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Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining. This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride (NaBH4) and Sudan black B (SBB) alone (p

Styrene and its metabolites are known to have serious adverse effects on human health and hence, strategies to prevent its release, eradicate it from the environment, and understand its route of degradation are being considered. A total of 18 strains were isolated from 4 samples of diesel contaminated soils. Among them 5 strains were selected for their ability to degrade styrene and use it as a sole carbon source to produce PHA. These strains were identified as Enterobacter spp. on the basis of 16S rRNA gene sequencing. Bacteria were screened for their ability to produce PHA by utilizing glucose and styrene as a carbon sources. Screening for PHA production was done by Nile blue A, Sudan black B, and phase contrast microscopy and the selected 3 strains showed positive results. Growth kinetics along with time profiling of PHA was performed for glucose and styrene as carbon sources. PHA extraction was done at equal intervals of 12 h by sodium hypochlorite method which showed that these strains accumulate maximum amount of PHA after 48 h in glucose (30.60%). FTIR analysis of PHA was done which revealed homopolymer PHB and copolymer (PHB-co-PHV) production in strains by utilizing glucose and styrene. Gas chromatography mass spectrometry was carried out to identify the metabolites produced by bacterial strains grown on styrene. Metabolites of styrene degradation included propyne and phenylalanine. Genomic DNA isolation was carried out to amplify phaC gene which encodes PHA synthase enzyme. The conversion of styrene to polyhydroxyalkanoates (PHA) provides a new and unique link between an aromatic environmental pollutant and aliphatic PHA accumulation.

Bacterial poly3-hydroxyalkanotes are the polyesters that are accumulated in conditions of un-balanced nutrients. Soil samples were collected from Hilly areas of Muzaffarabad, and oil processing area of Lahore, Pakistan to isolate PHA producing bacteria. The bacteria were screened on Sudan black B and Nile blue A staining methodologies. An efficient PHA producing strain AZR-1 was isolated and characterized morphologically and biochemically. The strain was identified as Bacillus sp. AZR-1 on the genetic basis of 16S rRNA gene sequence. Bacillus sp. AZR-1 produced 40% PHA contents when grown on N-limited mineral salt medium supplemented with glucose in shake flasks. Starch and sodium gluconate were found to be other putative carbon sources for PHA production as the bacterium produced 22 and 17% PHA on these sources. FTIR confirmed the presence of scl-PHA monomers in the polymer extracted from the bacterium. The PHA synthase gene (phaC) was partially amplified and sequenced which showed maximum similarity with the PHA synthase of Bacillus weihenstephanensis KBAB4.

Using a simple impregnation process, the present study has introduced a novel route for the photosensitization of TiO2 nanoparticles using Sudan black B dye as a sensitizer. DRS, XRD, FE-SEM, BET, UV–Vis, FT-IR and TGA–DSC analyses were considered for characterization of the synthesized purple powder as a visible light photocatalyst for removal of the phenolic pollutant. The band gap of the dye-sensitized TiO2 and corresponding BET surface area were 2.9 eV and 50.0 m2 g−1, respectively. Based on the results, Sudan Black sensitized TiO2 photocatalyst outperforms the pure commercial TiO2 (Degussa P25) in terms of phenol degradation under visible light. Under a 100-min photo-catalytic experiment, dye-sensitized TiO2 removed 96 % of the phenol content. The Sudan black B dye-sensitized TiO2 is expected to show further interesting physical characteristics related to potential uses.

