Iron-alum Haematoxylin Research Articles

Demonstration Of Entamoeba Histolytica Cyst From Urban And Rural School Children.

School children between 6 to 12 years of age were studied for the presence of Entamoeba histolytica cysts in a period of 6 months, form March to August 1980. Fecal specimens collected form urban and rural children were examined first by the Lugol stained technique and then by the Faust's iron alum hematoxylin method. A total of 731 specimens were examined. Of these 26.9 per cent, 197 specimens, harbored the cysts. The demonstration rate for the cysts by age group varied from 19.6 to 43.5 per cent. The demonstration rate in boys was 24.4 per cent and in girls 29.8 per cent. Similarily, the rate in urban and rural area was 32.4 and 24.1 per cent, respectively. It was found in the present study that there were no significant differences in the age and sex rates of demonstration between urban and rural children but there was a predominance of the small race of Entamoeba histolytica over the large race.

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.

On the antheridium development of Sargassum micracanthum and S. ringgoldianum

The antheridium development of Sargassum micracanthum Yendo and S. ringgoldianum Harvey was investigated with the iron-alum haematoxylin staining method and the aceto-carmine squash technique. Synapsis occurred at the prophase in the first division while the chromatin threads were still contracted, and the nucleus passed through diakinesis. The first two divisions in the antheridium were meiotic, and the four other divisions (mitosis) followed without cytokinesis. In S. micracanthum and S. ringgoldianum, 31 and 32 chromosomes respectively were counted at prometaphase in the first division, 64 sperm nuclei were organized in the final division. After the final division cytoplasmic cleavage occurred, and 64 sperm cells were finally completed in an antheridium. Centrosomes and asters could not be discerned at any of the divisions, because of limited magnification facilities. A peculiarly shaped small body was observed at the prophase of the first division, but not thereafter.

Infectivity, Growth, and Development of Leucochloridiomorpha constantiae (Trematoda) in the Chick and on the Chorioallantois

Leucochloridiomorpha constantiae metacercariae grew, attained sexual maturity, and produced eggs containing fully developed miracidia in the bursa of Fabricius of the domestic chick and on the chick chorioallantois. Chicks cloacally exposed to metacercariae remained infected up to 8 weeks although the bursa was still present after 12 and 13 weeks. Following cloacal exposure with metacercariae, a low percentage of worms was recovered from the rectum of chemically bursectomized chicks. Body and testicular growth were greater in chickthan in chorioallantoic worms although as development progressed both demonstrated similar testicular-to-body size ratios. Daily development in the chick and on the chorioallantois has been described through sexual maturity. Chorioallantoic worms became ovigerous and contained eggs with miracidia later than chick worms and frequently produced aberrant eggs and abnormal stages of spermatogenesis, suggesting the chick is a more favorable site for development. Leucochloridiomorpha constantiae (Mueller, 1935) parasitizes the bursa of Fabricius of the black duck, Anas rubripes, and has been maintained in the small intestine of raccoons, and in the bursa of domestic ducks and chicks in the laboratory (Gower, 1938; Allison, 1943; Fried and Harris, 1971). Free metacercariae obtained from the uterus of naturally infected Campeloma decisum snails are suitable for experimental studies since they are easily maintained in the laboratory, infect domestic chicks and the chick chorioallantois, and become ovigerous in these sites within 3 or 4 days. This paper reports studies on infection, growth, and development of this trematode in the chick and on the chorioallantois. MATERIALS AND METHODS As described previously (Fried and Harris, 1971), metacercariae obtained from C. decisum snails were used to infect 1and 2-day-old domestic chicks cloacally and worms were recovered from the bursa. Chicks exposed to 4 to 6 metacercariae were necropsied at intervals from 5 min to 13 weeks to determine infectivity. Growth and development studies were made on worms reReceived for publication 7 September 1971. * Supported in part by a research grant from the Lafayette College Committee on Advanced Study and Research. t Present address: Institute for Pathobiology, Lehigh University, Bethlehem, Pennsylvania 18015. + Present address: Cornell University Medical College, New York, New York 10021. covered daily from 1 to 7 days following exposure of chicks to either 3 or 5 metacercariae. Infectivity was also determined for 14 1and 2-day-old chicks exposed orally to a total of 116 metacercariae (5, 6, or 10 each) in Ringer's and necropsied 3 days later. Chemically bursectomized chicks were hatched from eggs which, on the 3rd day of incubation, had been dipped, pointed end down, into cold 3% testosterone propionate (Nutritional Biochemicals Co., Cleveland, Ohio) in 95% ethanol to a depth of approximately 40 mm for 5 sec (Glick and Sadler, 1961). Infectivity was determined for 14 1and 2-day-old bursectomized chicks exposed cloacally to a total of 94 metacercariae (3 to 17 each) and necropsied at intervals from 4 hr to 3 days. Metacercariae transferred through 3 changes of sterile Ringer's were placed on chorioallantoic membranes of 8to 10-day-old fertile white leghorn eggs (Zwilling, 1959). Eggs which received 5 or 6 metacercariae each were incubated at 37.5 C and flukes were recovered daily from 1 to 8 days postinoculation. Metacercariae, chick-, and chorioallantoic worms were pipetted into hot AFA (Fried, 1962a), stained in Gower's (1939) carmine, cleared in terpineol, mounted in Permount, and used for growth measurements. Worms stained intravitally with neutral red were examined under light coverslip pressure to assess development and observations were also made on Bouin's and Caroy fixed paraffin sections stained with either Harris' hematoxylin or Heidenhain's iron alum hematoxylin and eosin.

