Heidenhain's Hematoxylin Research Articles

Two New Amoebae from the Box Elder Bug, Leptocoris trivittatus Say

While examining specimens of the box elder bug collected (1936-1938) in Salt Lake City for flagellates several other protozoa were observed from time to time. Of these, two, both Endamoebidae, were found with sufficient frequency and in large enough number to make their identification possible. The descriptions which follow are based on the examination of specimens in smears fixed with Schaudinn's fluid or osmic acid vapor, stained with Heidenhain's haematoxylin and mounted in balsam.

Seasonal Changes in the Testes of the Passerine Bird, Phainopepla nitens lepida.

This study was undertaken to determine the histological changes which take place in the testes of the Phainopepla during an entire year. It was hoped that such changes which were noted could be correlated in some way with the behavior of the birds. The collection of Phainopeplas for this study was started on November 26, 1937, and has been carried on periodically up to the present time, December, 1938. The gonads were removed while still warm and fixed in Bouin's solution and then carried through the usual paraffin method. They were sectioned and stained, using Delafield's hematoxylin and eosin for some, and Heidenhain's iron hematoxylin for others. The testes showed a gradual increase in size from a minimum of one mm on November 26, 1937, to their maximum size of 8 mm, which was recorded on May 14, 1938. While the general trend throughout the spring was upward in size some individual variations were noted. The testes showed no rapid increase in size until March 24th, and few sperms appeared in the lumina of the tubules until May 12th. This is rather unusual for in most seasons on the desert breeding starts in February and is at its height by the end of March. Although the rains were late and it stayed cool much later than usual this year it does not seem that the reproductive cycle should have been slowed to such an extent. Yet this seemed to be the true condition, as it was further substantiated by extensive field excursions during that period. In the winter testis (Fig. 1) the size was about 1 mm, the tunica albuginea was thick, as was also the tunica propria. The seminiferous tubules were small and there was an abundance of intertubular material, composed chiefly of connective tissue cells with possibly a few interstitial cells among them.

Differential Staining of the Anterior Pituitary Gland of the Cat

The terms “acidophile” and “basophile” as applied to the two recognized types of chromophilic cells of the anterior pituitary have been sanctioned by long usage. Nevertheless, these cells do not always give a specific reaction to acid and basic dyes respectively. The difficulty of classification has been further complicated by the demonstration of an additional tinctorial type in the female rabbit and cat which has been provisionally classed as a modified acidophile. This distinction is based on the differential staining reaction obtained with Heidenhain's “azan” technic following sublimate-formalin fixation, the standard acidophile reacting with orange G, the modified type with azocarmine.Both of these cells are stained with copper hematoxylin, Heidenhain's iron hematoxylin, Mallory's phosphotungstic acid hematoxylin and Kultschitzky's acetic hematoxylin. However, Hansen's chroroalum hematoxylin stains the basophiles intensely and also reacts very slightly with the so-called modified acidophile.The orang...

The chromosomes in the maturation of the germ cells of two species of triclad Turbellarians

AbstractThe maturation of the germ cells of the two hermaphroditic species, Curtisia foremanii (Girard) and Bdelloura candida (Girard) has supplied the material for this chromosome study.Both species of flatworms are believed by the author to possess a diploid number of twelve chromosomes and a haploid number of six, although Curtisia foremanii has previously been reported as having a smaller and variable number of chromosomes.A tendency of the chromatids comprising individual chromosomes to separate from one another at certain times was noted in both species. This action results in giving the appearance of a larger number of chromosomes than the germ cells actually possess. A further source of apparent increase in chromosome number in the Curtisia oocytes, after treatment with the usual Allen's B3 and B15 and Heidenhain's iron haematoxylin, is the presence of some deeply staining, non‐chromatin material.No significant differences in number, form and behavior of chromosomes of male and female complexes were noted, with the possible exception of the tendency of the chromatids to separate from one another, to be greater in the female than in male germ cells.

