Microchemical Tests Research Articles

A cytochemical and ultrastructural study of the echinate cytoplasmic inclusion in Nitella flexilis (Characeae)

Echinate bodies most commonly 20–40 micrometers (μm) in diameter are conspicuous inclusions carried with the cyclosis in the internode cells of some charophytes. Those in Nitella flexilis were investigated with light and electron microscopes. The chemical nature of the inclusion identified through various cytochemical techniques in the present study disagrees with the generally held belief that the echinate body is almost entirely composed of proteins. Microchemical tests have convincingly revealed the presence of tannin. The inclusion's close spatial association to endoplasmic reticula and to vesicles apparently derived from the Golgi apparatus and the cytochemical demonstration of acid phosphatase and thiamine pyrophosphatase activities in the matrix of the inclusion suggest that both cell organelles are actively involved in the development of this bizarre body with unknown function.

Metabolism of a glutathione conjugate of 2-hydroxyoestradiol-17 in the adult male rat.

Adult male rats with cannulated or ligated bile ducts were given S-(2-hydroxyoestradiol-1-yl)[(35)S]glutathione, S-(2-hydroxy[6,7-(3)H(2)]oestradiol-1-yl)glutathione or S-(2-hydroxyoestradiol-1-yl)[glycine-(3)H]glutathione by intraperitoneal injection. The recovery of radioactivity in the bile of bile duct-cannulated rats was 33-86% and in the urine of bile duct-ligated rats was 54-105%. Oestrogen thioether derivatives of glutathione, cysteinylglycine, cysteine and N-acetylcysteine were isolated from bile; only the N-acetylcysteine derivatives could be identified in the urine. The steroid moiety was characterized by microchemical tests before and after treatment with Raney nickel: 2-hydroxyoestradiol-17beta was released from the glutathione conjugate, and 2-hydroxyoestrone and 2-hydroxyoestrone 3-methyl ether from the other conjugates. From intact rats the recovery of administered radioactivity was about 15% in the urine and 5% in the faeces over a period of several days and the radioactivity appeared to be largely protein-bound. The results demonstrate that injected oestrogen-glutathione conjugate undergoes conversion into N-acetylcysteine derivatives in vivo. Oestrogen-glutathione conjugates formed in the intact rat may be excreted in an apparently non-steroidal, possibly protein-bound form, which would not be detected by current analytical techniques.

Microscopical identification of water in crystals

Water can exist within solids in many forms : adsorbed on the surface or within micropores, occluded in voids, trapped within a molecular cage as in clathrates, or chemically bonded to species within the crystal lattice. Only in the latter case does it markedly alter the microscopical properties of a crystal. The water molecule possesses an unusually low molecular refraction coefficient. When bound to a crystal, it strongly depresses the principal refractive indices from those of the parent (anhydrous) compound. Examination of optical-crystallographic data from several hundred compounds shows a linear relationship between the per cent water and the mean refractive index for a given series of crystal hydrates. Moisture evolved on heating may be identified by its characteristictemperature and rate of evolution and by other physical and microchemical tests. Hot stage and micro DTA methods are discussed, along with special techniques for the examination of hydrated crystals in the scanning and transmission electron microscopes.

Revision of the Systematic Position of Saprochaete Saccharophila

SUMMARY Saprochaete saccharophila, described in 1939 and placed in the Chlorophyta, is validated and belongs in the Fungi Imperfecti based on fungal characteristics revealed by recent electron microscope, X-ray diffraction, and bio-assay studies. In 1939 Coker and Shanor described Saprochaete saccharophila Coker & Shanor as a saprophytic fungoid alga. On the basis of such morphological features as mode of branching (FIG. 1), rhizoidal holdfasts and cell wall composition, they placed it among the algae near the Chaetophoraceae. But the plant is devoid of chlorophyll and any trace of plastids (FIGS. 2-4). Smith (5) argued against placing Saprochaete in the Chlorophyta because of the presence of a glycogen-like substance which has been shown to be present by the positive reaction with I2KI (1, 7, 8). In 1955, Vischenevskaja (6) described a new species of the genus Saprochaete placing it in the Fungi Imperfecti. However, Lewin (3), in fact, suggested that Saprochaete should be examined biochemically before any definite assignment of its taxonomic position can be made. Dawes' (2) studies of S. saccharophila involved use of light and electron microscopes, X-ray diffraction, microchemical tests and biochemical assay with chitinase. He found the cell structure to be eucaryotic noting that mitochondria, nuclei, lipid bodies, endoplasmic reticulum, and micro

