Ehrlich's Hematoxylin Research Articles

Differentiation of two types of basophils in the adenohypophysis of the rat and the mouse.

Pituitaries are fixed for 24 hr. in Bouin's fluid containing 0.5% trichloroacetic acid instead of 5% acetic acid, or in a mixture of 9 parts SUSA and 1 part saturated aqueous solution of picric acid. They are embedded in paraffin and horizontal sections are cut at 3–4 μ. The staining method consists of 3 phases: (a) immersion in aldehyde-fuchsin for the selective demonstration of the beta cell granules, (b) staining of the nuclei with Ehrlich's hematoxylin and (c) a rapid one-step counterstain with light green and orange G dissolved in a phosphotungstic-acetic acid mixture for the differentiation of the acidophilic and the delta cell granules.

Mortality of Laemophloeus (Coleoptera, Cucujidae) infected with Mattesia dispora Naville (protozoa, schizogregarinaria).

It was noticed that some stock cultures of Laemophloeus minutus Olivier and L. ferrugineus Stephen were showing an abnormally high mortality. The numbers of living larvae appeared to decrease, many pupae died inside their cocoons and most of the few adults which emerged died within a few weeks. As adult Laemophloeus live for about 6 months at the conditions under which the cultures were kept (25° C. and 70% r.h.) it was apparent that something was killing the beetles at all stages in development. Microscopic examination of dead larvae and adults which had been chopped up and mounted in Berlese's fluid (Swan's formula) revealed huge numbers of the lemon-shaped spores (sporocysts) or pseudonavicellae of a sporozoan parasite. Living larvae from infected cultures were dissected in saline, and smears were fixed in Bouin's fluid (aqueous) and stained with Heidenhain's haematoxylin. Other larvae were fixed in Bouin's fluid, sectioned, stained with Ehrlich's or Heidenhain's haematoxylin and eosin, cleared in xylol and mounted in Canada balsam. These preparations revealed various stages of a schizogregarine which appeared to be Mattesia dispora which was described by Naville (1930) from Ephestia kühniella Zell. (Lepidoptera, Phycitidae) and recorded by Musgrave & Mackinnon (1938) from Plodia interpunctella Hbn. (Lepidoptera, Phycitidae). Careful examination of sections and measurement of various stages confirmed this identification. All stages were found, with the possible exception of young gamonts formed by macronuclear schizogony.

Cestodes of California Gulls

There is but little information available regarding the helminthology of the California coast; therefore the following account of some cestodes found in gulls of this region may be of interest. Sixty-six birds were examined, fifty-six of which contained one or more parasites, an infection of 84.8o%. The worms have been examined in both fresh and preserved material, in whole mounts and sections. The fixatives employed were alcoholic-sublimate-acetic and alcoholic Bouin, with a few specimens fixed in 10%o formalin. The stains used were aceto-carmine and Ehrlich's hematoxylin for whole mounts, and for sections the latter and iron alum hematoxylin. All measurements in this paper are expressed in mm. and were made on preserved material except as stated in the text.

Sanguinicola huronis n. sp. (Trematoda: Sanguinicolidae) from the Blood System of the Largemouth and Smallmouth Basses

During a parasite survey of northwest Wisconsin fishes, a species of blood fluke of the genus Sanguinicola Plehn, 1905, was reported by the author (1947) from the mesenteric blood vessels of the largemouth bass, Huro salmoides (Lac.) and the northern smallmouth bass, Micropterus d. dolomieu Lac. In the 1947 paper, without naming or describing the fluke, it was declared a new species because it differed morphologically from the single known North American species, Sanguinicola occidentalis Van Cleave and Mueller, 1932, from the heart of the walleye, Stizostedion vitreum. The genus Sanguinicola was established by Plehn (1905) to receive S. armata and S. inermzis from European cyprinids; she called them endoparasitic Turbellaria. Later (1908), she reviewed her earlier conclusions and reclassified the two species as Cestodaria. Odhner (1911) correctly placed these forms in the Trematoda and discussed his and Looss' observations on the life cycle of S. inermis. The life cycle of this species was further clarified by Scheuring (1922). Woodland (1923) described a species of Sanguinicola from a Nile silurid, but did not name it. Odhner (1924) redescribed this species and named it S. chalmersi. S. intermedia was described by Ejsmont (1926) from European cyprinids. He also presented information on S. armata, S. inermis and S. chahnersi as well as additional life cycle data. Van Cleave and Mueller (1932) described S. occidentalis from a percid fish, the walleye (Stizostedion vitreum), in Oneida Lake, New York. More information on the biology and ecology of this species was presented by Van Cleave and Mueller (1934). Fischthal (1947) found S. occidentalis fairly widely distributed in the walleyes of northwest Wisconsin, and also reported this species from an additional percid host, the yellow perch (Perca flavescens). The present species was first observed April 15, 1944, when six adults were recovered from one of 11 Huro salmoides collected from Bear Lake, Barron County. On September 20, 1944, seven of 12 Huro salmoides from Lost Land Lake, Sawyer County. were found parasitized, two with one worm each and five with two each. Also on this date, seven worms were recovered from one of two Micropterus d. dolomieu from Lost Land Lake. On October 4, 1945, a fingerling Huro salmoides from Spring Creek, Washburn County, was infected with one worm. The author recalls finding in 1940 a single, typically triangular Sanguinicola egg in the ureter of Huro salmoides from the Huron River, Washtenaw County, Michigan. In all probability it belonged to the species herein described. The name Sanguinicola huronis is now proposed for this species. Its description is based on whole mounts of specimens fixed in AFA, stained in Ehrlich's acid haematoxylin, and mounted in balsam. The interpretation of the morphology presented many difficulties as encountered by other workers on the genus Sanguinicola.

