APPENDIX
TECHNIQUE USED IN THE PREPARATION OF THE PATHOLOGICAL TISSUES FOR STUDY
HEMATOXYLIN AND EOSIN STAINING
Sections, affixed to the slide, were brought through xylene and alcohols to water and stained two to five minutes in hematoxylin solution prepared as follows:
Hematoxylin ............................................... gram... 1.0
Alcohol ....................................................... mil......10.0
(Dye dissolved in the alcohol.)
Alum (ammonium or potassium) ...........grams... 20.0
Distilled water ............................................mil......200.0
The alum was dissolved in the water with the aid of heat, and the alcoholic solution of the dye added. The mixture was brought to a boil rapidly and 0.5 grain of mercuric oxide added. The solution at once assumed a dark purple color, and as soon as this occurred it was cooled by plunging the flask into cold water. For use, 4 percent glacial acetic acid was added, since this increases the precision of nuclear staining.
Sections were then passed through 1 per cent hydrochloric acid in 70 per cent alcohol for 10 to 15 seconds, rinsed in water, placed in 0.5 of 1 percent aqueous solution of lithium carbonate for 5 minutes, washed in water for 5 minutes, stained in 0.5 of 1 percent eosiin in 25 percent alcohol for 30 seconds to 1 minute, dehydrated in alcohols, cleared in xylene, and mounted in euparal or balsam.
Euparal a often was employed as a mounting medium in preference to balsam.
ELASTIC TISSUE STAINING
Sections affixed to the slide were brought through xylene and alcohols to water, and stained in the following elastic tissue stain b for one-half to two hours:
Grams
Crystal violet ................................................................2.0
Dextrin............................................................................0.5
Resorcinol .....................................................................4.0
Water, distilled ..........................................................200.0
This mixture was boiled in a flask and when boiling briskly 25 mil. of a 29 percent aqueous solution of ferric chloride was added. Boiling was continued for two to five minutes. A heavy precipitate formed, and the mixture assumed a greenish cast. It was then cooled and filtered. The precipitate was dissolved by boiling with 200 mil. of 95 percent alcohol over a water bath or electric hot plate. After cooling and filtering the volume was made up to 200 mil. with 95 percent alcohol and 4 mil. of strong hydrochloric acid (33.8 percent) added.
Sections were differentiated in 95 percent alcohol, washed in water and stained for 10 to 15 minutes in Weigert iron hematoxylin prepared as follows:
Mil.
30 percent aqueous ferric chloride ............................ 1.0
Strong hydrochloric acid ............................................0.25
Distilled water to make ...............................................25.0
1 percent alcoholic solution of hematoxylin.............25.0
This mixture kept satisfactorily for several weeks in a Coplin jar.
a A trade name for a mixture of camsal, sandarac, eucalyptol, and paraldehyde.
b For further details consult: Elastic fiber stain," by L. H. Prince. United States Veterans’ Bureau Medical Bulletin, Washington, 1927;iii, 83.
572
Sections were washed in water and stained in the following Van Gieson mixture for one to two minutes:
1 percent aqueous acid fuchsin -------------------------2.0
Saturated aqueous picric acid --------------------------38.0
The nature of the acid fuchsin is important; a detailed description of it is given below in the discussion of dyes.
Sections were then differentiated in 95 percent alcohol, dehydrated through alcohols, cleared in xylene, and mounted in euparal or balsam. Euparal was found to preserve a Van Gieson stain better than balsam.
Difficulty was experienced in the consistent preparation of an elastic stain, until it was noted that certain pre-war samples of dye were satisfactory while pure dyes produced since the war often failed. It formerly had been customary to weight dyes with dextrin as a diluen it was found that the presence of dextrin was necessary for the production of the stain.
Prepared with crystal violet as the basic dye, this elastic stain shows elastin as a bright grass green, while the original technique of Weigert with basic fuchsin resulted in a bluish black elastin. It was noted further that by using both of these dyes in combination, in various proportions, a varying shade of green could be obtained in the elastin. For photography, a mixture of equal parts of basic fuchsin and crystal violet proved to be excellent.
