Aug.1934] IRWIN: SILICEOUS MATERiAL

135~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

coccal pus was drained from an abscess in the arm. Noother metastatic abscesses occurred, and the patient 'stemperature reached normal on the twenty-fourth day,or about five weeks after operation. He left hospitalthree weeks later with no apparent residual disability.

COMMENTSSince this paper was accepted for publication

fuirther laboratory evidence has been reportedto which attention may relevantly be drawn.By injecting rabbits with a series of sub-cutaneous injections of living toxigenic staphy-lococci, followed by injections of staphylococcustoxoid, Connor and McKiel7 have been able toestablish complete immunity in rabbits tostaphylococcal toxin and to massive doses ofliving toxigenic staphylococci introduced intra-venously. This acquisition of immunity wasaccompanied by a marked rise in the staphy-lococcus antitoxin content of their serum,as measured by the anti-haemolytic method.Parish, O 'Meara, and Clark,18 report that activeimmunization with toxoid confers on guinea-pigs and rabbits a very high degree of resis-tance to the pyogenic lesions which normally

follow injection of living cultures of staphylo-cocci; and they further state that rabbits im-munized with toxoid have survived intravenousinjection of many lethal doses of staphylococcusculture. Moreover, a definite degree of pro-tection against infection by living staphylo-cocci, as well as against the effects ofstaphylococcus toxin, was conferred on miceand rabbits by passive immunization with anti-toxic serum.

This recent work clearly supports the maincontention of the present series of papers, thatto confer antitoxic immunity upon a patient bypassive immunization with antitoxic serum, andlater by active immunization with toxoid, offersthe most hopeful and reasonable type of specifictreatment at present available for acute staphy-lococcal infections and toxaemias.

REFErENcEs(Oontinwd from Pt. II)

15. MATHEW, R. Y., M. J. AXtralia, 1930, 1: 34.16. RoUMANN, H. A., J. Am M. A88., 1933, 101: 514.17. CONNOn, J. I. AND McKiE, M., Brit. T. Derm. & Syph.,

1934, 46: 20.18. PAaSH, H. J., O'MEARA, R. A. Q. AND CLARN, W. H. M.,

The Lomoet, 1934, 1: 1054.

THE HISTOLOGICAL DEMONSTRATION OF SILICEOUS MATERIALBy MICROINCINERATION*

BY DUDLEY A. IRwIN, M.B.,

Department of Medical Research, Banting Institute, University of Toronto,

Toronto

THE demonstration of siliceous material inhistological preparations has been most un-

satisfactory. This has been due to (a) thetranslucency and minuteness of the, siliceousparticles; (b) the close approximation of therefractive indices of histological mounting mediaand the siliceous particles; (c) the presence of"occult" (hydrated) siliceous material; (d)the presence of an opaque covering on the silice-ous particles, such as carbon (Fig. 9).In the past, siliceous material, being doubly

refractive, has been demonstrated in histologicalsections of tissue by the use of crossed Nicolprisms (Fig. 2). This method is unsatisfactory,as a great many substances apart from siliconcompounds, including fibrous tissue and smoothmuscle, are doubly refractive. One-half of the

* Read before the Section of Medicine, TorontoAcademy of Medicine, on March 13, 1934.

total number of finely divided doubly refractiveparticles, when viewed at one time betweencrossed Nicol prisms, are in the extinction phaseand are not visible. Histological mountingmedia usually contain a few small, doubly re-fractive particles.The incineration of histological preparations

has been used extensively by Policard,l Cowdry,2Scott3 and Schultz-Brauns6 to correlate inorganicmaterial with histological structure. The methodconsists of mounting the unstained histologicalsection on.a glass slide and heating it until allthe organic material has been removed by oxida-tion (Fig. 3), leaving only the inorganic ash.This ash is left so remarkably in situ that mosttissues can readily be recognized from their ashpattern. Policard7 et at., and Scheid8 have usedmicroincineration to demonstrate siliceous ma-terial in histological sections. The method de-

IRWIN: SILICEOUS MATERIAL 135Aug. 1934]

136~ ~TH CAADA MEIAAsolo JORA [Ag 193

scribed in this communication can be usedroutinely in any pathological laboratory by mak-ing use of the ordinary formalin-fixed paraffin-cut sections of tissue. The author has found theash of an incinerated section of tissue to besoluble in strong mineral acids, with the excep-tion of siliceous material which is practicallyinsoluble (Figs. 4, 7, 10). Such material canbe related to the histological structure in whichit was contained by means of serial sections(Figs. 5, 6, 7 and 8, 9, 10).