A FIBER APPARATUS IN THE NUCLEUS OF THE YEAST CELL

The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope.

Retarded Embryo Sac Development in Irradiated Clones of Malus silvestris Hort. cv. ‘Golden Delicious’

SEVEN ‘Golden Delicious’ apple clones propagated from buds irradiated at 5,000 r. in a cobalt-60 source were selected for high, intermediate, and low pollen abortion. Pollen abortion was 4.9 per cent in a typical check clone, and varied in the irradiated clones from 10.4 per cent to 84.8 per cent. Blossoms collected at anthesis were sectioned serially at 10µ thickness and stained with Heiden-hain's iron alum haematoxylin for embryo sac examinations.

INDUCTION OF POLYPLIODY BY KINETIN IN TRITURUS VIRIDESCENS.

IN 1962 Buckley, Witkus and Berger1 reported that kinetin induced mitotic divisions throughout the digestive tract of the salamander, Triturus viridescens. It was also reported that these were diploid divisions. The effect followed exposure for 9 days to kinetin and was most evident in the duodenum. Since this was one of the first clear cases of mitotic stimulation in animal tissues by kinetin, a more detailed examination was made of the duodenum. As in the previous work1, the animals were immersed in a 10 mg per cent solution of kinetin for 9 days. During the period of treatment the animals were not fed and the solutions were changed every third day. After 9 days exposure the animals were returned to spring water and allowed a 9-day period of recovery. Immediately after treatment and after daily periods of recovery the tissue was fixed, sectioned and stained using the standard iron alum haematoxylin technique. Acetoorcein smears were also made of the same regions.

Cytology of Rhodotorula glutinis.

Thyagarajan, T. R. (Cornell University, Ithaca, N. Y.) and H. B. Naylor. Cytology of Rhodotorula glutinis. J. Bacteriol. 83:127-136. 1962.-The structure and manner of division of nuclei in actively dividing cells of Rhodotorula glutinis were studied with the phase contrast microscope. The nucleus consists of a dense central body, surrounded by a shell of optically uniform material of low density. The entire structure is enclosed within a nuclear membrane. Various fixation and staining techniques were employed to confirm the observations made from living cells. Since the dense central body is Feulgen-negative and is readily stained by iron alum hematoxylin, it is identified as the nucleolus. The material surrounding the nucleolus has no marked affinity for hematoxylin but is Feulgen-positive and stains intensely with Giemsa and basic fuchsin. The nucleus appears to divide by a process of elongation and constriction during which roughly half of the nucleolus, along with the surrounding chromatin, passes into the bud. The nuclear membrane was found to persist during all stages of division. Vacuoles were seldom observed in actively dividing cells. The nucleus of R. glutinis is similar in structure to the nuclei of higher organisms, but its behavior during division is quite different.