Three adult Holstein-Friesian dairy cows (4 to 6 years old) which had been raised at three different farms were culled with a long history of locomotor dysfunction attributed to dislocation or subluxation of the hip joint (two cows) or foot rot (one cow). They revealed dark brown discoloration of multiple sites of the carcasses at postmortem examination. On histopathological and histochemical investigations, lipofuscin pigments showing autofluorescence, sudanophilia, argyrophilia, periodic acid-Schiff (PAS) positiveness, and acid fastness were stored within otherwise normal cells of various sites in the body, including many thoracic and abdominal viscera, skeletal muscle, diaphragm, and tongue. In the central nervous system, some neurons with massive lipofuscin deposition were prominently shrunken but associated with neither necrotic changes nor glial reaction. Such a generalized pigmentary condition was distinguished from neuronal ceroid lipofuscinosis and thus was diagnosed as generalized lipofuscinosis. Furthermore, distinct proteinaceous eosinophilic inclusions were simultaneously observed to a varying degree within myofibers of the diaphragm, heart, rumen, or small intestine. These inclusions stained weakly positive with PAS and negative with Nile blue, Sudan black B, periodic acid-methenamine silver (PAM), and acid-fast stains. Autofluorescence was not seen. Staining properties demonstrated by phosphotungstic acid-hematoxylin (purple blue) and Masson’s trichrome (bright red) stains were compatible with those of myofibrils, presumably suggesting that the inclusions have chemical composition similar or identical to that of myofibrils. It remains to be elucidated whether the occurrence of such sarcoplasmic inclusions was related to lipofuscinogenesis or a nonspecific phenomenon indicating an incidental finding.

In tissue engineering, autofluorescence of polymer scaffolds often lowers the image contrast, making it difficult to examine cells and subcellular structures. Treating the scaffold materials with Sudan Black B (SBB) after cell fixation can effectively suppress autofluorescence, but this approach is not conducive to live cell imaging. Post-culture SBB treatment also disrupts intracellular structures and leads to reduced fluorescence intensity of the targets of interest. In this study, we introduce pre-culture SBB treatment to suppress autofluorescence, where SBB is applied to polymeric scaffold materials before cell seeding. The results show that the autofluorescence signals emitted from polycaprolactone (PCL) scaffolds in three commonly used fluorescence channels effectively decrease without diminishing the fluorescence signals emitted from the cells. The pre-culture SBB treatment does not significantly affect cell viability. The autofluorescence suppressive effect does not substantially diminish during the culturing period up to 28 days. The results also show that cell migration, proliferation, and myogenic differentiation in pre-culture SBB-treated groups do not exhibit statistical difference from the non-treated groups. As such, this approach greatly improves the fluorescence image quality for examining live cell behaviors and dynamics while the cells are cultured within autofluorescent polymer scaffolds.

In this study, nine heterotrophic yeast isolates were tested for their ability to assimilate crude glycerol and consecutive conversion to triacylglycerides (TGAs). All the organisms were initially screened on crude glycerol-based selection media, and those producing lipid globules were further evaluated for lipid production. Sudan Black B staining of eight isolates showed lipid globules. These strains were further studied at different C/N ratio. The molecular identification revealed that the isolates belonged to the genera of Yarrowia and Candida. Among these isolates, SKY7 (Yarrowia lipolytica) produced up to 42.04 ± 0.11 % of lipid w/w) with a C/N ratio of 100 and fermentation time of 72 h. The other strains produced 5.82 ± 0.4 to 34.57 ± 0.44 % lipid (w/w). The GC-flame ionization detector (FID) lipid profile showed that the lipid produced by the strains had close resemblance with vegetable oil and could serve as a feedstock for biodiesel production. Biolog test of the isolates revealed a wide spectrum of carbon utilization.

A contaminating bacterium growing along with the stock culture of Saccharophagus degradans ATCC 43961 (Sde 2-40) on marine agar plate was isolated and investigated for its ability to produce polyhydoxyalkonates (PHA). Preliminary screening by Sudan black B and Nile blue A staining indicated positive characteristic of the isolate to produce PHA. The isolate was able to grow and produce PHA in minimal sea salt medium broth. PHA quantification studies with gas chromatographic analyses of the dry cells derived from culture broths revealed accumulation of PHA in bacterial cells. PHA production started after 20 h and increased with cell growth and attained maximum values of 61 % of dry cell weight at 70 h of cultivation. After 70 h, a slight decrease in the level of PHA content was observed. The nature/type of PHA was found to be poly(3-hydroxybutyraye) by Fourier transform-infrared spectroscopy. Microbiological and 16S rRNA gene sequencing analyses suggested that the PHA producing bacterial isolate belongs to Bacillus genera and shows 100 % nucleotide sequence similarity with Bacillus cereus species in GenBank. This study is a first report for ability of Bacillus species to grow in marine sea salt media and produce PHA. The media used for the polymer production was novel in the context of the genus Bacillus and the production of PHA was three-fold higher than Sde 2-40 using same growth medium. This study shows that the contaminant bacteria once properly investigated can be used for advantageous characteristic of metabolites production in place of original cultures.