VEGETATIVE REPRODUCTION IN CHLOROPHYTUM ELATUM

THE PLANT commonly known as Chlorophytum elatum R. Br. is easily propagated by small plants which develop at the tips and nodal regions of stolons. Scores of such individuals may be found on one mature plant, and no doubt such ease of propagation has helped to make this plant a favorite greenhouse subject. Other members of the Liliaceae, especially the closely related genus Anthericum, commonly known as St. Bernard's lily, illustrate this same method of rapid vegetative reproduction. This study was made to determine the origin of these new plants and of the stolons upon which they are produced. Bailey (1925) states that the scapes of C. elcatum are sometimes transformed into stolons which bear leafy offshoots. Fairburn (1936) in a general study on plant propagation mentions that Anthericum is reproduced by offsets attached to the parent by a short stem. Gravis and Donceel (1900) in speaking of the flower-stalk of C. elatum say that sometimes an axis corresponding to the peduncle is transformed into a foliated shoot capable of taking root. Laurie and Chadwick (1931) list Anthericum Liliago as reproducing by offsets and stolons. Most of the investigations have been concerned with practical methods of propagation and little with the histology of the origin of new plants. Detailed studies would give a more nearly complete understanding of cellular changes as related to the formation of new structures in vegetative reproduction. EXPERIMENTAL PROCEDUJRE.The plants used for the investigation were grown from a single original plant obtained through the courtesy of the Missouri Botanical Garden. Thirty young plants, from four to six inches high, were removed from the stolons in late September and placed in ordinary potting soil in the greenhouse. Later plantings were made at about four-week intervals. Early growth was rather slow, but after two months all the plants showed very vigorous growth and many new stolons. Material was removed for study at various intervals as soon as the young stolons first made their appearance. Such sections included the embryonic stolon, fips and nodes of the young stolon, and different stages of development of the young plants on the stolon. Material was fixed in the usual formalinacetic-alcohol mixture, dehydrated with alcohol, and cleared in xylene. Paraffin sections were cut 8-12 microins in thickness and stained in Heidenhain's iron-alum haematoxylin. Free hand sections were also generally employed throughout the investigation. GENERAL MORPHOLOGY. Chlorophytum is herbaceous, with a much shortened stem, long linear leaves, and rather fleshy roots. The flower scape is long and slender and considerably branched. It is almost erect when young but soon becomes procumbent, and from it the flowers and fruits as well as the new plants are

The Microtechnic of the Eye, with Suggestions as to Material

Fresh eyes are fixed 24 hours in Kolmer's fluid and dehydrated slowly, being opened only in absolute alcohol by a clean razor blade cut and the lens then removed if desirable. Embed in nitrocellulose by the hot-dry process, giving large eyes an extended hot or cold infiltration. Mallory's triple and Heidenhain's hematoxylin are the most useful stains. The combination of tree squirrel or chipmunk (for the globe as a whole) and copperhead (for the rods and cones) is urged in place of the mammals usually used, since it affords a closer simulation of human material where the latter is not available. The collared lizard is suggested for the study of the fovea, and the use of frog material in medical courses is criticized.

Dissection, Staining, and Mounting of the Embryos of Conifers

A statement is given of the advantages of this special technic and its place in embryological investigations, including directions for selecting the proper stages in collecting conifer cones and ovules, their methods of dissection from living material and their preservation for later dissection. The choice of dissecting microscopes and dissecting instruments, as well as directions for staining embryos with phloxine which may be combined with slow dehydration in glycerin, or for staining with Delafield's or Heidenhain's hematoxylin which may be followed by the glycerin dehydration are described. Glycerin affords a convenient break for a temporary stopping place in this technic.Directions are given for transfer from glycerin thru 95% and absolute ethyl alcohol into other solvents such as diaphane solvent, essence of euparel or an easily prepared sandarac solvent. Other mounting media which have been used for conifer embryos are discussed—glycerin jelly, Venetian turpentine and Canada balsam—emphasizing the ...