MacCALLUM'S STAIN
Paraffine sections affixed to the slide were passed through xylene and alcohols to water, and stained for 10 minutes to one-half hour in the following mixture:
30 percent alcohol ......................................... mil....100.0
Basic fuchsin ................................................gram......0.59
Aniline ..............................................................mil......1.0
Phenol, crystals................................................mil......1.0
They were then washed in water and differentiated in 40 percent formaldehyde. This required only a few seconds, the bright red color being washed away and replaced by a clear rose. Sections were next washed in water and counterstained in a saturated aqueous solution of picric acid. The sections remained in this until they assumed a purplish color (three to five minutes); then they were washed in water and differentiated in 95 per cent alcohol, the red reappearing and some of it being washed out as was some of the yellow of the picric acid. The sections then were washed in water and stained for three to five minutes in the following solution:
Crystal violet ............................................... grams.....5.0
Alcohol. .............................................................mil....10.0
Aniline ...............................................................mil......2.0
Water .................................................................mil....88.0
Following this, the sections were washed in water and immersed in iodine solution for one minute.
Iodine .............................................................gram.......1.0
Potassium iodide ..............................................do.......2.0
Water .................................................................mil...300.0
The slides were finally blotted dry without washing, treated in equal parts of xylene and aniline until no more color came away, passed through two changes of xylene, and mounted in balsam or euparal.
Gram-negative organisms were stained red; Gram-positive, blue. The stained preparations are easily bleached by the intense lights used in photomicrography, particularly by the carbon arc.
GIEMSA TISSUE STAIN
Paraffine sections affixed to the slide were brought through xylene and alcohols to water, and placed in the following stain, for two to three hours to overnight, in the incubator at 37°C.
573
Mil.
Giemsa substitute (see below ....................................5. 0
Distilled water pH 7.5 phosphate buffer.................45. 0
They were differentiated in 15 percent solution of colophonium (rosin) in acetone for 20 to 30 seconds (until the free blue was completely washed out). A second change of 15 percent colophonium in acetone for a few seconds and placed in the following mixture for 30 seconds to 1 minute:
Mil.
Acetone .......................................................................30.0
Xylene ...........................................................................70.0
Cleared in xylene and mounted in euparal or balsam.
GIEMSA SUBSTITUTE c
Methylene blue, zinc free ..............................gram....1.0
Eosin, yellowish .................................................do.....1.0
Azure C .................................................................do.....0.6
Methylene violet (Bernthsen) ...........................do.....0.2
Anhydrous glycerin ..........................................mil..100.0
Absolute methyl alcohol, acetone free............do..300.0
After the addition of the dry dyes to the glycerin-alcohol, the mixture was allowed to stand in the thermostat at 54°C. for from 12 to 24 hours or in the incubator at 37°C. for several days. This was necessary to permit of a chemical reaction between the basic and acid dyes and the subsequent solution of the reaction products in the alcohol-glycerine mixture.
While azure C d was employed, similar results were obtained with azure Ac or a good grade of toluidine blue. Azure C stained a slightly deeper shade of blue than was obtainable with the other dyes.
Suitable buffers were employed to control the pH of the staining solution thereby affording control of the relative intensity of the acid and basic dyes (eosin and methylene blue, azure, etc.). Buffering on the acid side accentuated the eosin staining, while with reactions towards the alkaline side the blue staining was enhanced. For bacillary staining the alkaline reaction, pH 7.5, was employed as a more intense bacillary stain was desired, while the eosin staining was relatively unimportant.
Satisfactory buffer solutions were prepared with an M/15 solution of primary potassium phosphate containing 9.087 grams of KH2 PO4 in the liter of distilled water and an M/15solution of secondary sodium phosphate which contained 11.876 grams of Na2HP02-2H20, in the liter of distilled water. The primary phosphate as received was of sufficient purity for the work while the general supply of sodium phosphate had 12 molecules of water of crystallization. This was readily changed to the two-molecule form by drying in an opened dish in the incubator at 37? C. for three or four days. For varying values of pH the following proportions of the two solutions were employed:
CHART
c Consult: "Polychrome stains. 1. A substitute for Giemsa’s stain," by R. W. French. The Journal of Laboratory and Clinical Medicine. St. Louis, 1926, xi, 352-354.
d For method of production, consult: " The Oxidation Products of Methylene Blue," by W. C. Holmes and R. W. French. Stain Technology, Geneva, N. Y., 1926, I, 17-26.
e For method of production, consult: "Methylene Violet and Methylene Azure A and B," by Ward J. MacNeal. Journal of Infectious Diseases, Chicago, 1925, xxxvi, 538-546.