The Life History of Scaberia Agardhii Greville

Scaberia agardhii is an endemic species in southern Australia. Growth is initiated by means of a three-sided apical cell which segments and forms the meristoderm, the cortex, and the medulla. Slender, scale-like, and verruciform segments occur. The plant is monoecious and the conceptacles are bisexual. On germination two primary rhizoids are formed. Scaberia is included within the Cystoseiraceae. ent parts of southern Australia. No complete account of the genus is available. Gruber's observation on the presence of a three-sided apical cell was based on a fragmentary specimen (1896). This study, based on adequate fresh, liquid-preserved, and dried material, was undertaken to obtain detailed morphological, anatomical, and embryo- logical information of the genus, and so to clarify its systematic position. Material and Methods In most cases the material was preserved in Karpechenko solution (Papen- fuss modification) or in 4 per cent, formal in sea water. After washing five or more times in sea water the material was dehydrated in the usual manner in seawater-distilled water-alcohol mixtures. Johansen's method (195 1) of em- bedding was carried out after dehydration. Microtome sections of various thicknesses between 4 and 12 fx were cut and stained in 0-5 per cent, iron alum haematoxylin. Material collected for embryological studies was brought into the laboratory either immersed in sea water or in plastic bags. Within an hour from the time of collection or immediately conceptacles (fertile segments) were washed thoroughly in sea water and placed in Petri-dishes, or on slides in filtered sea water. The conceptacles were removed after 2 hours and the water was changed gently. Either three times filtered sea water or Schreiber or Erd- schreiber solutions were used as culture media. The zygotes, attached on the dishes or on slides, were rinsed with the culture solution to remove debris.

XYLEM ANATOMY OF SOPHORA (LEGUMINOSAE) IN RELATION TO HABIT

CUMBIE, B. G., and DAN MERTZ. (U. Missouri, Columbia.) Xylem anatomy of Sophora (Leguminosae) in relation to habit. Amer. Jour. Bot. 49(1): 33-40. Illus. 1962.-A comparison of 8 tree, shrub, and herbaceous species of Sophora showed considerable variation in structural organization of the secondary xylem in relation to habit. A reduction in duration of cambial activity has been accompanied by a decrease in conspicuousness of storied structure, more frequent arrangement of vessels in multiples, the formation of narrower rays, and a striking reduction in amount of axial xylem parenchyma. On the other hand, relatively minor changes in size of xylem elements have resulted from decreased cambial activity, as shown by comparing the first-formed secondary xylem elements of woody and herbaceous species. In many structural features, shrubs were found to be more or less intermediate between the tree and herbaceous species, at least in the early stages of development. These observations indicate that the often repeated generalization that the organization of herbaceous stems resembles that of the first year's growth in closely related woody species is not always applicable. AN EARLIER investigation of stem anatomy in herbaceous Leguminosae (Cumbie, 1960) provided a basis for comparing the structural organization of secondary xylem among plants of different habits. Although only limited comparisons were possible, a number of differences in xylem structure between woody and herbaceous species indicated that the often repeated generalization that the organization of herbaceous stems resembles the first year's growth in closely related woody species may not always be valid. The observations suggested, however, that intensive studies of individual genera would permit the modifications accompanying decreased cambial activity to be defined more precisely. This investigation of the genus Sophora extends the earlier study. Sophora is one of several genera in the Leguminosae which includes species of widely varying habits, ranging from tall trees to small, herbaceous plants. Although usually described as a woody genus, there are a few perennial herbs among approximately 70 species (Turner, 1960) of Sophora. The species are distributed sporadically over a large part of the world, mainly the New World subtropics, warmer temperate regions of North America and Asia, and many islands in the Pacific area. Because of its widespread distribution, the genus is usually considered to be an old one (Andrews, 1914). MATERIALS AND METHODS-Since a comparison of xylem anatomy of species of different habits was the main objective of this study, 8 representative tree, shrub, and herbaceous species were included. These are listed in Table 1 with collection data cited according to the method proposed by Stern and Chambers (1960). Most of the material was pro. 1 Received for publication May 29, 1961. This investigation was supported in part by a grant to the first author while on the staff of Texas Technological College. Sincere appreciation is extended Professor Charles Heimsch for his helpful advice and constructive criticism of the manuscript. vided by Professor B. L. Turner, The University of Texas; Dr. W. L. Stern, Smithsonian Institution; and Professor R. H. Wetmore, Harvard University. The assistance of these individuals is gratefully acknowledged. Except for S. japonica, young twigs for all woody species as well as older material were available. Following the procedures outlined by Wetmore (1932), stems and blocks of wood were sectioned by the celloidin method. Sections were stained in Heidenhain's iron-alum hematoxylin and safranin. Macerations were prepared following Jeffrey's method as outlined by Johansen (1940). In the anatomical descriptions, the diagnostic features believed most significant for this investigation were selected from among those listed by Tippo (1941) . Cell dimensions were determined by measuring 100 elements for each character. Most descriptive terminology adheres to that advocated by the Committee on Nomenclature of the International Association of Wood Anatomists (1957). Since many xylem characteristics in woody plants undergo ontogenetic changes associated with growth in diameter, the development of each character was considered in plants of this habit. In referring to xylem in different stages of development, the secondary xylem at the outer margin of the primary xylem is designated as first-formed secondary xylem or wood near the pith, and the most recently formed xylem as outer or later-formed wood. It seemed inadvisable to use the term adult wood or mature wood for the outer wood in this study, because it was not possible to determine if most samples available were actually mature, and in some specimens it was readily apparent that the outer wood was not mature on the basis of cell dimensions. ANATOMICAL DESCRIPTIONS-Storied structureStorying of vessel elements and axial wood parenchyma occurs in all trees and shrubs examined