REGENERATION IN CRASSULA MULTICAVA

CRASSULA MULTICAVA Lem. (C. quadrifida Bak.) is a native of South Africa. According to Figdor (1918) this plant may be propagated not only by buds which are formed regularly in the axils of the bracts of the inflorescences but also by buds induced to form on leaves. The latter are formed after severance of the main vein both on leaves still attached to the parent and on those detached and placed on moist sand. Figdor reported that the new structures formed on the leaves arise from callus, although he failed to give the histological details. The purpose of this investigation is to make a histological study of the origin of the new plants formed on the leaves. EXPERIMENTAL PROCEDUJRE.-Material for this study was taken from cuttings obtained from the Missouri Botanical Garden and from the New York Botanical Garden. Entire leaves and portions of leaves were placed on moist filter paper in Petri dishes. Others were placed with the wounded surface in moist sand. At intervals of one or two days the material was examined with the binocular microscope, and basal portions of leaves or parts of leaves were removed and placed in chromo-acetic solution. The paraffin technique was used; the material was sectioned at 8 microns and stained with Heidenhain's iron-alum haematoxylin. Both transverse vertical and longitudinal vertical sections were made. GROSS MORPHOLOGY.-Crassula multicava is a low herb with more or less decumbent base. The stems are suicculent and bear opposite, decussate leaves (fig. 1). The petioles are short, sometimes so short that the leaves appear almost sessile, and petioles of a pair are joined. The fleshy, glabrous leaves are obovate with very obtuse tips and a tapering base. Numerous hydathodes give the leaves a more or less spotted appearance. The maximum size of the leaves is approximately 7.5 by 4.0 by 0.2 cm. Both lamina and petiole are composed largely of parenchymatous tissue. In this tissue there are occasional cells which are filled with tannin and are stained black in the preparations. Characteristic of both petiole and lamina is a layer of subepidermal cells almost all of which are filled with tannin (fig. 5, 8). This layer of cells aids in the study of the slides. The cells of the upper and lower epidermis are afike in cross section. The mesophyll is composed entirely of spongy tissue; there is no palisade layer (fig. 11). In the lamina these cells contain many chloroplasts, especially near the margins of the leaves; in the petiole the chloroplasts are less numerous. Commonly there are five leaf traces, one large central vein with two smaller lateral veins on each side of it. Occasionally there are seven traces. The phloem is arranged in small groups of cells on the

A New Cestode, Raillietina centrocerci, from the Sage Grouse Centrocercus urophasianus

In the parasitology collections at the University of Wyoming are two lots of cestodes from the sage grouse. One of these lots was collected by George L. Girard in the region of Daniel, the other by Dr. John W. Scott and Ralph F. Honess in the region of the Sweetwater River, both in Wyoming. In the lot from Sweetwater River both Rhabdometra and Raillietina are to be found, in the other only Raillietina. From this abundance of material, twenty specimens were selected and stained by various techniques. Paracarmine, cochineal, and Delafield's haematoxylin were found to be equally good for general purposes; however, testes and eggs were best brought out by Heidenhain's haematoxylin. Several scoleces were stained and so mounted as to present an anterior view, which insured more accurate observations on rostellum, hooks, and suckers. This work was done under the general direction of Dr. John W. Scott. Diagnosis. Raillietina: Genital pores irregularly alternate. Eggs singly dispersed in capsules; average diameter of eggs 28.5,u in capsules. Length of mature worms 135 to 450 mm.; maximum width 1.5 to 3.0 mm. near middle of worm. Number of proglottids 429 to 516. Average length of scolex 413u; width 431u. Length of neck 2.64 to 3.85 mm. Rostellum roughly hemispherical, bearing double crown of somewhat unstable hooks which number 198 to 205; average diameter of rostellum outside of crown 110u; length of hooks 15.7u. Suckers armed with several rows of unstable hooks of length 15u; average diameter of suckers 120u. Cirrus sac extends slightly less than half way to excretory canal, occasionally half way; length of cirrus sac 149 to 169g; width 65 to 88u. Length of cirrus 64 to 99,u; width 14 to 17,u. Testes number 63 to 118, average 85; diameter 42 to 73u. The ovary is a