A Dicrocoeliid Trematode, Eurytrema vulpis n. sp. provis., from the Pancreatic Ducts of the Red Fox

About one hundred trematodes were found in the pancreatic ducts of a female red fox, Vulpes fulva, killed in Rockland County, New York, in February, 1947. The carcass had been frozen for four days before dissection and the worms were dead when removed. All of them were well extended, remarkably uniform in size and shape, and sexually mature. Specimens fixed in various fluids and stained with Ehrlich's haematoxylin and Mayer's paracarmine were mounted entire and others, sectioned serially in transverse, frontal and sagittal planes, were stained with haematoxylin and erythrosin. Freezing and death of the parasites before fixation had produced disintegrative changes in the tissues and partially obliterated cytological and histological details. The worms (Fig. 1) are widest at the zone of the vitellaria, in the posterior half of the body; this region is flattened dorso-ventrally with thin, slightly wavy edges. The anterior portion, which tends to bend dorsally (Figs. 2, 3), may be almost circular in cross section, or thicker than wide. There is a conspicuous narrowing of the body at the level of the posterior ends of the ceca, forming the so-called caudal appendage of previous authors. The specimens, fixed and mounted, varied from 1.4 to 2.1 mm in length and from 0.5 to 0.8 mm in greatest width. The cuticula is thin, without spines, but a few small papillae which may represent sensory structures were observed. The musculature is very weak and the medullary portion of the body consists of a loose parenchyma in which the organs are embedded. The suckers are large but their musculature is weak. The acetabulum is situated at the posterior end of the anterior third of the body and measures from 0.21 to 0.29 mm in diameter. It is bounded by a fibrous capsule, within which there are weak muscular elements, but the greater part of the organ is filled with loose, vacuolated parenchyma. Accordingly, the lumen of the sucker is restricted to a small, shallow depression (Fig. 3). In most of the specimens, possibly as a result of muscular contraction and possibly as a result of previous freezing, the sucker is protruded and the ventral portion is further protruded to form a large, hemispherical projection, (Fig. 2). In such specimens there is no lumen of the sucker, and they can be prepared as whole mounts only by supporting the cover-glass. Otherwise, the weight of the cover-glass causes the sucker to bend over, or if the specimen is well hardened, it turns partially on one side. Such a condition is shown in Fig. 2, which is an oblique view, intermediate between lateral and ventral aspects. In sectioned specimens where the acetablum is protruded, the parenchymatous reticulm is replaced by a large, fluid-filled cavity in the center of the organ. Since the worms were not observed in the living condition, it is not possible to state positively how the acetabulum functions, but study of fixed specimens gives the impression that the sucker is constructed in two sections and that the ventral portion

A New Anoplocephalid Cestode, Andrya neotomae, from the Wood Rat Neotoma fuscipes

Thirteen cestodes, which I am describing as Andrya neotomae n. sp., were collected on June 23, 1941, from the small intestine of a male Neotoma fuscipes caught at the Hastings Natural History Reservation, Monterey Co., California. The worms were fixed in formalin and stained with Ranvier's picro-carmine, Grenacher's borax carmine, or Ehrlich's haematoxylin. The author is indebted to Dr. Harold Kirby for helpful suggestions in the preparation of this paper, and to Dr. Jean M. Linsdale for lending the material from which this study was made.

Cytological observations on fertilization in the carp, <i>Cyprinus</i> <i>carpio</i> L.