Chromosomal elimination induced by 6-aza-uracil.

DURING preliminary work in this Laboratory dealing with the effects of some antimetabolites on cell division in Allium cepa, it was noted that 6-aza-uracil brings about chromosomal elimination. This elimination occurred at metaphase and anaphase. Root tips from bulbs after reaching a length of 2–3 cm. were treated for 6 hr. with a 1 per cent aqueous solution. The solution was prepared by first grinding the powder and dissolving it in warm tap water. In order to clarify the location of the spindle attachment region of the chromosomes the root tips were treated with a 1 per cent aqueous solution of colchicine for a half-hour prior to fixation. On each preparation the number of normal metaphases, anaphases and telophases and the number of these stages showing elimination were counted. The eliminated chromosomes were studied in detail to determine whether or not the same chromosomes were always eliminated. Aceto-orcein smears and sections stained in iron alum haematoxylin were used.

Fungus Nuclei in the Diagnosis of Mycoses

SUMMARYThe morphologic integrity of the fungus nucleus is lost in histologic preparations, unless all stages of fixation, dehydration, infiltration and staining are carefully conducted. Fixation with a modification of Bouin's fixative, dehydration and infiltration by slow and graded steps and staining with Heidenhain's iron-alum haematoxylin demonstrate small nuclei in the tissue phases of some pathogens of man with a minimum of apparent distortion and shrinkage.Coccidioides, Blastomyces and Paracoccidioides are multinucleate. Histoplasma and Cryptococcus are uninucleate. The nuclei of these fungi are characteristic of those described and illustrated in many other fungi and consist of a delicate nuclear membrane, a vacuolated nucleoplasm and a densely stained single nucleus.

Morphological and Physiological Studis on Nodule Formation in Leguminous Crops : VI. On the factors detemining the location of nodule formation on root system in kidney bean and sword bean.

In a previous paper (KAMATA 1958a), the author reported that eighteen leguminous crops might be classified into two groups which belonged to the following types ; i. e. I-A type--nodules formed on both the primary and all the lateral roots, and I-B type--nodulation took place only on the lateral roots. This experiment was carried out to know the mechanism of nodule formation in I-B type using kidney bean (Phaseolus vulgaris L.) and sword bean (Canavalia gladiata D. C.) as materials. By the histological study, it was clearly seen that the number of the cells in primary root, which were infected with penetration of Rhzobium were similar in both cases, I-B type (kidney bean and sword bean) and I-A type (soybean and peanut), even though the nodule scarcely formed afterwards on the primary root in the former. The weak staining with HEIDENHAIN'S iron-alum hematoxylin, when examined microscopically, proved no proliferation of nodule bacterium in the first infection cells of I-B type plants. Cutting off a part of cotyledon from seed before sowing resulted to stop the development of nodule on a primery root in I-B type crops until flowering season. The nodule bacterium however, succeeded to grow, when it was cultured on RAGGIO'S complete cultural medium added with the different amount of the extract obtained by the systematic extraction method from primary, secondary and tertiary roots of I-B type plants. The fact proved that the extract contained no inhibitor, which might be a toxic or promotive substance for the growth of bacterium in primary roots of I-B type plants. In comparison of the amount of reducing sugar, non-reducing sugar and crude starch in root and rootlets of I-A type plant (soybean) and I-B type plants (kidney bean and sword bean) measured by Somogi's method, a complete parallelism was found between the concentration of available sugar, which was carbon source for growth of nodule bacterium in vitro, and the degree of nodule development. The amount of available sugar in the primary root of I-B type plants was lower and the amount of unavailable carbohydrate such as starch was higher as compared with those contained in other rootlets. Therefore, the nodule bacterium in the first infection cell might be inhibited to grow by the lack of available sugar in the primary roots in I-B type plants. If the above mentioned supposition is appropriate, nodules must appear on primary root of I-B type crops, when unavailable carbohydrate in the root is changed into available sugar. The wilting treatment was thought to be one of the useful methods for changing carbohydrate to available sugar, and such results were obtained after treatments of wilting given three times during seedling stage as incease of available sugar, decrease of crude starch and the development of nodules on the primary roots in both the case of kidney bean and sword bean.