CHROMOSOME MORPHOLOGY AND NUMBER IN TULIPA

IT HAS been shown by Newton (1927), De Mol (1925, 1928), Hall (1931), Newton and Darlington (1929), and recently by Upcott and La Cour (1936) that most of the species and varieties of Tulipa have a diploid number of 24, although there are triploids, tetraploids, and a pentaploid. Aneuploids have also been reported (Newton, 1927; Upcott and La Cour, 1936). In such a genus the chromosome number alone can be of little aid in helping to determine the probable relationships of species and varieties. Newton (1927), who recognized this, was able to separate certain groups of tulips on the basis of chromosome morphology. This was done by comparing the position of the spindle attachment constrictions and the relative lengths of the chromosomes. Upcott and La Cour (1936) have recently added to the studies of Newton (1927) and in addition have described secondary constrictions and satellites, but only in certain tulips. Similar features of chromosome morphology have been recognized in a number of other plants (Sakamura, 1920; S. Nawaschin, 1927; M. Nawaschin, 1925; Heitz, 1931; Lewitsky, 1931; Taylor, 1925, et a-l). During hybridization studies begun in 1932 it seemed desirable to study the morphology of the tulip chromosomes. MATERIALS AND METHODS. -The species and varieties were obtained from growers in this country and in Europe duiring 1932 and later. All but one, T. ocutis-solis, were allowed to flower in the greenhouse or garden and were compared with the descriptions of the species given by Hall (1928), Dykes (1930), and Bailey (1935). Bulbs were planted out of doors in the fall, and as soon as root growth was well started, they were dug and the root tips fixed in either Allen and Wilson's modification of Bouin's fluid or in Flemming's medium solution. Both fixatives gave good results, but in general, preparations from the former were slightly superior. The usual paraffin embedding technique was followed, sections being cut at thicknesses varying from 15 to 22 microns. Heidenhain's iron-alum haematoxylin stain gave excellent results and was used exclusively. All studies of chromosome morphology were made on chromosomes in the root tip. The details of chromosome morphology have been worked out not only from the chromosomes that were measured, but from a great many other metaphase and anaphase chromosomes as well. While the metaphase stages are particularly favorable to demonstrate secondary constrictions and satellites, length determinations at that stage are likelv to be misleading because it is very difficult to get perfectly flat chromosomes in the section. In determining lengths of chromosomes, anaphase stages were used exclusively. Measurements were made with a camera lucida and a pair of sharply pointed dividers and

An Improved Method for the Study of Chromosomes

It has been felt strongly that the current methods for the fixation of chromosomes are inadequate, inasmuch as the reagents are unduly long in reaching the important elements of the cells, particularly the nucleus. A realization of this situation has been at the bottom of the development of the so-called smear method. This method can, however, be advantageously employed only where loose cells are available, as in the reproductive elements of plants and animals. The present author has found that cutting up the material in very thin slices of almost microscopical tenuity, and passing them immediately into the best fixing solutions, namely those containing osmic acid, gives excellent results. The material is then assembled on cards in accordance with the present author's mass method (for which see the original article), and after embedding in nitrocellulose is sectioned 5μ or even thinner. Practically all methods of staining may be used, but the most satisfactory results have been achieved with Heidenhain's hematoxylin bleached almost to disappearance and followed by prolonged treatment with an aqueous safranin. This differentiates the chromosomal structures extremely well. It is clear by this method that chromosomes in all cases consist of a ground substance in which are situated two chromatids. In the somatic tissues these chromatids are apparently generally spiral and run in opposite directions. The crossing points of these spirals are responsible for the optical illusion which has been designated the gene.

CYTOLOGICAL PHENOMENA IN ALEURIA SP

THREE COLLECTIONS of Aleuria species, growing in damp soil on rotting wood, were made at Hayward, Wisconsin, during August, 1931.2 One of the collections of about thirty maturing apothecia, at first noted as a variant of Aleuria wisconsinensis, according to Seaver (1928), was found, upon closer examination, to vary from the type,3 and all apothecia were fixed in alcohol-formalin-acetic.4 The preliminary studies suggested possibilities for some worth-while observations. It was decided to make a complete study of various stages in the development of the fruiting body. Especially good material for the study of the developing ascus was available, anid it was hoped that an investigation might aid in settling some of the debatable questions as to chromosome number and behavior. Sections of apothecia, mostly seven microns thick, were stained in the following ways: At first Flemming's triple stain was used and proved unsatisfactory. Other stains were tried: Heidenhain's haematoxylin, counterstained with fast green, light green, cotton blue, or Orange G; Delafield's haematoxylin with the same counterstains, also dilute safranin; and aceto-carmine with Erlich's haematoxylin. Aceto-carmine preparations proved unsatisfactory. Heidenhain's haematoxylin counterstained with dilute safranin was used to good advantage; but the best preparations were, obtained with Delafield's haematoxylin and dilute safranin. Observations were made with a Bausch and Lomb 1/16 achromatic oil-immersion objective, and all drawings were made with the aid of a Spencer camera lucida so as to give a magnification of about twenty-one hundred. In reproduction these have been reduced one-fifth. OBSERVATIONS.-The largest apothecium studied was 4 mm. in diameter, the smallest less than 1 mm.; yet the size of the apothecium did not indicate the stages to be fouind within. Even in the smallest, abundant ascogenous hyphae and asci in all stages of development were present and, in some cases, stages of ascogonia. In the youngest apothecia, the

Proteolytic digestion and the problem of the pancreas in the ammocoete larva ofLampetra planeri