THE TECHNIQUE OF MICROINCINERATION

When the more soluble compounds (sodium,calcium, etc.) are being investigated, a 10 per

cent solution of formalin in absolute alcohol3 isnecessary as a fixative. Some of the tissues usedin this study had been fixed in 10 per centformalin for some months or years.- Formalinfixative, on standing, slowly liberates formicacid. The siliceous material, being relativelyinsoluble in acid, was apparently little disturbed.

Histological preparation.-The tissues weredehydrated in ascending alcohols, cleared inxylol and were imbedded and cut in paraffin.Sections 5 microns in thickness were quite satis-factory. If a tissue contains an excessive amountof inorganic material, sections thicker than 5microns are not satisfactory for incineration,due to "spitting" of the ash during the process

I If'- .

FIG. 1.-Serial section 1. Early silicotic nodule from the lung of a gold miner. Haematoxylin andeosin. Mag. lOOx.

FIG. 2.-Serial section 1. Same nodule as seen between crossed Nicol prisms. Mag. lOOx.FIG. 3.-Serial section 2. Appearance of the same nodule after incineration. Mag. 1oox.FIG. 4.-Serial section 2. The same incinerated section after treatment with concentrated hydrochloric

acid. Mag. lOOx.

THE CANADIAN MEDICAL AssocIATION JOURNAL136 [AUg. 1934

IRWIN: SILICEOUS MATERIAL

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138 THE CANADIAN MEDICAL ASSOCIATION

of heating. White-edged slides are found toresist heating better than green-edged ones.

Incineration of sections.-The preparationswere incinerated on a chromium-nickel tray ina specially constructed quartz electric oven.They were heated gradually to 550-6000 C.(dull red in daylight) and this heat was main-tained for one hour. When cool, the incineratedsections were examined microscopically by directoblique illumination for the presence of anycarbonaceous residue, a sign of incomplete com-bustion. With this illumination the dark car-bonaceous material stands out prominentlyagainst the white background of ash. Some sec-tions required further incineration. When in-cineration was complete the preparation wasprotected by a coverslip attached to the slide bymeans of wax at the corners.Acid treatment of incinerated sections.-Con-

centrated hydrochloric acid (c.p.) was found tobe the most satisfactory solvent of the non-siliceous material. One c.c. of the acid at roomtemperature was delivered to the slide adjacentto the ash and allowed to flow gently over theash. The acid was allowed to remain 30 min-utes and was drained off. When excessiveamounts of iron were present in the ash theslide was gently heated to accelerate the reactionafter the acid had been added. Following acidtreatment the preparation was carefully rinsedwith flowing distilled water, and the slide placedin a vertical position and allowed to dry spon-taneously.

Serial sections.-Serial sections facilitated themicroscopic orientation and study of similarareas of tissue. As a routine all tissues were cutserially (Figs. 5, 6, 7 and 8, 9, 10) and threeconsecutive sections treated as follows:

Section 1-Incinerated only (Figs. 3, 5, 8).Section 2- Stained with hsematoxylin and

eosin (Figs. 1, 6, 9).Section 3 -Incinerated, followed by hydro-

chloric acid treatment (Figs. 4, 7, 10).When special stains were required, serial sets

of five were used; slides 1, 3 and 5 were stainedand slides 2 and 4 incinerated.The microscopic examination of incinerated

sections. - Direct illumination by transmittedlight was found to be the most satisfactory forthe study of the morphology of the ash particlesat high magnification. Dark-field illuminationwas fairly satisfactory but had the disadvantageof illuminating all particulate matter to much

the same intensity. Reflected illumination wasmost satisfactory for routine examination, asthe inherent colour shades of the ash particlescould be observed. With this lighting the in-tensity of illumination of the ash particles wasan indication of their relative translucency. Aspecial apparatus* was used to provide this typeof illumination.

DESCRIPTION OF INCINERATED SECTIONSNormal tissues.-The general topography of

the ash enables recognition of the tissue. Thefixed cells of most tissues present an ash thatdelineates the cell boundaries and nucleus.Small irregular deposits of ash are scatteredthrough the cytoplasm and nucleus. Red bloodcells and most leucocytes cannot be recognizedindividually, but when present in numbers pre-sent a characteristic collective ash. Three typesof ash are present.

1. Greyish white ash.-This makes up a largepercentage of the total ash. The particlesare small (2 ,u or less) and amorphous.This ash is readily and completely solublein concentrated hydrochloric acid, and inall probability represents the chlorides,carbonates and phosphates of magnesium,calcium, sodium and potassium which werepresent in the tissue.

2. Red ash.-The red ash is confined usuallyto the lumina of blood vessels and corres-ponds to collections of red blood cells. Theindividual particles are very small (2microns or less) and have a characteristicred rust colour. This ash is quite solublein concentrated hydrochloric acid, yieldinga yellow solution in which iron is demon-strable. It is probably iron oxide.