A standardized method for the identification of lichen products

A procedure for the routine identification of the products of lichen-forming fungi by thin-layer chromatography is described. Microextracts of plant fragments are chromatographed in three solvent systems. The spots of unknowns are assigned to R F classes defined by the R F values of marker controls of two lichen substances (atranorin and norstictic acid) chromatographed on every plate. The unknowns are tenatively identified by sorting (by R F classes) punched cards summarizing microchemical data for all compounds previously studied. The preliminary identification is then confirmed by additional microchemical tests. The open-ended system can incorporate new and unknown compounds as well as information from other chromatographic systems. Data obtained by the standardized procedure are given for 104 products.

Microcrystalline Identification of Phenothiazine-Type Tranquilizers. I. Development of the Method

Abstract Microcrystalline reactions are described for the identification of twelve related phenothiazine-type compounds with six reagents (1% ammonium reineckate, 5% ammonium thiocyanate, gold chloride, picric acid, platinum bromide, and stannous chloride). The procedure involves the diffusion of a drop of reagent into a drop of a solution of substituted phenothiazine in HC1 ( 1 : 9 9 ) . The resultant crystalline derivatives are described, and photomicrographs are included as an additional identification guide. By the use of these microchemical tests, it is possible to detect and identify microgram quantities of promazine, chlorpromazine, trifiupromazine, mepazine, prochlorperazine, trifluoperazine, methoxypromazine, promethazine, propiomazine, fluphenazine, acetophenazine, and chlorprothixene. The method was successfully applied to ten trade-name drug products. A collaborative study is underway

Effect of the removal of cell surface sialic acids on cell aggregation in vitro.

THE extensive investigations of Gottschalk1 and Warren2 have established that sialic acids (9-carbon sugars) occur widely in all orders of the Vertebrata. In many species histochemical studies using both the light and the electron microscope have shown that these acids constitute part of a carbohydrate-rich substance found at the surfaces of both normal3,4 and tumour cells5,6. The utilization of the specific enzyme N-acetylneuraminate glycohydrolase15 has enabled investigators to demonstrate by microchemical tests that sialic acids occur at the surfaces of Ehrlich ascites tumour cells7,8, of red blood cells9,10 and of normal and malignant rat liver cells11. Studies employing cell electrophoresis have resulted in the detection of sialic acids in many other cell types12, and have demonstrated that the increased negative mobilities of some transformed cell lines13 and of RPMI No. 41 cells (derived from human osteogenic sarcoma) in mitotic peak phase14 are caused by an increased amount of cell surface sialic acids.

A MUCILAGINOUS ROOT SHEATH IN ERICACEAE

A prominent mucilaginous sheath surrounding the root tips of plants in the family Ericaceae is described. The sheath was ubiquitous in all species examined. These included representatives of both subfamilies and all tribes of the family. Anatomical studies indicated that the sheath was not the breakdown product of root cap cells but rather that it was formed exterior to a thickened cell wall or cellular layer at the root tip. Although the mucilaginous sheath stained readily with ruthenium red (a specific test for pectins), it was not possible to show it to be one of the common pectins by microchemical tests. Relationships between the soil environment and the extent and condition of the sheath are discussed. Possible significance or functions of the sheath are suggested.