Remarkably little experimental work has been carried out upon the digestive system of the Cyclostomata. Alcock (1899) described the occurrence of peptic digestion in the ammocoete, but in view of the early date of this work and of the fact that the protease of the Tunicata is now known to be of the tryptic rather than the peptic type (Yonge, 1925; Berrill, 1929) the matter seems to demand reinvestigation. Moreover, recent histological studies on the “ pancreas ” and intestine of lampreys, to be described below, have led to conclusions regarding the distribution of the zymogen cells which have not yet been tested by experimental methods. The present work deals with these two problems, to the significance of which attention has already been drawn elsewhere (Barrington, 1935), and provides also a basis for a comparison of proteolytic digestion in the Cyclostomata with that in the Tunicata and in the Pisces. The ammocoete larvae ofLampetra planeriused in this work have been collected from the River Itchen in Hampshire at various times between April and September. Material for sectioning has been fixed in Bouin’s solution, Lane’s fixative, mercuric formol, and in Susa’s solution, and subsequently stained on the slide with Mallory’s stain, Heidenhain's haematoxylin, and by Lane’s method for zymogen granules. The experimental methods will be described later.

CHROMOSOME STRUCTURE AND BEHAVIOR IN SPHAEROCARPOS TEXANUS

In a previous paper (Tinney, 1935) the structure and behavior of the chromosomes in Sphaerocarpos Donnellii have been described. At that time sufficient material was not available to permit a thorough study of S. texanus Aust. The present study deals particularly with the structure of the resting nuclei in respect to heteropycnosis in differently active cells, and the structure and behavior of the chromosomes during the prophases in the female gametophytes of S. texanus. The literature dealing with the occurrence of sex chromosomes and of heteropycnosis in bryophytes was discussed in the previous paper. Schacke (igiga) found in American material of S. texanus, 8 chromosomes in both male and female. One chromosome (X) in the female is much larger than the other 7 and corresponds to a small chromosome (Y) in the male. In a few preparations an unstained region appeared near the middle of the X-chromosome (Schacke, igigb, unpublished). Wolfson (I927) figured and described a similar clear region in the X-chromosome of European material of S. texanus a distance from one end equal to about 2/5 of the entire length. He postulated that the differentiated region with slight affinity for stains is always present, but that it is frequently unobservable because of the position or the curving of the chromosome. Material received from Prof. J. C. McKee, collected near the campus of the Mississippi State College, was planted on soil in the botanical greenhouses at the University of Wisconsin, and allowed to attain an active state of growth before it was fixed. The fixative was Karpechenko's solution; the stain Heidenhain's iron-alum haematoxylin.

The neuromotor system of oxytricha

AbstractThe neuromotor system and associated organelles of Oxytricha have been studied in living specimens viewed with dark field illumination, and in whole mounts and sections stained with Heidenhain's hematoxylin or Mallory's stain. Microdissection has also been used in some cases.The neuromotor system has been found to consist of the adoral membranelles and coordinating fibrils, the undulating membrane and its basal fibril, two sets of cytostomal fibrils and two sets of postesophageal fibrils. The anal cirri may possibly be involved in the complex. No neuromotorium seems to be present.Motor and feeding organelles not morphologically connected with the above system are eight frontal and five ventral cirri, and a varying number of marginal cirri. The anal cirri, if not connected with the neuromotor system, would be included here. The minute structure of each type of cirrus has been studied, and the action and probable function has been determined from studies on normal, anesthetized, and dissected individuals. A brief comparison is made between the neuromotor system and organelles of Oxytricha and those of Euplotes and Paramecium that have previously been described in the literature.

Iron Hematoxylin Stain for Differentiation of Sclerites from Membranous Areas

In my work in comparative insect morphology I have encountered much difficulty in studying the sclerites of the exoskeleton of the mantis, Stagmomantis carolina , especially those of the genitalia. In this work the specimens were boiled in potassium hydroxide until the internal organs and tissues became sufficiently disintegrated for removal with forceps and a stream of water generated by a pipette. However, potassium hydroxide bleaches, and some insects, such as the carolina mantis, have little cuticular pigment, which means that the student experiences much difficulty in identifying sclerites that are invisible, or practically so. Gage stain Ent. News, May 1919) is of little value for morphological work, since the exoskeleton has to be preserved in 80 or 85 per cent alcohol, and Gage stain washes out in alcohol. The only thing left to do was to try some of the common stains. Heidenhain's iron hematoxylin produced excellent results in distinguishing the sclerites from the membranous portions.