Histological studies on pollen degeneration in male-sterile Japanese radish.

(1) Previously the author (1951) reported on the spontaneous occurrence of the male-sterility in Japanese radish (Raphanus sativeis L.) and also explained that it was caused by a Mendelian reccssive gene. Results of histological observations carried out with anthers of both the sterile and its comparable fertile individuals are reported in this present paper. Flower buds or anthers were fixed with Carnoy's fluid. Usual methods of dehydrating and embedding in paraffin were employed. Sections were cut 10, h thick and stained with Heidenhain's iron-alum haematoxylin. (2) Development of the anther and formation of the pollen grains in the normal fertile radish : Tissues of a very young, anther consist of two parts, i.e., archesporial and parietal tissues. Archesporium situated in the center is surrounded by three or four layers of parietai cells. Cells of the former tissue are somewhat larger and rounder, and stained more densely with haematoxylin than those of the latter (Fig. 1). With the progress of auther development one layer of tapetal cells differentiates in the innermost part of the parietal tissues, enclosing the archesporium. Tapetal cells are clearly larger and angular or rather irregular in shape, and appear densely stained with haematoxylin than those of other neighboring tissues (Figs. 2 and 3). Such normal tapetal tissue begins, in turn, to degenerate when the pollen grains are formed, and it even disappears completely before the blooming time. On the other hand, the endothecium of the parietal tissue becomes thick and fibrous, so it can play an important rale for the dehiscence of anther walls (Fig. 4). Microsporgenesis proceeds quite normally and the mature pollen grains are seen well dispersed at the flowering.

牛馬胎児生殖腺の組織学的所見

The gonads of 9 male and 9 female fetuses of the cattle were used in this study. The fetuses are 5.7 to 45.0 cm in "crown-rump length" in males and 16.3 to 38.0 con in females. The gonads were fixed in 10% formalin and ZENKER-formol. Routine alcohol dehydration and paraffin embedding were practiced. Sections were cut at 7μ. stained with hematoxylin-eosin or HEIDENHAIN's iron alum hematoxylin, and after MASSON's trichrome method, VAN GIESON'S method and BIELSCHOWSK Y-MARESCH reticulm method. The following observations were made.Testis: There is a cuboidal layer of germ epithelium in the testis of a 5.7-cm-long fetus It becomes flattened gradually with ages. With the vascularization in the tunica albuginea, there is a remarkable increase of connective tissue fibers in the vascularized areas. The seminiferous tubules are distinctly limited by a membrana propria, exterior to which is a thin layer of small connective tissue cells forming a capsular investment. The tubules consist of two kinds of cells, i. e., indifferent cells and germ cells. The former is numerous and the latter a few. Both cells in the tubules have a tendency to increase in number, the numerical ratio between them being scarcely altered in all testes used. Interstitial cells are found sparsely at first and then form cluster-like masses with gradual vascularization.Ovary: In 16.3-cm-to 18.5-cm-long fetuses, the sex cords having a broad hand-like appearancee contain many small indlfferent cells and a few germ cells. Primary follicles increase gradually in the inner part of the cortex. In the bodies of 25-cm-and 27-cm-long fetuses, the sex cords manifested a degenerated appearance. Meanwhile, in a fetus grown 30.0-cm-long, the remarkable change is noticed with the increase in replenished sex cords. Therefore, it is presumed that the sex cords have newly been formed descending from the germ layer, or reconstructed from the parenchymal tissues in these growing stages. No interstitial cells have been found in any ovaries used.The origin of the interstitial cells in the testisand of the medullary cords in the ovary is also. discussed briefly.