Employing the carp, Cyprinus carpio L. as the meterial, the processes of maturation of the egg and the phenomena of fertilization were microscopically observed in this study. Observations were carried on in the sections of the eggs, fixed at different intervals ranging from five minutes to 2 hours after fertilization. The majority of eggs were fixed in Carothers' solution and some were preserved in Gilson's fluid. For sectioning the Gilson material proved to be better than the Carothers material. Sections were made following the usual paraffin method. They were stained with Ehrlich's haematoxylin. The results of observations are briefly outlined as follows:1) The first maturation division of the egg is completed prior to ovulation within the ovary. After the extrusion of the first polar body, the chromosomes remaining in the egg are immdiately drawn into the equatorial plate forming the metaphase spindle of the second maturation division (Fig. 4).2) The spawning and fertilization take place in the water at the same time.3) At the time of insemination the second maturation division of the egg persists in the stage of metaphase, and then it gradually advances in further course of division (Fig. 8). About 25 to 30 minutes after insemination the second division is completed, resulting in the formation of the second polar body (Fig. 9).4) During the ovulation process the course of the second division does not proceed being arrested in metaphase. Stages subsequent to the metaphase of the second division take place only after the entry of the spermatozoon into the egg. This fact emphasizes that insemination is a necessary antecedent to the second maturation division to advance further from metaphase.5) Only a single spermatozoon enters an egg (Fig. 7). Thus the carp egg is monospermic in fertilization under the normal condition.6) About 40 to 50 minutes after insemination there is found at the animal pole of the egg the already metamorphosed female pronucleus. It assumes a spherical shape lying in naked state in the ooplasm (Fig. 13).7) As early as 50-60 minutes after entrance, the head of the spermatozoon is converted into the male pronucleus attaining nearly spherical form (Figs. 10-12). At this stage there is no visible demarkation between the male and female pronuclei, so far as their structure and size are concerned. But there is present a ramarkable character peculiar to the male pronucleus. That is the pronounced appearance of the sperm-aster.8) After completion both the pronuclei begin to move towards the deeper part of the animal pole of the egg to conjugate with each other (Fig. 14). The conjugation of the pronuclei generally takes place about 1 hour after insemination occurs (Figs. 15-17).9) After the two pronuclei conjugate together, they do not actually fuse, but lie side by side in closs contact with the nuclear membrance intact (Figs. 15-16). Thus the maternal and paternal chromosome materials are separated in distinct groups during the stages prior to the first cleavage division. The chromosomes are formd independently in each vesicle (Fig. 17).10) In the egg about 11/2 hours after fertilization, the first cleavage spindle is generally formed (Fig. 18).

Triganodistomum hypentelii n. sp. (Trematoda: Lissorchiidae) from the Hog Sucker, Hypentelium nigricans (Le Sueur)

Examination of the small intestine of a hog sucker, Hypentelium nigricans (LeSueur)2 collected on April 3, 1941, from the Saline River near Milan, Monroe County, Michigan, revealed three specimens of a new species of trematode belonging to the genus Triganodistomum Simer, 1929. In the fall of 1941, two of nine hog suckers taken from the Saline River near Saline, Washtenaw County, Michigan, contained three and four adult trematodes respectively. The following description is based upon the study of living specimens, and of whole mounts and transverse sections of specimens fixed in hot Bouin's fluid, stained in Mayer's paracarmine (whole mounts) or Ehrlich's acid haematoxylin (sections), and mounted in clarite X (Nevillite no. 1).

INCREASE OF CORTICAL CALCIUM WITH AGE IN THE CELLS OF A ROTIFER, EUCHLANIS DILATATA, A PLANARIAN, PHAGOCATA SP., AND A TOAD, BUFO FOWLERI, AS SHOWN BY THE MICROINCINERATION TECHNIQUE

A rotifer, Euchlanis dilatata, a planarian, Phagocata sp., and toad, Bufo fowleri, were selected for an analysis of calcium localization and of possible calcium increase with age. Samples of animals of different ages were sectioned, incinerated and examined with a darkfield microscope. Calcium was identified by means of sodium alizarin sulfonate and the gypsum reaction. It was demonstrated that calcium increased with age in the cell membranes of all the cells studied. Calcium also increased with age in the nuclei and cytoplasm of nerve and interstitial cells of Phagocata and the cells of Euchlanis. Because of the large amounts of calcium present in the nuclei it was difficult to determine the degree of calcium increase.The iron content of the various cells appeared to vary considerably but inconsistently with age.Control slides stained with Ehrlich's haematoxylin showed a. correlation between the intensity of staining of structures and the calcium content of those structures. The suggestion was made that Ehrlich's haematoxylin may be used as an indicator for bound calcium in cells.

A New Astomatous Ciliate from the Newt, Eurycea bislineata (Green)

In the course of examination of the intestinal contents of 21 specimens of Eurycea bislineata (Green) collected at Oxford, Mass., during the summer of 1939 a single newt was found of which the small intestine was heavily infested with an astomatous ciliate. Observations and measurements were made on the living organisms which were later killed with hot saturated aqueous mercuric chloride solution containing 2% of acetic acid. These specimens were ultimately stained, some with acid carmine, others with Ehrlich's acid haematoxylin with or without a counterstain of eosin.