The reproductive cycle of Trophon geversianus (Pallas 1774) population from Golfo San Jose´ (Chubut, Argentina, 42330 S, 64330 W) was studied using histochemical methods and digital image analysis. For such purpose, ovary samples were taken monthly between July 2006 and August 2007 and stained with hematoxylin and eosin, Sudan black B (to identify lipids) or Schiff’s ninhydrin (to identify proteins). Four different gametogenic stages were described: oogonias, previtellogenic oocytes, early vitellogenic oocytes, and late vitellogenic oocytes. Two spawning events were registered; one between September and October 2006, and a second one between February and March 2007. Oocyte quality was determined by the changes in lipid and protein composition during gametogenesis. Through digital image analysis, a lipid and a protein indexes (LI and PI) were calculated. Significant differences were observed in LI over month sampled, contrarily to what PI showed. A significant and positive correlation was found between lipid and protein content and oocytes areas, indicating that these nutrients accumulate during the entire vitellogenesis. Also, when dividing the oocytes into two size groups, analysis indicates a positive correlation between LI and oocyte area for smaller area oocytes. This demonstrates that while proteins accumulate linearly throughout vitellogenesis process, lipids accumulate in two steps: first at a growing rate, secondly at constant rate. This information is essential to determine the nutritional requirements of brood stock individuals at hatcheries in this potential fishery resource that inhabits patagonian waters. Fil: de la Barra, Paula María. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Invertebrados Marinos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Giménez, Juliana. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Invertebrados Marinos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Knack de Almeida, Henrique. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Invertebrados Marinos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Arrighetti, Florencia. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Invertebrados Marinos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The Malayan tapir (Tapirus indicus) is classified as a preserved animal of Thailand. It is listed as an endangered species on the 2008 IUCN red list and also by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Blood samples from captive Malayan tapirs (n = 20, 10 males and 10 females) from five zoos under the Zoological Park Organization of Thailand were examined and measured for complete blood counts, serum chemistry and electrolyte analysis. Cytochemical staining including Peroxidase, Sudan Black B, Periodic acid Schiff's reaction (PAS), α-naphthyl acetate esterase (ANAE) and β-glucuronidase (BG) was also analysed. Statistically significant differences were found between sexes including alkaline phosphatase, total protein and red cell distribution widths. Red blood cells were slightly anisocytosis, (range 4.5–8.5 μm) with 6.3-μm mean diameter. Eosinophils and monocytes were positive with all five cytochemical stains. Lymphocytes showed a variable reaction for ANAE and BG. PAS was positive for all type of blood cells except red blood cells. Ultrastructurally, eosinophils had the largest round shape with three-capsule layer granules which were prominent and distinguishable from other species. This investigation provides basic haematology and serum chemistry analyses as well blood cells identification guideline for the Malayan tapir and serves as endangered mammal reference values.

Egg development in Pteridium aquilinum var. latiusculum was studied using ultrastructural and cytochemical methods to examine structural features influencing fertilization in leptosporangiate ferns. Ultrastructural observations indicate a separation cavity is first formed above the egg during oogenesis with a pore region persistently connecting the egg and the ventral canal cell. The egg envelope is formed by deposition of amorphous materials in the separation cavity on the outer surface of plasmalemma. The egg envelope was not formed across the pore region; instead, a fertilization pore was formed. During oogenesis, the egg nucleus produced extensive evaginations containing osmiophilic bodies. Cytochemical experiments revealed that the egg envelope displays strong periodic acid-Schiff reaction indicative of polysaccharides, with negligible Sudan black B staining for lipids, suggesting that the egg envelope is composed principally of polysaccharides, and not lipids. The present manuscript provides new insights into egg structure and development of Pteridium, including discovery and characterization of the fertilization pore and observations on the chemical nature of the egg envelope, thus contributing to the understanding of the cytological mechanism of the sexual reproduction of ferns.