574
Ten percent of the buffer mixture in distilled water was employed with the Giemsa substitute.
Differentiation in the acetone-colophonium mixture was accomplished through the action of the abietic acid, the active ingredient of rosin. By increasing the percentage of the colophonium the eosin staining was improved, but at the expense of a good blue stain which was most important in studying bacteria. In general the stain was manipulated to obtain a strong blue stain as this was found to be specially necessary in the staining of Gram-negative, weakly staining organisms such as B. influenzae.
Euparal was employed almost exclusively as a mounting medium following the Giemsa stain as it had less bleaching effect than the average balsam.
RETICULUM STAINING f
Paraffine sections affixed to the slide were passed through xylene and alcohols to water and placed in 0.25 percent aqueous solution of potassium permanganate for 5 minutes, washed in water, placed in 5 percent aqueous solution of oxalic acid for 10 minutes, washed in tap water, washed in distilled water and incubated at 37°C. for 10 to 15 minutes in the following freshly prepared silver-ammonium carbonate mixture:
To 10 mil. Of a 10 percent aqueous solution of silver nitrate were added 10 mil. Of a saturated solution of lithium carbonate in distilled water. The resulting heavy white precipitate of silver carbonate was allowed to settle and the supernatant fluid poured off. The precipitate was then washed several times in from 25 to 30 mil. Of distilled water, allowed to settle each time, and the supernatant fluid poured off. After the addition of 25 mil. Of freshly distilled water the washed precipitate was almost dissolved by the addition of 25 mil. Of freshly distilled water the washed precipitate was almost dissolved by the addition of strong ammonia water while the container was constantly agitated. The precipitate turned a grayish brown as the ammonia was added. From 8 to 15 drops of ammonia were required depending upon the strength of the ammonia. Extreme care was taken not to overstep depending upon the strength of the ammonia. Extreme care was taken not to overstep the end point of the solution, and it was preferable to leave a few granules of the precipitate undissolved. The entire solution was then made up to 100 mil. With distilled water and heated to about 50°C. The slides were immersed in this solution and placed in an incubator at 37°C. Care was exercised lest the section become detached from the slide in the warm alkaline bath. It was found that sections properly affixed to the slide and thoroughly dried did not come off.
The sections were next rinsed quickly in distilled water, placed in 20 percent neutral formalin (8 percent formaldehyde) for 2 minutes; washed in tap water, placed in 1 to 500 gold chloride for 2 minutes, washed in tap water; placed in 5 percent aqueous solution of hyposulphite of sodium 2 minutes and washed in water.
Following the above reticulum stain, section were stained 3 to 5 minutes in Harris’s hematoxylin, washed in tap water until blue, counterstained in Van Gieson’s mixture for 45 seconds, differentiated in 95 percent alcohol, dehydrated, cleared in xylene, and mounted in balsam or euparal.
IRON HEMATOXYLIN AND PICRO-EOSIN
To show certain types of muscle degeneration the Weigert iron hematoxylin, as mentioned under elastic tissue staining, was employed, followed by a weak picric acid-eosin mixture. One part of saturated aqueous picric acid was mixed with nine parts of a 0.25 percent aqueous eosin. If certain tissues had a tendency to show too much of a yellow stain the staining in the picro-eosin mixture was followed by a few seconds in the standard eosin described under hematoxylin and eosin staining. This technique afforded an excellent stain for the photography of muscle degeneration; fibers staining in tint from a rich red of the eosin through orange to a brown; the more alkaline portions taking the picric acid stain and the more degenerated acid fibers showing a pure eosin color.
f Based on: “Rapid Method for Silver Impregnation of Reticulum,” by N.C. Foot, and M.C. Menard. Archives of Pathology and Laboratory Medicine, Chicago, 1927, iv, 211-21.