Hymenolepis scalopi n. sp.

The worms were studied both alive and preserved. They were fixed either in Gilson's or Bouin's fluid, stained in borax carmine, cleared in toluol, and mounted in balsam. Some of the pieces were later removed from the balsam, sectioned, and stained in Ehrlich's acid haematoxylin. All of the measurements recorded below, except the length and width of the entire strobila, were taken from preserved specimens.

MICROSPOROGENESIS AND THE DEVELOPMENT OF THE MALE GAMETES IN PORTULACA OLERACEA

Dahlgren (I9I6), Rocen (I927), and Tjebbes (I928) have described the development of the anther and microsporogenesis in Portulaca; grandflora. Rocen also examined material of P. oleracea and found that it does lnot differ materially from P. grandiflora. in the development of the floral structures. The haploid chromosomne number is reported by Tjebbes as 9 in P. graudiflora and by Hagerup (1932) as 27 in P. oleracea. Buds of various ages were collected during the early summer of 1932 from plants growing along the roadsides near the campus of the University of Wisconsin and fixed in Licent's solution, La Cour's chrom-acetic-osmic solution, and Karpechenko's modification of Nawaschin's solution. The material was imbedded in paraffin, and sections io to I2y in thickness. were cut, mounted serially, and stained in Ehrlich's and in Delafield's haematoxylin. For stages in pollen-grain development, Ehrlich's haematoxylin was particularly useful because of the transparency of the stain.

Some Notes on Dibothriocephalus Perfoliatus

The specimens described in the following paragraphs were collected from material secured by the German Soutlh Pole Expedition of 1901-1903. They were sent to Professor Pintner, head of the department of invertebrate zoology in the Un-iversity of Vienna, who placed the material at my disposition for determination and description. The specimens, though now over a quarter of a century old, are in a remarkable state of preservation. Aside from a slight bleaching and some brittleness due to the action oL -the preservative fluid, the specimens are quite suitable for investigation. Mention must be made here of the procedure follbwed in staining sections. The ordinary paraffin method was used, and the sections cut to a thickness of ten micra. Borax carmine as a staining agent proved unsatisfactory. The best results were obtained by using Ehrlich's hematoxylin preceded by iron alum. The sections were takeut through the alcohol series and then treated with distilled water. They were then immersed in a bath of iron alumr for about twenty minutes, washed in distilled water and stained for about five minutes with Ehrlich's hematoxylin. The stain is washed off under running tap water. Distilled water does not give results. Sections prepared in this way show every detail of structure, especially the nucleus. Up to the present, about fourteen species of Bothriocephalidae have been described. But this number must be reduced to eleven because three specific names are evidently synonyms. The species under consideration is Dibothriocephalus perfoliatus, (Diphyllobothrium clavatum, Railliet.) It was founid in sevent specimens of a species of seal,-Leptony-

Prof. Paul Ehrlich

ONLY recently we lost Löfler, one of the pioneers of modern bacteriology, and now Paul Ehrlich has passed away. He died on August 20, as all good workers might wish to die suddenly in his laboratory, in full harness, and before the rust of age had dimmed his powers. Born in 1854 at Strehlen, in Silesia, of Jewish parents, he was one of the many distinguished Hebrews who have contributed to make Germany's fame what it is in the world of science and art. He was educated at the Gymnasium at Breslau, and afterwards at the Universities of Breslau and Strasburg, where he graduated in medicine. From the outset of his career he took the deepest interest in the chemical relationships of living matter and in the affinities of various reagents for living cells. One of his earliest researches was upon the effects of certain aniline colours upon living tissues, and he devoted much attention to staining methods, devising new stains, and Ehrlich's haematoxylin and Ehrlich's triacid stain are stock solutions in the present-day biological laboratory. An investigation on staining methods for the tubercle bacillus led to the discovery that certain dyes possessed a peculiar affinity for this bacillus, and this fact tinged his whole philosophy, and suggested the conception of the specific affinity of certain chemical groups for particular cells and tissues.

THE INVARIABLE BLOOD-STAIN

For years I have used in my laboratory a method for the staining of blood-films which has proved so satisfactory from all points of view as to have earned the term invariable. The idea is not original with me, but is based on two well-known principles, combined, modified and finally perfected. First of all, single films are best fixed by burning in absolute alcohol; a proper mixing of eosin with Ehrlich's acid hematoxylin gives an excellent stain, one which proved its worth before the panoptic formulas became so popular. This method is especially suited to the needs of the general practitioner, the man who desires results and who cannot become proficient in technic. Certain men, who do considerable blood work, are now using the invariable almost routinely. Several of these have prevailed on me to publish the formula and technic; and this will perhaps justify the addition of another