Sudan black B (SBB) was used to investigate as the nonionic diazo dye-doped in polymer dispersed liquid crystal (PDLC) display, by polymerization-induced phase separation (PIPS) method. The maximum absorbance, contrast ratio, dichroic ratio and the order parameter of nonionic diazo dye in nemetic host (TL203) were investigated using UV–Vis polarized spectroscopy. The orientation of the dye molecules was controlled by electric field, which enabled the contrast ratio of the dye to be obtained by electrically switching. The change occurring on droplet morphologies and electro-optical properties of PDLC film with the change in contents of Sudan black dye and liquid crystals (LC) contents was investigated. We found an increase in LC droplet sizes with the increase of diazo dye and LC contents. Moreover the addition of small amount of nonionic diazo dye reduced the threshold voltage (V\(_{\boldsymbol{\rm th}})\), increased off-state transmittance, enhanced the contrast ratio and decreased the response time of dye-doped PDLC. Additionally the change in transition temperature of LC and changes in LC droplet morphologies with the addition of dye were also observed. Such changes were observed with the images taken by polarized optical microscope (POM). The detail discussions on such behaviours were also made.

In the present study, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]-producing bacterial strain was isolated from trinitrotoluene (TNT)-contaminated soil using Sudan black B and Nile blue A staining as a screening method. The bacterial strain was identified as Bacillus cereus FA11 on the basis of biochemical tests and 16S rRNA sequencing. Culture conditions, such as pH, temperature, time and carbon sources, were optimized for maximum production of the copolymer. B. cereus FA11 was found capable of producing a maximum of 48.43% poly(3HB-co-3HV) at pH 7, with glucose as the carbon source, after 48 h of incubation at 30°C. Fourier transform infrared analysis of the extracted polymer revealed similarities in the spectra of the microbial and commercially available poly(3HB-co-3HV) at 1720.2, 1278.5 and 2932.1 cm−1 wavelength. 1H-nuclear magnetic resonance analysis of the polymer confirmed it as a copolymer composed of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units.

A total of 20 different strains were isolated, purified and screened for polyhydroxyalkanoate (PHA) production. PHA-producing strains were screened by Nile blue staining and confirmed by Sudan Black B staining. Strain 1.1 was selected for further analysis due to its high PHA production ability. PHA production was optimized and time profiling was calculated. PHA production on various different cheap carbon sources, i.e., sugar industry waste (fermented mash, molasses, spent wash) and corn oil, was compared. Cell dry weight and PHA content (%) were calculated and compared. The 12.53 g/L is the CDW of bacterial strain when grown in medium containing corn oil. It was found that corn oil at 12.53 g/L medium can serve as a carbon source for bacterial growth, allowing cells to accumulate PHA up to 35.63 %. The PhaC gene was amplified to confirm the genetic basis for the production of PHAs. Moreover, 16S rRNA gene sequence analysis showed that strain 1.1 belongs to Pseudomonas species.

The jungle cat (Felis chaus subsp. fulvidina) of Thailand is a protected animal. Haematological and morphological characteristics of blood cells in eight captive jungle cats (three males and five females) were investigated using complete blood cell counts, cytochemical stains and scanning and transmission electron microscopy. Haematological results were reported as mean, median and range (minimum–maximum). Neutrophils stained strongly positive for Sudan black B (SBB) and peroxidase (PO). Basophils were strongly positive for α-naphthyl acetate esterase (ANAE) and β-glucuronidase (BG) while eosinophils were negative for all cytochemical stains. Monocytes were negative for SBB and had fine granular reactivity for PO, ANAE and BG. The ultra-structure of eosinophils and basophils were different from that reported for other wild cats in Southeast Asia. The haematology, cytochemistry and ultra-structure of blood cells in the jungle cat may be useful in evaluating and monitoring the health of this species in captivity.

Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3–100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.