Some Microchemical Tests for Two New Lichen Substances, Scrobiculin and 4-O-Methylphysodic Acid

Scrobiculin, a recently described meta-depside, is a constant com- ponent of Lobaria amplissima and L. scrobiculata, and it is identifiable in fragments of herbarium specimens by microcrystal tests and thin-layer chroma- tography. In L. scrobiculata usnic acid is probably an accessory substance, and two unidentified substances are present in addition to stictic acid and norstictic acid. 4-O-Methylphysodic acid can be identified in Parmelia livida by thin-layer chromatography, but other constituents present interfere with microcrystal tests for this depsidone. Atranorin, lividic acid, and two unidentified substances were also detected as constant components of this species, and two additional, un- known compounds were found in variable concentration. In 1947 Asahina found stictic acid and an unknown substance, recrystallizing as needles in the GE solution, by microcrystal tests on Lobaria verrucosa Hoffm. (=L. scrobiculata (Scop.) DC.). Recently, the unknown substance, named scrobiculin, was isolated and its chemical structure (Fig. 1) determined (C. F. Culberson, 1967). Scrobiculin was the only phenolic compound obtained from Lobaria amplissima, but it occurred with (+)-usnic acid, stictic acid, and norstictic acid in L. scrobiculata. 4-O-Methylphysodic acid is a second new lichen substance which has recently been studied chemically (C. F. Culberson, 1966). The only known natural source of 4-0-methylphysodic acid is Parmelia livida Tayl., from which it was first isolated along with atranorin and a major constituent of unknown chemical structure, which I shall call lividic acid. Earlier, Hale (1958) found atranorin and an unknown substance crystallizing in the GE solution by microchemical tests on the type specimen of Parmelia livida. W. L. Culberson (1961) obtained similar results, described the microchemical test in more detail, and gave a good photograph of the crystals ob- served in the GE solution. These crystals are now known to be formed by the major constituent, lividic acid. The purpose of the present report is to describe microchemical tests which may be useful for the identification of the two new lichen substances scrobiculin and 4-O-methylphysodic acid. These methods were employed in microchemical surveys of the three lichens, Lobaria amplissima, L. scrobiculata, and Parmelia livida, which in- dicate that the new substances are constant components of the species in which they occur. In addition, two unidentified substances were detected microchemically in L. scrobiculata and four in P. livida. Some chromatographic properties of these un- identified compounds are described.

Aerosol OT solution--an effective softener of herbarium specimens for anatomical study.

Dried plant parts are cut into convenient sizes and soaked in a solution containing 2.5–3.3% Aerosol OT in distilled water for 5 or more hours until well penetrated by the Aerosol. After brief washing in distilled water the material can be embedded, can be sectioned freehand or, if the nature of the material permits, with a sliding microtome, without embedding. Although it is a good wetting agent, Aerosol is chemically neutral; therefore, microchemical tests can be performed successfully on material treated with it. Refractory plant tissues embedded in paraffin can be successfully softened if one face of the block is trimmed to expose the tissue, then soaked in an Aerosol solution before sectioning.

Some Microchemical Tests for Two New Lichen Substances, Scrobiculin and 4-O-Methylphysodic Acid

Some Microchemical Tests for Two New Lichen Substances, Scrobiculin and 4-O-Methylphysodic Acid

The Identification of Imbricaric Acid and a New Imbricaric Acid-Containing Lichen Species

