation in alcohol formalin and in alcohol formalin acetic acid.

Contrast is good in buffered formalin at either pH 3-5 or pH 7-0, but thepreparations suffer from poor general fixation and lack of homogeneity.

Fluids containing mercuric chloride gave greatly inferior results. In Mallorypreparations the staining of the 'yellow' nuclei is good after fixation in theacid mercuric chloride fluids (Zenker, Susa), but the blue staining of the'blue' nuclei is strongly depressed. In Helly, this is reversed. In both casesthe preparations lack contrast. In iron haematoxylin preparations, tolerablecontrast was observed after Susa fixation. The general staining of the nucleiis very poor after Helly fixation, and differences between nuclei can hardlybe seen. The very strong staining of the cytoplasm after Zenker fixationprohibits the study of the nuclei.

In conclusion, the same basic differences between nuclei may be observedafter all the fixatives used. However, some fixing fluids interfere with thegeneral staining properties of the tissues, so that the differential staining ofthe nuclei may be obscured.

Integrity of the nuclear membrane as the factor for the differential stainingof nuclei

The study of sections of different thicknesses mounted on the same slideshowed extraordinary differences between the proportion of the differentlycoloured nuclei. In sections at 4 /x, no 'yellow' or 'dark' nucleus could be seen;in sections at 6 fi, a few were present; in thicker sections, their numberincreased rapidly with increasing thickness. This led me to try to find outin what position in the section the nuclei are situated which are differentiatedby the staining. Parr, Mossberg, Rosenzweig, Breslar, and Clark observedthat differently coloured nuclei may lie at the same depth in the section.This is undoubtedly true. However, careful inspection of the preparationsrevealed the following fact: those nuclei that stain 'yellow' in Mallory pre-parations or 'dark' with iron haematoxylin are those that are completelyincluded in the section, with the nuclear membrane intact. When the nuclearmembrane is divided by the razor, so that a part of the nucleus is cut away,the nucleus stains 'blue' in Mallory preparations, or 'light' in iron haema-toxylin preparations.

Fig. 1 shows three untouched photomicrographs of the same field takenat different levels, under oil-immersion lens, in a very thick (16 fx) section ofliver stained with iron haematoxylin. In the upper level of the section(fig. 1, A), four nuclei of the 'light' type are focused. Microscopic examinationshowed that none remained untouched by the razor. The 'dark nuclei presentin deeper layers of the section are seen as spots with vague outlines. In B,taken in the mid-part of the section, ten 'dark' nuclei may be seen. Only one


'light' nucleus is present, which is actually the upper part of an incomplete,more deeply situated nucleus. In c, taken at the deepest level of the section,the 'dark' nuclei are out of focus; three 'light' nuclei are visible, their mem-branes having been partly cut away. These photographs show that only intactnuclei are able to stain 'dark'.

This circumstance explains the fact that 'dark' or 'yellow' nuclei are muchmore numerous in thick sections than in thin ones. The probability that anucleus will be intact increases with increasing thickness of section. For a

FIG. 2. In a section of thickness t, a nucleus of diameter d is contained in the section (left inthe figure) if its centre occupies a position between A and B; this corresponds to a distancet+d. The same nucleus (right in the figure) is contained entirely in the section if its centre

is located between C and £>; this corresponds to a distance t — d.

given diameter of the nucleus this probability may be calculated. If t is thethickness of the section and d the diameter of the nucleus, the centre of anucleus which is included (partially or entirely) in the section may occupy allthe positions along a line whose length is t + d (see fig. 2). This nucleus willbe included entirely in the section if its centre falls on that part of the linewhich is marked C—D and whose length is t-d. Hence the probability p of anucleus being included entirely in the section is:

t—d , s( )

FIG. I (plate). A, liver of rat stained with Heidenhain's iron haematoxylin. Section 16/ithick. The focus is on the upper plane of the section. The nuclei located near the surface ofthe section are partly cut and are stained 'light'. The nuclei lying in deeper layers are out offocus and are seen as ill-defined dark spots.

B, same preparation as A, but the focus is on the central part of the section. In this layer thenuclear membranes are untouched, and for this reason the nuclei stain 'dark'. Only one nucleusin this figure is of the 'light' type; this one is lying in a deeper layer, as may be seen in c.Microscopical examination showed that the membrane of this nucleus has been partiallycut away.

c, same preparation as A and B, but the focus is on the deepest layer. The 'dark' nuclei areout of focus. Three 'light' nuclei are visible, all of them with the membrane partly cut away.