Viverra zibetha, also known as the large Indian civet, is found from Indochina to southern China. This species is listed as Near Threatened. Blood samples from four adult (two males and two females) large Indian civets in Khao Kheow Open Zoo, Thailand, were prepared for morphological, cytochemical and ultra-structural observation. The red blood cells showed marked anisocytosis, with a mean diameter of 5.0 µm. The cytochemical staining (Sudan black B, peroxidase, α-naphthyl acetate esterase and β-glucuronidase) patterns of leukocytes and haematology were summarised. Eosinophils contained numerous short rod, red refractive granules. Eosinophils lacked sudanophilia and peroxidase activity. Under scanning electron microscopy (SEM), eosinophils showed rod-shaped granular surfaces. Ultra-structurally, these granules contained lamellar components. Basophils had dull grey, round to oval granules. Under SEM, the basophil surface was smooth with pleomorphic granular contours. Ultra-structurally, the basophil granules contained crystalloid components. Neutrophil engulfed red blood cells, and a sac of eosinophil granules was observed under a transmission electron microscope. These results provide more information on morphological, cytochemical and ultra-structural characteristics of blood cells in large Indian civets.

Bacterial isolates from sludge samples collected at a local municipal sewage treatment plant were screened for bacteria producing polyhydroxyalkanoates (PHA). Initially Sudan black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen limitation E2 medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent staining with Nile blue A. The positive isolates were quantified initially with a u.v. spectrophotometer, for a very large number of isolates (105) and among them high PHA-producing isolates (15) were selected and were confirmed by gas chromatographic analysis. The GC analysis showed the polymers produced by 13 of the selected isolates to be polyhydroxybutyrate (PHB), and the remaining two isolates produced polyhydroxybutyrate-co-hydroxyvalerate (PHB-co-HV) copolymer. The proportion of the PHA-positive bacterial isolates showed variability in the number of PHA accumulators during various months. The correlation of PHB production with the cell dry weight (CDW) was found to be statistically significant. The metabolism of PHB in these selected 15 isolates was studied using the Nile blue A staining, which showed an initial increase in the fluorescence followed by a decline, on further incubation. All the selected 15 isolates were classified to genus level by studying their morphological and biochemical characteristics. There were seven Bacillus species, three Pseudomonas species, two Alcaligenes species, two Aeromonas species, and one Chromobacterium species.

Sclerosing lipogranuloma (SLG) is a rare disease that occurs in the male genital region. We report here three cases of SLG, including two of Y-shaped lesions in the penile base forming an intrascrotal mass, as well as a patient with a mass in the epidydimal region. All three lesions histologically exhibited formation of multiple granulomas consisting of multinucleated giant cells and epithelioid cells, in the fibroadipose tissue or around the epidydimis, in association with eosinophil infiltration. Membranocystic changes were found within the cytoplasm of multinucleated cells. In the two patients with scrotal lesions, membranocystic changes were positive for diastase-PAS reaction and on Sudan black B staining in dewaxed sections. CD68 staining was strongly positive in multinucleated giant cells and epithelioid cells. Most of the lymphocytes infiltrating the lesions were T cells associated with some S-100-positive dendritic cells. T-cell mediated immune reaction appears to be important in the histogenesis of SLG. The histogenesis is generally considered to be a foreign-body reaction to degenerated or damaged fatty tissue or lipids, but no apparent causative factors can be identified in some reported cases. All three patients in the present study had a good clinical course after biopsy or surgical excision. We thus report three cases of SLG including an unusual case in the epidydimal region, with some considerations regarding the histogenesis of SLG.

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage.

Thirty-seven soil isolates and mutants of Azotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2%) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 micrograms/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 micrograms/g.