Cetraria alaskana, a new, endemic, tundra-inhabiting species from western Alaska, is described. It differs from its nearest relatives morphologically in lacking soredia and chemically in producing the depsides imbricaric acid and atranorin. Pure imbricaric acid was isolated from the new species and its identity proven chemically. The pure sample was used for microchemical comparisons with the chemically very closely related but much more common depsides perlatolic acid and divaricatic acid, both of which frequently occur as constituents of species closely allied taxonomically to species producing imbricaric acid. Early in our monographic study of the species traditionally assigned to the genus Cetraria and of some obviously closely related plants in Parmelia, we had difficulty in distinguishing microchemically between the closely related depsides perlatolic acid and imbricaric acid. Although perlatolic acid is relatively widespread in Cladonia and Parmelia, imbricaric acid is absent from Cladonia and in Parmelia is known with certainty from only a single species. Imbricatic acid, however, is not uncommon in species of Cetraria (in the broad sense). 1 This study was supported in part by research grants GM-08345 from the Division of General Medical Science of the National Institutes of Health, U.S. Public Health Service, and GB-1239 from the National Science Foundation. 2 Department of Botany, Duke University, Durham, North Carolina 27706. This content downloaded from 157.55.39.35 on Fri, 02 Sep 2016 04:05:53 UTC All use subject to http://about.jstor.org/terms 1966] CULBERSON & CULBERSON: IMBRICARIC ACID 198 In Cetraria sens. lat. the problem of determining perlatolic and imbricaric acids is restricted exclusively to the broad-lobed species, the parmelioid 'Cetrariae', a series of some two dozen species to be treated in two genera in our monograph (for a taxonomic characterization of these groups, see Culberson and Culberson 1965). (The parmelioid 'Cetrariae also include a few species previously classified in Parmelia, for example P. cetrarioides in which imbricaric acid was originally discovered.) While both perlatolic acid and imbricaric acid are represented among the parmelioid 'Cetrariae', imbricaric acid is more common. Moreover, it was noted that many of the plants suspected of containing imbricaric rather than perlatolic acid also produce a pink coloration when the medulla is treated with KOH followed by Ca(OC1) 2. The problematic chemical indentifications were further complicated by these anomalous spot tests also noted by Krog (1951) and many earlier workers since from the structural formulae neither perlatolic acid nor imbricaric acid should give a positive KC test. One of the parmelioid 'Cetrariae' containing imbricaric acid and giving an anomalous KC test is a new species, Cetrarir a alaskana. In view of the difficulties previously encountered in identifying imbricaric acid and in explaining the KC test that is often associated with species containing imbricaric acid, it was thought desirable to prepare a chemically complete proof of the identity of the major constitutent of the new species in the hope that the information found and the tests used would have an even broader application in the chemical elucidation of the other, related, and obviously chemically similar species in the same group. These aims were only partially realized, however, in that while imbricaric acid was extracted and determined chemically beyond question, the substance or substances responsible for the KC test could not be identified. It seems desirable, however, to record the chemical analysis of the new species with its description because it is usually not practical to undertake chemical tests of the extensive sort carried out here in ordinary taxonomic studies where, if chemical information is sought at all, microchemical tests alone are used for the determinations.

Les laves recentes de l'Etna

Abstract The recent lavas of Etna are tephritic trachybasalts always showing the same mineral composition: olivine phenocrysts; phenocrysts and microlites of strongly zoned calcic plagioclase, slightly titaniferous and aluminous augite, and titanomagnetite; [and] interstitial sanidine and nepheline accompanied by a minimum amount of sodalite. These minerals have been studied by chemical analysis, as well as by optical and x-ray diffraction methods. Analyses of plagioclase, augite, and olivine have again been carried out and compared with those already published. Sanidine, nepheline, and sodalite which, although implicit in the total chemical analysis of the rock, had never been explicitly described, have been identified by selective staining, microchemical tests, and x-ray diffraction. This has been followed by chemical and modal analysis of the most representative lavas. An average type has been defined and its place in the classification of volcanic rocks discussed.

Observations on the Mineral Composition and Staining Properties of the Inclusion Bodies of Infectious Canine Hepatitis

SUMMARY The hepatic intranuclear inclusion bodies of infectious canine hepatitis were examined by staining, by refractometry in phase-contrast illumination, and by microincineration techniques. The refractometric investigation of fixed but unstained cells revealed the complex structure of these inclusion bodies much more readily than did several staining methods. An improved method for measuring and recording the refractive index of fixed and unstained cells is described. Thin sections of injected liver were incinerated and the ash residues of inclusion bodies were examined in incident light (Leitz Ultropak). Their structure appeared to be very similar to non-incinerated, stained specimens. They possessed an abundant ash. Microchemical tests for sodium/potassium, calcium and iron were performed. Sodium/potassium crystals were demonstrated; the test for iron was negative and for calcium doubtful. When water-soluble salts and calcium were dissolved, abundant mineral residues still persisted,consisting, most probably, of phosphates, but technical difficulties prevented a more definite identification.

Studies on Andropogon gayanus Kunth

Nectar secretion by grasses is rare. Andropogon gayanus var. bisquamulatus has been observed to secrete nectar from two localized areas: (a) near the sheath margin immediately below the pseudopetiole; (b) the pseudopetiolar ridges adjacent to the ligule. Microchemical tests with Fehlings solution revealed high concentrations of reducing sugars at these two sites. Some evidence is given for supposing that the macrohair basal cells may secrete the nectar.