Some Physical Properties of the Nuclear Membrane

During the last 50 years several investigators have described processes which bring into evidence such physical properties of nuclear membranes as resistance to deformation, extensibility, elasticity, etc. (Albrecht, Mottier, Kite, Chambers, Nemec). Our observations made on resting nuclei of the embryonic region of the onion root tip, centrifuged at 30,000 g., (35,000 r.p.m., radius: 22 mm.) are a contribution to the stock of facts already accumulated by these authors. The root tips, cut off at a length of 18 mm. were fastened by their cut end in a brass tube 15 mm. long and slightly wider than the roots; their free ends were in the centrifugal direction. The tubes were fixed firmly to the rotor of a Sharpies electric super-centrifuge. The rotor contained some Knop's plant medium in order to keep the roots steadily immersed. The centrifugation lasted from 10 minutes to 2 hours. The roots were then fixed in Flemming's fluid; and stained either with Heidenhain's iron haematoxylin or Flemming's triple stain. After a short centrifugation, the nuclear content is concentrated at the bottom of the nuclei and a clearer space appears at the top. Then the whole nuclei are thrown to the bottom of the cells. (Fig. 1 A.) So far these results are the same as those obtained by previous investigators working with a much lower centrifugal force. After a longer centrifugation the nucleus flattens against the floor of the cell, taking a more or less hemispheric shape, eventually forming an angle of less than 90° between the horizontal diameter of the hemisphere and the vertical portion of the nuclear membrane (Fig. 1B). The diametric line is perfectly straight and shows no evidence of any folding or shrinking. At the top of these hemispheric nuclei one can almost always observe an elevated portion of the membrane which encloses a transparent region and reminds one of a blister.

Heidenhain's Hematoxylin Used with the Smear Technic

Heidenhain's hematoxylin, according to the following formula, may be used for studying chromosomes in temporary smears of pollen mother cells: 1 part 0.5% hematoxylin and 4% iron-alum (equal parts ...

The Number of Chromosomes in the Cultivated <i>Brassica</i>

The number of chromosomes in Brassica has been reported by several authors. Since 1929 present writers also examined the chromosome number in 13 species of Brassica covering about one hundred cultivated varieties as the aid to the genetical work of the senior author.The plants that provided the materials were raised from the seeds obtained from home and abroad. The smear method and sometimes the paraffin method were used. In the case of smear methed, the staining of BELLING's iron-aceto-carmine and HEITZ's aceto-carmine with acetic alcohol were used. Staining of the chromosomes was satisfactory with these stains.For permanent preparations, CARNOY's fluids mostly and in a few cases NAWASCHIN's fluid were used as fixing reagent and HEIDENHAIN's iron-alum haematoxylin as stain.The different varieties within the same species exhibited invariably an identical chromosome number. The haploid number of chromosomes is given as follows:Brassica nigra, KOCH 8 Brassica arvensis, RABH. 9B. oleracea, L. 9 B. campestris, L. 10B. pekinensis, RUPR. 10 B. chinensis, L. 10B. narinosa, BAILEY 10 B. rapa, L. 10B. nipposinica, BAILEY 10 B. alba, RABH. 12B. juncea, COSS. B. napiformis BAILEY 18(including B. cernua, HEMSL.) 18B. Napus, L. 19

Reactions of Subcutaneous Tissue to Sodium Ricinoleate and Other Foreign Substances.

The purpose of this study was to ascertain whether there is a specific cytological response of subcutaneous tissue to detoxified toxin as compared with the reaction to a pure toxin. For further comparison of the reactions, one of the least toxic colloidal dyes was used.Adult rabbits were injected subcutaneously with single doses of 1 cc. of 1% trypan blau in distilled water, 1 cc. of 1% sodium ricinoleate in distilled water, 1 cc. of diphtheria toxin in 1/500 dilution, and 1 cc. of diphtheria vaccine, i. e., a combination of sodium ricinoleate and diphtheria toxin as prepared by Dr. W. P. Larson of the University of Minnesota. Subcutaneous tissue spreads were made by the spread method used by Maximow, von Mollendorf and others, and were fixed immediately in Zenker-formol. The stains used were Dominici's Eosin-Orange G and Toluidine Blue, Maximow's Hematoxylin Azure II-Eosin, Heidenhain's Iron Hematoxylin, and Weigert's Iron Hematoxylin. Tissue was taken at 45 minute, 1, 2, 3, and 4 day intervals for direc...