タマネギの雄性不稔個體に於ける花粉退化の細胞學的研究

Male-sterile plants were obtained from a variety of the onion, Senshu-Hiragata-Ohtamanegi (a strain Yellow Glove Danvers), in 1944 and 1948. The pollen degeneration of the male-sterile plants was investigated cytologically in 1949 and 1950. The flower buds and anthers of the normal and the male-sterile plants were fixed in CARNOY's fluid. The usual methods of dehydrating and embedding in paraffin were employed. The sections were cut 10μ thick and stained with HEIDENHAIN's iron-alum haematoxylin. The results obtained may be summarized as follows:1. The maturation divisions of P.M.C's in the male-sterile plants were normal up to the liberation of microspores from tetrads, but the nuclear divisions of microspores were not observed.2. The cells of the anther tapetum began to behave in an abnormal manner, from late tetrad stage to the liberation of microspores (fig. 3). The boundaries of the tapetal cells broke down, and the contents flowed together to form a periplasmodium (fig. 4).3. After the periplasmodium has attained maximum development, it began to degenerate (fig. 5). With the complete disintegration of the periplasmodium the microspores were finally destroyed. The anther cavity became empty, except for a dark strand of disintegrating microspores (fig. 6).

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.

MEGASPOROGENESIS AND DEVELOPMENT OF MEGAGAMETOPHYTE IN ULMUS

MEGASPOROGENESIS AND DEVELOPMENT OF MEGAGAMETOPHYTE IN ULMUS

HISTOLOGICAL EFFECTS OF GROWTH‐REGULATING SUBSTANCES ON SUNFLOWER TISSUE OF CROWN‐GALL ORIGIN GROWN IN VITRO

VARIOUS GROWTH-REGULATING substances, each at progressively increasing concentrations, have shown marked effects on the development of sunflower and tobacco callus in tissue cultures (Hildebrandt and Riker, 1947). The sunflower tissue came from crown-gall but was bacteria free; the tobacco tissue came from a natural gall on a hybrid. Except for the amount of growth produced, no conspicuous macroscopic differences appeared in these tissues. However, the possibility remained that histological differences might be present. The inhibition of growth of plant tissue with a strong concentration of growth substance has been reported (K6gl et al., 1934; Marmer, 1937; Levan, 1939). Kraus et al. (1936) have shown the increased activity and proliferation of cells of bean resulting in the formation of adventitious roots when treated in certain ways with indole-3-acetic acid. Since then, many investigators have descrihed responses made by different types of plant cells following applications of different growth substances (reviewed by Skoog, 1947, who includes other reviews). The microscopic structure of tobacco callus cultured in vitro has been described by White (1939) and by Caplin (1947). White considered the two main requirements for a true tissue culture are an undifferentiated state and the unlimited capacity for growth. The effects of growth-regulating substances on normal and pathological plant tissue growing in vitro have already received some attention. Gautheret (1945, 1947a) for example. in a series of papers has shown the concentration to be a critical factor in callus development and differentiation. Similarly the effects of indole-3-acetic acid, indole-butvric acid and alpha-naphthalene-acetic acids, respectively, on normal plant tissue and bacteria-free tissue of crown-gall origin of sunflower and periwinkle. grown in vitro, was reported by de Ropp (1947). The normal tissues responded at high and low concentrations by proliferation of cells of the cambial zone and by root formation. Tissue of crowngall origin remained unchanged in structure, except at the high concentrations where growth of the tissues was reduced. Gautheret (1947a, 1947b) '-Received for publication December 12, 1948. This work was supported by the Donner Foundation and by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Valuable suggestions were received from Dr. J. B. Torrie on the statistical analysis and from Prof. Emma L. Fisk on the manuscript. studied normal plant tissue cultures grown in vitro. At a low concentration of indole-3-acetic acid, considerable cell division appeared; whereas cells without the growth substances failed to grow. As the concentration was increased, the frequency of cell division decreased but a swelling of the cells in all directions followed. The present studies were made to determine what, if any, histological changes were induced in the bacteria-free crown-gall tissue of sunflower by the added growth substances. This extends the work already reported by Hildebrandt and Riker (1947). Representative masses of tissues were fixed, sectioned, stained and examined. MATERIALS AND METHODS.-The sunflower used, the cultural procedures, and the amount of growth secured has been described bv Hildebrandt and Riker (1947). Representative tissues from 13,000 individual pieces were selected for examination. They explained that the sunflower tissue was originally isolated in 1941 from a secondary petiolar crown-gall but no longer contained crown-gall bacteria. The weak concentrations of growth-regulating substances on which sunflower tissue cultures examined here were grown, were 1 X 10-11 g./l. for indole-3-acetic acid and for alpha-naphthaleneacetic acid; I X 10-9 g./l. for indole-butyric acid; and 1 X 10-7 g.jl. for parachlorophenoxy-acetic acid. These concentrations were selected because they represented high points on the growth curves already described. The strong concentration for all of the growth substances was 1 X 10-3 g./l. Control cultures on media lacking a growth substance were also examined. After 6 weeks incubation on the media, fragments of the tissue were weighed and then fixed in formalin-aceto-alcohol. The tissue fragments were dehydrated with n-butyl alcohol and embedded in paraffin. Sections 10 , in thickness were cut on a rotary microtome. The stains used were either ironalum hematoxylin and safranin, or fast green and safranin. Microchemical tests (Johansen, 1940) were made for membrane substances. For statistical analysis, the total number of cells and the number of tracheal elements per unit area (approximately 1/4 sq. mm.) were counted. Cells were counted in ten fields in each of five different tissues at each of the concentrations (control, weak and strong) used for the four growth-regulating substances. OBSERVATIONS.-The kinds of cells present and their development were relativelv simple in this