The histochemical characteristics of the black-brown pigment granules accumulated during the lecitotrophic stage in the digestive tract of Siberian sturgeon, Acipenser baeri larvae, were studied in order to identify the nature of such pigments. Pigment granules appeared just after hatching in contact with the endodermal epithelium and in the periphery of the yolk-sac matrix, and increased in number with digestive system differentiation. At 3 days post hatch, the accumulation of pigment granules in the spiral valve formed a pigmented plug, which was ejected with first faeces after first feeding. Pigment granules were insoluble in all solubilising solutions tested (distilled water, H2O2, NH4OH, KMnO4, absolute methanol and different acid, basic and oxidising solutions). Pigment granules were completely bleached with the oxidising processes of Gomori and Casella (15 min) and with H2O2 (20%) during 48 h. Histochemical characteristics of bleached pigmented granules revealed that they were basophilic (Eosine negative), presented a moderate methachromasia (Toluidine Blue positive) and argentaffinity (Masson Fontana method positive), and contained carboxylated and sulphated (strongly and weakly ionised) glycoconjugated residues (Alcian Blue pH 0.5, 1.0, 2.5 positive). Bleached pigment granules also contained reductor groups (Ferric ferricyanide-Fe III positive), while they were negative for Bromophenol Blue, Perl's Prussian Blue, Red Oil O and Sudan Black B, indicating the absence of proteins, iron and lipidic substances respectively. According to such results, the black-brown pigment granules observed in the alimentary canal of Siberian sturgeon larvae during the lecitotrophic stage correspond to melanins.

Sezary syndrome was diagnosed in a dog with epidermotropic lymphosarcoma and a leukaemic blood picture wherein 73.5% of the leucocytes (15582 of 21200 leucocytes /µl of blood) were large mononuclear cells with markedly convoluted nuclei. The dog had a history of multiple skin tumours progressing to hepatosplenomegaly and lymphadenopathy. Alpha-naphthyl acetate esterase staining of blood smears and splenic imprints revealed a focal to multifocal pattern of cytoplasmic enzyme activity that resisted sodium fluoride inibition, indicating that the neoplastic mononuclear cells were T lymphocytes. The neoplastic cells were negative for Sudan black B staining, myeloperoxidase activity, and chloroacetate esterase activity. Ultrastructurally, these cells had convoluted to cerebriform nuclei, 1 to 2 nucleoli, scattered mitochondria, numerous free ribosomes, and a few profiles of rough endoplasmic reticulum. Histological study of tissues collected at necropsy revealed infiltration of the skin, lymph nodes, spleen, liver, and bone marrow with neoplastic lymphocytes.

Triacylglycerol is accumulated by Streptomyces spp. when grown in submerged culture. Ultrastructural studies using transmission electron microscopy (TEM), staining and freeze-fracture/freeze-etch procedures, and light microscopy confirmed the accumulation of neutral lipid by S. lividans and S. coelicolor during the stationary phase and its storage within membrane-bound globular structures within the cytoplasm. These structures were of various sizes and occupied up to approximately 80% of the total cell volume at that time. There was no evidence of such material within cells examined during the early exponential phase of growth. The globules visualised by TEM were electron-transparent since they comprised lipids containing saturated fatty acids that did not react with osmium tetroxide. The globules appeared to be bounded by a single membrane.

A case of acute myeloblastic leukaemia with unusual cytochemical reactivity in a dog is reported. Blood and bone marrow examination revealed predominantly highly undifferentiated leucocytes lacking peroxidase, sudan black B, and alpha-naphthyl acetate esterase activities. Most of the immature cells in blood and bone marrow smears showed strong alkaline phosphatase activity. Transmission electron microscopy of bone marrow cells revealed features of myeloid differentiation particularly secondary granules. These findings supported the diagnosis of acute myeloblastic leukaemia without differentiation (Ml) under the FAB system of classification of acute myeloid leukaemas in humans.

Bacterial isolates from two environments, an integrated-farming pond in the university and palm-oil mill effluent (POME) ponds at a local palm-oil-processing factory, were screened for polyhydroxyalkanoates (PHAs). Initially Sudan Black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen-limiting medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent Nile Blue A staining. The PHA extracted from positive isolates was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The proportion of PHA-positive bacterial isolates was higher in the POME ponds compared to the integrated-farming pond.