3. White ash.-This brilliantly white ash isseldom seen in the normal tissues, exceptof the skin and rectum, and there only invery small amounts. It is insoluble in con-centrated hydrochloric, nitric and sulphu-ric acids. It is readily soluble in hydro-fluoric acid. Optically, it is doubly refrac-tive. This material consists of silica andsilicates.

Pathological tissues.-Microincineration wasused most advantageously in sections that con-tained excessive amounts of siliceous material.In these tissues the siliceous material for themost part was localized to areas scattered through

* Carl Zeiss Epi-Mirror.

138 THE CANADIAN AhDIcALAssoCIATION JOURNAL [Aug. 1934

Aug.1934] IRWIN: SILICEOUS MATERIAL139-the section. This concentration greatly facili-tated its demonstration and relation to histologi-cal structure. Observations on the siliceousmaterial present in pathological tissues will bepublished in the near future.

THE ACID TREATMENT OF ASH

The strong mineral acids were tried, and con-

centrated hydrochloric acid (c.p.) was found tobe the best solvent of the non-siliceous materialin the ash. This treatment removes all mineralconstituents of normal tissue except compoundsof silicon. The ash of a silicotic lung treatedwith coneentrated hydrochloric acid consists al-most entirely of silica and silicates.4 Othersubstances are insoluble in concentrated hydro-chloric acid, but are very unlikely to be present.Gold and platinum, if present, would not beremoved, but need only to be mentioned. Silverchloride is also insoluble in hydrochloric acid,but is not doubly refractive. This substance, ifpresent, could be quickly dissolved by ammoniumhydroxide. Barium sulphate, being relativelyinsoluble in concentrated hydrochloric acid,would remain on the slide. This substance iswhite, and, being doubly refractive, would beinterpreted as being of a siliceous nature.Barium sulphate in appreciable amounts is al-most never present in tissue. If present, it can

be removed by treatment with warm concen-

trated sulphuric acid.

FUSION OF ASH TO SLIDEWhen excessively heated (7000 C. +) much

of the ash fused to the slide, which showed signsof warping and pitting of the surface. The ashcould not be physically wiped from the surfaceof such a preparation or completely removed byacid. Such preparations were discarded. In-cineration at 550-6600 C. did not change theappearance of the glass slide, and the ash couldbe removed by wiping with a dry cloth.

RELATION OF THE HYDROCHLORIC ACID INSOLUBLERESIDUE TO THE CHEMICAL ASSAY OF

TOTAL SlLICAIt was observed that the acid insoluble ma-

terial of incinerated sections from varioustissues seemed to be proportional to the assay

of that tissue for siliceous material. To investi-gate this observation ten blocks of tissue were

selected from five silicotic lungs. In each case

there had been a history of long exposure tomine dust containing siliceous material. Theblocks were cut from areas that had as uniforma consistency throughout as possible. The blockswere halved. One-half was assayed by Dr. E. J.King for total silica by his own method.5 Theresult is expressed in mg. of silica per 100 g.of dried tissue. The other half was cut inparaffin and serial sections were prepared andincinerated, as previously mentioned. The acid-treated incinerated sections were arranged, with-out knowledge of the chemical assay, in orderof the descending magnitude of the acid in-soluble residue per unit area. The results areshown in the following Table.

Arrangement of incineratedsections in lessening Mg. Sio, per

amo,unts of acid insoluble 100 g. ofBlock No. residue per unit area dried tissue

1 ................ 2.............. 2,6402 ................,2.............. 3003 ................,4.............. 12404 ................,5.............. 11505 ................,3.............. 11076 ................6 .............. 9767 ................7 .............. 475S ............ 8 .4209 ........... 9.400

10 ............ 10.370

The author does not suggest that an acid-treated incinerated section of tissue containingsiliceous material should supplant chemical assayfor the quantitative estimation of that material.This method can be used as a quantitative guideof the siliceous content, and as such is frequentlyhelpful. The minimum amount of siliceousmaterial that can be recognized in an incineratedsection of tissue and related to histologicalstructure depends upon the concentration of thatmaterial at focal points. Siliceous material tothe extent of 100 mg. per 100 g. of dried tissuemay be present, but if uniformly dispersedthroughout the tissue it cannot be identified withany histological structure. Very small amountsmay be recognized if the local concentration issufficient, though the total assay may be low.The siliceous material present in a single alveo-lar phagocyte of an animal exposed to silicadust can readily be demonstrated.

SUMMARYA method is described for the demonstration

of siliceous material in histological sections oftissue. This consists in the incineration of the

IRWIN: SILICEOUS MATERIAL 139Aug. 1934]

140 THE CANADIAN MEDiCAL ASOCIATION JOURNAL [Aug. 193

histological section and the subsequent treat-ment of the ash with acid to remove the non-siliceous material.