575
DYES
Acidfuchsin. g - This dye wasthe disodium salt ofsulphonated rosanilin. It was found thatthe corresponding sulphonated salts of para-rosanilin and new fuchsinwere unsatisfactory,though sulphonated mixtures of para-rosanilin and rosanilin could beused when the relativeproportion of the latter dye was 75 percent or more.Spectrophotometrically the dye showed aclean cut maximum of absorption at a wave length 545 millimicrons witha specific absorptiveindex of 1.385 at 540 millimicrons. Satisfactory samples of sulphonatedrosanilin with a dyecontent of 70 percent showed an absorptive index of 0.969 at 540millimicrons and consumed2.53 mil. of normal titanous chloride per gram of dye.
Basicfuchsin. - This dye was the hydrochloride of rosanilin, or amixtureof the hydrochlorides of rosanilin and para-rosanilin containing atleast 50 percent of the former. Examinedspectrophotometricallv in solution in 50 percent ethyl alcohol itshowed a maximum ofabsorption at a wave length of 548 millimicrons. A solution of the dyeof 10 parts in a million of50 percent ethyl alcohol when examined with the spectrophotometershowed absorptive indicesof from 1.49 to 1.77 at 545 millimicrons and of from 1.36 to 1.60 at540 millimicrons. One gramof the dye consumed not less than 4.15 mil. nor more than 4.23 mil. ofnormal titanous chloride.
Crystalviolet. - Thisdye was the hydrochloride of hexamethyl para-rosanilin. Inasmuch asthis dye was a definite chemical entity, it was uniformly substitutedin all formulae calling forgentian violet. Examined spectrophotometricallv in a concentration of10 parts of the dye in amillion parts of 10 percent acetic acid in water a well-definedmaximum of absorption occurredat a wave length 591 millimicrons and such a solution showed anabsorptive index of 1.87 and590 millimicrons plus or minus 0.046 when examined in a layer of 1 cm.depth.
Hematoxylin.h - This dye was the glucoside obtained through the etherealextraction oflogwood (haematoxylon campechianum). Satisfactory samples consisted ofwell-defined crystalsof a sandy or light brown color and contained less than 0.1 of 1percent ash.
Methyleneblue.i - This dye was thehydrochloride of tetra-methylthionin with a slightadmixture of lower homologues (azures). Examined spectrophotometricallyin a concentration of10 parts of the dye to the million of M/150 phosphate buffer, pH 7, awell-defined maximum ofabsorption occurred at 660 millimicrons with a distinct secondarymaximum at 610 millimicrons.The ratio between the absorptive indices at 610 millimicrons and 660millimicrons was as 25 isto 39. Quantitatively absorptive indices of 1.575 at 660 millimicronsand of 1.01 at 610millimicrons were determined.
AzureA. - This dye was the asymmetrical dimethyl thionin. It reacted asathiazine dye onsulphuric acid and spectrophotometrically in aqueous solution showed aclean-cut maximum ofabsorption at 625 millimicrons.
AzureC. - This dye was monomethyl thionin. It reacted as a thiazine dyeonsulphuric acidand spectrophotometrically in aqueous solution exhibited a clean-cutmaximum of absorption at615 millimicrons.
Methyleneviolet. - This dye was the dimethyl thionolin ormethylene violet(Bernthsen).
EosinY. - This dye was the disodium salt of tetrabromfluorescein.Spectrophotometricallyin aqueous solution it showed a sharp absorptive maximum at 516millimicrons.
g For further details, consult: "Acid Fuchsinas a Stain: A Refinenient in Manufacture," by John T. Scanlan, R. W.French, and W. C. Holmes. Stain Technnoloqy, Geneva, N. Y.,1927, ii, 50-55.
h For further details, consult:"Standardizationof Biological Stains: III, Eosin and Hematoxylin," by R. E. ScottandR. W. French. Military Surgeon,Washington, 1924, lv, 617-624.
i For further details, consult:"Standardization of Biological Stains: 11, Methylene Blue, by R. E.Scott and R. W.French. Military Surgeon,Washington, 1924, lv, 337-352.