Studies on Andropogon gayanus Kunth

Nectar secretion by grasses is rare. Andropogon gayanus var. bisquamulatus has been observed to secrete nectar from two localized areas: (a) near the sheath margin immediately below the pseudopetiole; (b) the pseudopetiolar ridges adjacent to the ligule. Microchemical tests with Fehlings solution revealed high concentrations of reducing sugars at these two sites. Some evidence is given for supposing that the macrohair basal cells may secrete the nectar.

Sensitivities of some microchemical tests for usnic acid and atranorin

Sensitivities of some microchemical tests for usnic acid and atranorin

A Summary of the Lichen Genus haematomma in North America

A Summary of the Lichen Genus haematomma in North America

CERCIDIUM FLORIDUM SEED COAT, LIGHT AND ELECTRON MICROSCOPIC STUDY

Scott, Flora Murray, B. G. Bystrom, and E. Bowler. (U. California, Los Angeles.) Cercidium floridum seed coat, light and electron microscopic study. Amer. Jour. Bot. 49(8): 821–833. Illus. 1962.—The structure of the seed coat of the desert tree Cercidium is typical of the family Leguminosae, but the inner integument is mucilaginous. The characteristic palisade cells of the epidermis and the many‐armed cells of the mucilaginous zone are discussed in detail. The minute wax rodlets on the surface of the young seed fuse later into a film of wax. The epidermal palisade cells are polygonal in transection and the walls are strengthened by cellulose flanges. The palisade cells are enclosed in a thin suberin‐like sheath which connects with the film which lines all air‐filled intercellular spaces in the outer integument. Plasmodesmata extend from the protoplast through the subcuticular zone to the cuticle and also interconnect with adjacent cells through numerous pits in the interflange areas of the main wall. The cells of the inner integument are many‐armed. Intercellular spaces at all stages of growth observed are occluded with mucilage; nevertheless, they are lined with a suberin‐like film similar in reaction to that of the air‐filled intercellular spaces of the outer integument. The distribution of wall materials and ergastic substances was determined by microchemical tests. For electron microscopic study, the epidermis and the mucilaginous zone were fragmented ultrasonically, after chemical treatment when necessary. Cuticular fragments, frequently polygonal in outline, are dense to the electron beam at photographic illumination. When exposed to the electron beam, full‐strength, volatile components are driven off and condense on other wall fragments or on substrate. Unique structures occur in the subcuticular zone, termed here, collar, cone and paddle structure. The basic material of the cell wall as a whole is cellulose impregnated with matrix materials, mainly non‐cellulosic polysaccharides. In addition, lipid‐like substances are conspicuous particularly in the flanges of the palisade cells. Under the electron beam, full‐strength, these substances volatilize and leave the flanges with a blistered outline except in the region of the light line. The volatile substances may be absent from this zone or the microfibrillar structure may prevent distortion. The microfibrillar pattern throughout the length of the flanges is dominantly parallel, but a change to reticulate arrangement occurs in the light‐line region. Numerous pits mark the paths of the plasmodesmata in the subcuticular zone and also through flange and interflange areas of the wall. The dominant microfibrillar pattern in the mucilage cells is reticulate in young and old cells, except for helical orientation in extending arms. Pits occur on arm ends but are infrequent elsewhere. The loose microfibrillar structure presumably allows continuous secretion of mucilage.

Preparations of Cell-Walls of Some Fungi

LITTLE is known of the qualitative and quantitative composition that may occur in the cell wall of fungi. Cochrane1 has claimed that the most serious defect in our knowledge of cell-wall composition in the filamentous fungi is the fact that no ‘pure’ wall material is yet available. The major sources of error are the inclusion of extraneous substances in the preparation, and the loss of wall material during purification. Several workers have described results of the cell-wall composition based on X-ray diffraction patterns. Results from other methods (isolation of derivatives from a hydrolysate, microchemical tests, etc.) are usually in agreement with those of X-ray analysis, but are in most cases unsatisfactory.