Lison—Staining Differences in Cell Nuclei

and the probability of not being included entirely is, of course:

__ idg~J+d'q= i-p

233

(2)

If the staining method is able to differentiate the untouched nuclei fromthose which are cut, differential counts in sections of various thicknessesmust give ratios identical to these probabilities.

In order to verify this hypothesis, counts were made in sections of the sameliver, made at 4, 6, 8, 10, 12, and 14 ju,, mounted on the same slides and stainedwith the modified Mallory method. The results are expressed in fig. 3,which gives the percentage of the 'yellow' cells found in the preparations.

4 0 -

3 20

A

2 12 14 164 6 8 10Thickness of section in JJ

FIG. 3. Results of differential counts of nuclei in sections of the same rat liver stained bythe modified Mallory stain. The percentage of nuclei stained yellow is plotted as a functionof the thickness of the section. The broken line represents the theoretical percentage of the'yellow' nuclei calculated from the regression equation (4). The points indicate the valuesfound. The upper and lower limits of the confidence-interval for a probability P = 0-05are indicated as vertical lines. (Note the asymmetry of the confidence-interval for the lowest

percentages.)

The vertical lines indicate the upper and lower limits of the confidence-interval at a probability level of 5 per cent. The broken line represents thetheoretical values calculated as indicated hereafter.

The above expressions (1) and (2) for p and q do not suit very well for thecomputations. However, if we let Y=pjq, then we obtain

t—d , , t , .—r- or Y = — — 0-5. (3)zd zd 3 K0JY =

When studying the same liver, d is constant and Y is a straight-line functionof the thickness of the section. The best fit for the parameters of the equation


(3), which represents the regression of t on Y, may easily be calculated fromthe experimental data by the least squares method. This was done for thedata obtained, with the following result:

Y = o-oo.28<—0-529. (4)

It may be verified that the constant term 0-529 is only slightly differentfrom the theoretical value, 0-500. From the values of Y calculated fromequation (4) the percentages expected for the 'yellow' or 'dark1 nuclei were

Ycalculated from the formula: p = • The comparison of the values

found with the values expected from the theoretical data is given in thefollowing table.

TABLE 2

Theoretical and experimental values of the regression: percentage of 'yellow' or'dark' nuclei/ thickness of the preparation

Thicknessin /x

468

1 0

1 2

14

Yfound

-0-1580-027650-21920-3987O-58430-7698

Percentagecalculated

0//o0

2 71 7 62 8 536-943 -5

Percentage found, withstandard deviation

0//o0

2-o±o-6i6z±i-33 O I ± I - 937-7±2-o43"7±2-i

As the table shows, the fit between the experimental data and the expectedvalues is as good as can be expected.

I conclude that both histological evidence and statistical analysis demon-strate that the integrity of the nuclear membrane is the factor causingdifferential staining of the nuclei.

Differential staining of nuclei in other organs than liver

Sheinin and Davenport, and Parr and his associates, described thedifferential staining of nuclei only in the parenchymal liver cells. I investigateda variety of other organs by the same methods. I found that the nuclei ofsome cell-types are able to give a differential stain in the same conditions asin the liver, when other cell-types, sometimes in the same organ, do not showany differences at all. Table 3 summarizes these results.

DISCUSSION

My findings are contrary to the view expressed by Sheinin and Davenport,and by Parr and others, that there exist two types of nuclei in the parenchymaof the liver which differ by some histochemical character or which representnuclei in different physiological states. I found that it was possible to stain


the nuclei differentially with Mallory's stain, and demonstrated that Heiden-hain's iron haematoxylin allows the same differentiation. However, it has beenshown by the use of appropriate techniques that the factor responsible forthe differential staining is the integrity of the nuclear membrane. When themembrane is intact, the nucleus stains 'blue' with the modified Mallorymethod, or 'light' with the Heidenhain iron haematoxylin; when it is partiallycut away, the nucleus stains 'yellow' or 'dark'. This phenomenon is notrestricted to the liver cells, but is shown also by a number of other cell types,but not by all.