The author wishes to thank Dr. F. G. Banting forhis many helpful suggestions, and Dr. T. L. Walker, Pro-fessor of Mineralogy and Petrography, and membersof Iiig Departmient for advice in those subjects.

REFERENCES1. POLICALRD, A.: Sur une methode de microincine,ration ap-

plicable aux recherches histochimiques, Bull. Soc. Chimn.de France, 1923, 33: 1551.

2. COWDRY, E. V.: Microincineration of intranuclear inclusionsin yellow fever, Amt. J. Path., 1933, 9: 149.

3. SCOTT, G. H.: Distribution of mineral ash in striatedmuscle cells, Proc. Soc. Exp. Biol. d Mled., 1932, 29: 349.

4. JEPHCOTT, C. A.: Silicate contenit of silicotic lung ashes,Canad. Chewb. Met., 1934, 28: 32.

5. KING, E. J. AND STANTIAL, H.: Biochemistry of silicic acid;microdetermination of silica, Bioche.ns. J., 1933, 27: 990.

6. SCHULTZ-BRS-AuNS, 0.: Histo-topochemische Untersuchunigenan kraiikhaftverinderteni Organieni uniter Aniwedunigder Schnittveraschuing, Ar chiv. f. Path. Anat., 1929,273: 1.

7. POLICARD, A., DO-UBROW, S. AND BOuCHARLAT: A propcsdu mecanisme, de la silicose puliiionaire, Bull. d'hist.appliq. a la physiol., 1929, 6: 371.

8. SCHEID, K. F.: Uber die MIethodik der Darstellunig undBestisnuimng des iu pneumonokoniotischen Ge(vebenabgelagertein Staubes, Beitr-. z. Path. A oat. ut. z. aUg.Path., 1932, 89: 93.

MICROINCINERATION AS AN AID IN THE DIAGNOSIS OF SILICOSIS*

BY DUDLEY A. IRWIN, A.B.,

Department of Medical Research, Banting Institute, University of Toronto,

Toronto

TIIE pathologist is frequently the last courtof appeal in the diagnosis of pulmonary sili-

cosis. The period of dust exposure, the amountand type of dust breathed, the clinical course ofthe disease, the presence or absence of an ac-companying tuberculous infection, the physicaland roentgenological chest findings, and thechemical assay of the siliceous content of thelung tissue, are very important in the diagnosisof pulmonary silicosis and in the determinationof the amount of siliceous fibrosis present. Im-portant as these findings are, however, it is thepathologist who must ultimately determine bymicroscopic examination the amount and dis-tribution of siliceous fibrosis in the lungs, andfrom his findings give an opinion on the im-portance of the sequelwe of this fibrosis in theultimate cause of death.

Siliceous fibrosis can be defined as a fibroustissue reaction in which can be demonstratedrelatively large amounts of siliceous material,distributed diffusely throughout that tissue in acharacteristic manner. On account of the as-sociation of siliceous material with the fibrosisin human silicotic lungs; from the fibrous reac-tion constantly produced by the injection offinely particulate silica into experimental ani-mals at the site of the silica; from the inabilityto produce similar fibrous lesions with manycompounds other than silica, one must infer thatthe siliceous material is responsible for the

* R.ead before the Section of Medicine, TorontoAcademy of Medicine, on March 13, 1934.

fibrous reaction and that the harmful siliconcompound is silica.

Clinically, siliceous fibrosis is limited almostentirely to the lungs and to the course of thelymphatic channels draining these organs. Pul-monary tuberculosis is almost always the im-mediate eause of death in pulmonary silicosis.Tuberculous fibrosis can so simulate siliceousfibrosis that, at times, even microscopically, theycannot be separated by their morphological char-acteristics or by special staining. Fibrosis notof siliceous or tuberculous origin may be presentto further complicate the picture.The chemical assay of the total siliceous ma-

terial in a peribronchial or mediastinal lymphaticgland may be quite high without the presenceof siliceous fibrosis. Unfortunately, the silice-ous compounds present in silicotic lungs are insuch a finely particulate state that their identifi-cation, chemically or petroffraphically, leavesmuch to be desired. Scheid,' of Germany, andJones,2 of England, have claimed that they canspecifically identify these particles by petro-graphic methods. Most petrologists on this con-tinent claim that the identification of such fineparticles is a petrographic impossibility. Knopf3states: "When the individual particles are ofthe order of magnitude of 5 microns in diameter,petrographic examination of the material mustbe supplemented by other methods because theminimum grain size that can be conclusivelyidentified under the petrographic microscope isabout 10 microns in diameter."

140 THE CANADIAN MEDICAL AssocIAT-ION JOURNAL [Aug. 19-3-4