Correlated light and electron microscope histochemistry was performed on the oil bodies (OB) ofMarchantia paleacea. It was revealed that they contain significant quantities of material positive to PAS and periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactions. Ag deposits were localized on the matrix surrounding the globules and on the distinct one close to the inner face of the OB membrane. In addition, the lipophilic globules were peripherally impregnated with staining product. Polysaccharidic material appears to be elaborated in dictyosomes and then advanced into the OB by their vesicles. The OB show a lipophilic character and are intensely stained with Sudan black B, with an aqueous solution of osmium tetroxide and with the Nadi reaction as modified byDavid andCarde (1964). The performance of mercury bromophenol blue, aniline blue black, coomassie brilliant blue R and acid fuchsin stainings showed that the OB do not contain structurally detectable proteins. Besides, a positive reaction of the inner OB was obtained in fresh tissue with a variety of tests sensitive to phenolic or other aromatic ring compounds. On the contrary, the epidermal and scale OB containing small globules are obviously stained only with Gibbs' reagent. The colours obtained with the above reactions, were almost the same,i.e. brown with a red shade, except for the blue one of the Gibbs' reagent. The presented observations favour the conclusion that the OB matrix does not contain proteins, but substances “polysaccharidic” in nature. Phenolic and possibly other aromatic compounds appear to be present at least in inner OB.

Following reports of a Reye-Iike syndrome in children resulting from Margosa oil (MO) ingestion, we administered MO to laboratory rats in an attempt to produce an animal model of Reye's syndrome. Male rats were injected intraperitoneally with either MO or corn oil and observed for clinical signs of a toxic response. After 15 h the animals were administered a second dose and the MO-treated animals developed florid neurological symptoms. The animals were then sacrificed and blood samples were analyzed for glucose, ammonia, aspartate aminotransferase, and alanine aminotransferase. Sections of liver, kidney, and brain were examined by light microscopy after Sudan black B, hematoxylin and eosin, and periodic acid- Schiff staining. Liver was additionally examined by electron microscopy. Liver samples were analyzed for hepatic enzyme levels and brain samples were analyzed for water content. There were greatly increased levels of ammonia, aspartate aminotransferase, and alanine aminotransferase and decreased glucose levels in the blood of MO-treated animals. Light microscopy of MO-treated livers revealed fatty infiltration, granularity of the cytoplasm with normal nuclei, and glycogen depletion; electron microscopy revealed mitochondrial pathology in the livers of MO-treated animals. There were no significant morphological changes in brain or kidney specimens although the kidneys did show some fatty infiltration. Hepatic mitochondrial enzyme levels were unchanged and there was no increase in brain water content in the MO-treated animals. Thus, many of the abnormalities seen in Reye's syndrome were seen in this model; however, there were no hepatic enzyme changes despite altered mitochondrial morphology and no evidence of cerebral edema despite a florid encephalopathy. Nonetheless, this model may have important implications for the understanding of the pathogenetic mechanisms of this Reye-like syndrome and, perhaps, Reye's syndrome.

The use of the Romanowsky staining technique, Sudan Black B, the periodic acid-Schiff reaction and methods for revealing peroxidase, acid and alkaline phosphatases and butyrate, acetate or chloroacetate esterases for identifying and discriminating subvarieties of acute leukaemia at the light microscope level is reviewed and the results of their application in a recent study of the first 720 cases admitted to the Medical Research Council's 8th Acute Myeloid Leukaemia trial summarized. The distribution of varieties of acute myeloid leukaemia and the relevance of age and cytochemical findings to clinical prognosis is presented. Identification of the predominant primitive cell - myeloblast, promyelocyte, monoblast, and others - appears to have little prognostic significance. In fact, the presence of periodic acid-Schiff positive erythroblasts is a bad prognostic sign. The association of certain cytochemical findings with the 15;17 translocation and acute promyelocytic leukaemia, especially the patterns of esterase positivity in Auer rods and the Sudan Black, peroxidase, periodic acid-Schiff and esterase findings characteristic of the 8;21 translocation are illustrated. Cytochemical features helpful in distinguishing acute megakaryoblastic leukaemia, notably the periodic acid-Schiff reaction, differential esterase reactivities and 5'-nucleotidase, are discussed and illustrated. Brief reference is made to the cytochemical differentiation of lymphoblastic leukaemias. Details of a technical method for the demonstration of 5'-nucleotidase are given.