TABLE 3

Occurrence of differently stained nuclei in different rat organs

pancreas

thymus andlymphnodes

testis

seminalvesicles

connectivetissue

acinar cells +pancreatic ducts —islets of Langerhans —

lymphocytes —reticular cells +macrophages +

all cells except —spermatozoa +

epithelial cells +

fibrocytes +adipose cells +

kidney

ureter

supra-renal

submaxillarygland

sublingualgland

glomerular epithelium +proximal convoluted

segment -j-loop of Henle —distal convoluted

segment —collecting tubules —

mucous membrane +

cortex +medulla —

tubular segments +acinar segments +excretory ducts +

all parts +

+Nuclei differently stained —All nuclei similarly stained

I am not able to explain entirely the mechanism of the differential staining.However, there is some evidence that the main factor may be the relativelylow permeability of the nuclear membrane. This is strongly suggested by aconsideration of the mechanism of the staining methods which are able togive a differential stain of the nuclei.

In the Heidenhain's iron haematoxylin method, a highly insoluble dye-lake is formed in the tissue by successive prolonged treatment with ironalum and haematoxylin solutions. During differentiation the dye-lake isslowly extracted by further treatment with iron alum, in which the lake issoluble. It may be admitted that the relatively large dye-lake molecule doesnot pass readily through the nuclear membrane during the destaining. If themembrane is cut away, the solution of the lake under the action of the ironalum is much more rapid. For this reason, the nuclei with intact membranesare much darker than the other ones.


The Mallory method makes use of a mixture of the acid dyes orange Gand aniline blue. Von Mollendorff demonstrated long ago (1924) that anilineblue is much less diffusible in protein gels than orange G. When one uses amixture of the two dyes, the penetration of the less diffusible stain throughmembranes of low permeability is likely to be much less than the penetrationof the more diffusible. When the nuclear membrane is intact, the orange G isthe only dye of the mixture that is able to pass readily through the membrane.The orange G acts as if it were alone and stains those nuclei yellow. Whenthe nuclear membrane is partially cut away, both stains enter the nuclei andcompete for the staining of the nuclear components. As aniline blue is moreadsorbable than orange G, it excludes the latter to a large extent, so that thenuclei appear blue.

It may seem rather strange, to speak of the permeability of the nuclearmembrane in dead cells after histological fixation, for it appears questionablewhether a killed cell can retain any selective permeability. However, thesuggested differences of permeability are not necessarily of the same selectivetype as during life.

The fact that some types of nuclei do not give any differential stain isinteresting, for it suggests that the properties of the nuclear membrane inthe fixed state may vary according to the cell-type.

In the experiences of Parr and others, changes in the proportion of thetwo types of nuclei were observed when rats were submitted to starvationor to a protein-free diet. It is very doubtful whether this change representsactual modification of the chemical composition of the nuclei. It results fromtheoretical evidence and from my experimental studies that the proportionbetween the differentially stained nuclei depends upon the thickness of thesections and upon the diameters of the nuclei. It is to be expected that anytreatment capable of modifying the volume of the nucleus will change thisproportion. For this reason it is believed that the action of the starvation orof a protein-free diet is explained by modifications in the nuclear volume.In the counts of Parr and others striking differences were observed betweenthe means of the various untreated control groups. Three series of experi-ments gave the following values for the mean percentage of 'blue' cells innormal animals: 94-7 per cent., 84-4 per cent., and 61-2 per cent. The authorsspeculated about the explanation of such extreme variations. They suggested(without experimental proof) either genetic differences between the animalsused in the experiments, or 'slight, and perhaps unavoidable' variations instaining and differentiation of the slides. My opinion is that the three seriesof counts were made either on sections of varying thickness or on blocksembedded in paraffin under different conditions, with the result that theunavoidable shrinkage of the tissues during the embedding process wasdifferent.


REFERENCESFISHER, R. A. Statistical methods for research workers, n th edn., 1950. Edinburgh (Oliver

and Boyd).MOLLENDORFF, W. VON, 1924. 'Untersuchungen zur Theorie der Farbung fixierter Pra-

parate.' Ergebn. d. Anat. u. Entwickl., 25, 1.PARR, L. L., MOSSBERG, S. M.( ROSENZWEIG, M. W., BRESLAR, E. I., and CLARK, G., 1953. 'An

alteration in the nuclei of rat liver cells in starvation." Anat. Rec, 116, 457.SHEININ, J. J., and DAVENPORT, H. A., 1931. 'Staining differences in hepatic cells'. Proc.

Soc. exp. Biol. and Med., 28, 574.SNEDECOR, G. W., 1946. Statistical methods, 3rd edn. Iowa (State College Press).

staining differences in cell nuclei · 2006. 5. 26. · lison—staining differences in cell nuclei...

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