576
PHOTOGRAPHY
PHOTOGRAPHYOF GROSS SPECIMENS
Thegross specimens were photographed, using a commercial type camera inthe verticalposition. Illumination was obtained by nitrogen-filled bulbs of 75watts each. The specimen wasimmersed in water to eliminate high lights and to record on the platethe maximum of detail.Commercial orthochromatic films were used in this work. i
PHOTOMICROGRAPHY
Illuminant.-Threetypes of illuminant were employed, the carbon arc, the "Point-o- light"or "Tungsarc," and the ribbon filament. The source of power with thetwo former light sourceswas 110 volts direct current, while with the latter lamp 110 voltsalternating current wastransformed to 6 volts, the lamp employed requiring 108 watts of powerat 6 volts. The arc lampwas employed for higher magnifications, while the majority of the workwas accomplished withthe ribbon filament lamp. A water cell was employed with the are lampat all times. The arc lampafforded approximately six times as much illumination as either of theother lamps, which wereabout equal, but it was much more difficult to manipulate and dangerousto preparations onaccount of the heat and intense illumination.
Condensingsystem.-Immediately in front of the light source was a condensingsystem forthe collection of the converging rays from the light source into aparallel beam of sufficient sizeto fill the collecting lens of the microscope substage condenser. Withthe microscope, anachromatic substage condenser was employed. This condenser completewith an equivalent focusof 3 millimeters was employed with 2 and 3 millimeter homogeneous oilimmersion objectives.With the front lens removed, resulting in a condenser with anequivalent focus of 22 millimeters,the system was employed with the 8 and 16 millimeter objectives. Inlower power work withmicrotessars, special simple condensers of the same equivalent focus asthe objective were used.Careful centering and focusing of the condensers was found to benecessary at all times.
Objectives.-Inlow-power work microtessars, of 32, 48, and 72 millimeter equivalentfocus, were used without oculars. In higher power work achromatic dryobjectives with initialmagnifications of 3, 5, and 6 were used, as well as dry apochromaticobjectives with initialmagnifications of 10 and 20. Homogeneous immersion apochromaticobjectives, with initialmagnifications of 60 and 120, were used. For satisfactory color work itproved necessary to havean objective which brought all of the spectral colors into a focus inthe same plane.
Oculars.-Compensatingoculars giving magnifications of 3, 5, 10, 12, and 18 times wereused with the apochromatic objectives, while "homal" oculars with amagnification of about 12.5times were used with both achromatic and apochromatic objectives. A fewpictures also weremade with a "Periplan" ocular (semicompensating) with a magnificationof six times. Forautochrome photography it was necessary to employ compensating ocularswith properlycorrected apochromatic objectives.
Camera.-Bellowsextension up to 100 centimeters was available. Over-all magnificationwas determined with the aid of a stage micrometer, all objectives andoculars being calibratedand the magnifications determined for various camera extensions, asindicated on the camerabed, which was graduated in centimeters.
Photographicmaterials.-Commercial orthochromatic films and plates and processpan-chromatic plates were used for most of the photomicrographs. Whereextreme contrast wasdesired, Wratten and Wainwright M. Panchromatic plates were used.
i For details, consult: "The Photography ofGross Pathological Material," by Theodore Bitterman. Bulletin oftheInternational Association of Medical Museums and Journal of TechnicalMethods, No. viii, December, 1922.
577
Light filtration.-The following light filtersand filter combinations were used:
CHART
With the various stains the filters were usedas follows:
Well balanced hematoxylin and eosin stain: Band G.
Hematoxylin and eosin with weak eosin stain:B.
Hematoxylin and eosin with weak hematoxylinstain: G.
Elastic tissue stain: A.
Giemsa stain: E.
MacCallum stain: B.
Reticulum stain: A.
Iron hematoxylin and picro-eosin: H.
In autochrome photomicrography light filterswere not employed with the nitrogenfilled light sources whilewith the are lamp filters were employed as indicated by the platemakers.