ORIGIN AND DEVELOPMENT OF AXILLARY BUDS: SYRINGA VULGARIS L.

A SURVEY of botanical literature shows that the origin and the early stages in the development of axillary buds have been investigated a number of times, yet the later phases of development have, in contrast, received comparatively little attention. The works of Koch (1893), Schmidt (1924), and Louis (1935) are especially noteworthy for their detailed accounts of the initial stages in bud formation. Recently several other papers (Majumdar on Heracleum, 1942; Majumdar and Datta on Heracleum and Leonurus, 1946; Reeve on Garrya, 1943; Wardlaw on Matteuccia, Onoclea, and Dryopteris, 1943a, 1943b, 1946; Sterling on Sequoia, 1945a, 1945b, and on Pseudotsuga, 1947; Miller and Wetmore on Phlox, 1946) have further added to our knowledge on this subject, though on widely separated groups of plants. With few references to the later stages of development, especially vasculation, it has not been possible to make generalizations or to formulate a clear concept of bud ontogeny. The differentiation of xylem and phloem in axillary buds is considered in only one paper (Miller and Wetmore, 1946). Further, there has existed a marked divergence of opinion on some of the basic concepts of bud formation; the differences concern such fundamental problems as the origin of bud primordia, the origin of bud traces, and the differentiation of the procambium. With incomplete information on certain developmental stages on the one hand, and with contrasting descriptions and interpretations on the other hand, it seemed desirable that a comprehensive study of the main phases of bud development be made. In this first paper of a series which is concerned with axillary buds of plants with different phyllotactic patterns, different nodal connections, and different bud arrangements, attention is centered upon the origin, the development of general form, and the vasculation of axillary buds of Syringa vulgaris. Consideration is given to the timing and the orderly succession of the progressive developmental stages and to the nature of the vascular strands connecting axillary buds with the vascular tissue of the main axis. MATERIAL AND METHODS.-Syringa vulgaris (fig. 1) was selected as a species for this first study because the plant has opposite leaves, a condition favorable for sectioning and examination of nodal structure; and because the leaf traces are unilacunar, the least complex type of vascular connection. Collections were made every 3 or 4 days or weekly during March and April, 1946, at the time when the buds were opening and during the early period of shoot elongation. Additional material was 1Received for publication September 8, 1948. The writer wishes to express her sincere appreciation and gratitude to Professor Ralph H. Wetmore for his kindly interest, his guidance, and constructive criticism throughout the course of this investigation. obtained at intervals throughout the late spring, summer, and fall months of 1945 and during the spring and summer of 1946. Buds and young twigs were fixed in Craf solution, dehydrated in a series of n-butyl alcohol (Zirkle, 1930), and embedded in rubberized paraffin. Serial sections, transverse and longitudinal, were cut 8 and 10 , thick. Two staining procedures were used: Heidenhain's iron-alum haematoxylin with safranin as the counterstain (Jeffrey, 1917), ......~~~~~~.. ........ . . , J......,