We tested how long unstained and unfixed films of blood and aspirated bone marrow could be kept before special cytochemical staining. To exclude influences other than time on the outcome of the reactions, all the material was taken from hematologically normal persons. A simple score was used to evaluate the results. These may be summarized as follows: in most cells Naphthol-AS-acetate-esterase first shows a progressive decline in reactivity after a delay of 6 weeks, the reaction becoming very weak after 10 +/- 1 weeks. This does not hold for monocytes, megakaryocytes and platelets, which maintain reactivity (and susceptibility to NaF) for more than three months. With PAS staining, a slight decrease in reactivity after 8 +/- 2 weeks could be observed, whereas all other stainings tested (POX, Sudan black B and Naphthol-AS-D-esterase) were unaffected even by a delay of up to 16 weeks.

Modern methods of reparative surgery of the blood vessels enable the patency of the main arteries of the lower limbs to be restored comparatively quickly after acute occlusion. However, operative revascularization is not always effective, ischemic changes in the skeletal muscles are progressive, and under these conditions amputation of the affected limb cannot be avoided [3-6, 8, 15]. Development and implementation of timely ahd effective correction of acute ischemia of the limbs would undoubtedly be aided by knowledge of the dynamics: of the structural and metabolic changes taking place in skeletal muscles during acute occlusion of the main arteries.* Data in the literature on this problem do not deal with the problem as a whole, but only with certain of its aspects, and as a rule, they do not give an objective and quantitative evaluation of the morphological changes discovered [1, 7, 10, 12, 14]. The aim of this investigation was a combined quantitative morphological analysis of changes in the skeletal muscles during acute arterial occlusion of the limbs. EXPERIMENTAL METHOD Skeletal muscles from the leg of 36 dogs weighing 13-18 kg, with experimental acute occlusion of the terminal part of the aorta [2], were studied. The duration of ischemia was 0 (control), 3, 6, 9, and 12 h. Sections through the leg muscles, after embedding in paraffin wax, were stained with hematoxylin and eosin, by Van Gieson's Regaud's, and Lie's methods, and by the trichrome method of Goldner [9, 11]. Microscopic preparations were studied in ordinary and polarized light. Tissue stereologic analysis of volumes of intact and injured muscle fibers and the edematous stroma was carried out on sections stained by Regaud's method in 10 fields of vision on the "Struktura" apparatus (Central Design Bureau, Academy of Medical Sciences of the USSR). The following histochemical methods were used: the diaminobenzidine method for myoglobin [13], staining with Sudan Black B and Oil Red for phospholipids and neutral fats, the PAS reaction with amylase control to detect glycogen and neutral glycosaminoglyca ne. The enzyme-histologi cal investigation of the soleus muscle was undertaken on frozen section 10 # thick. Activity of the following enzymes was determined by the usual methods [11]: succinate (SDH), isocitrate (ICDH), malate (MDH), ~-giycerophospha te (~-GPDH), lactate (LDH), glucose-6-phosph ate (G-6-PDH), glutamate (GDH), and fi-hydroxybutyrate (fi-HBDH) dehydrogenases; NADand NADP-diaphorases , alkaline and acid phosphatases (AlP and AcP respectively), myosin ATPase, and phosphorylase. Changes in SDH, LDH, fl-HBDH, NAD, NADP, and ATPase were estimated quantitative on an MIF-7 integrating photometric microscope. In one transverse section 66 areas of red and white muscle fibers (R~F and WMF respectively) were measured photometrically. All numerical data were subjected to statistical analysis. The significance of differences between the values for the parameters was determined by Student's test. EXPERIMENTAL RESULTS After ischemia for 3 h damage to the muscle fibers was found. Tissue analysis showed changes in the ratio of the relative frequency of areas of intact and injured fibers and stroma compared with the control. In the control group, for instance, the ratio was 67:6:22%, and after ischemia for 3 h it was 6~.13:24%. The focal and more widespread increase in cross-striation of the muscle fibers will be noted (Fig. la), and in polarized light this was revealed as more intensive fluorescence of the A-disks. The time course of contraetura[ injuries to the muscle fibers could be discerned: localized areas of approximation of the A-disks, which merged *Academician of the Academy of Medical Sciences of the USSR.

Balanus kondakovi an operculate barnacle was chosen as an experimental material forin-situ characterisation of the adhesive material of its cement apparatus by standard histochemical techniques. Reactions of PAS, LTAS or Alcian Blue with cement were generally negative indicating the absence of polysaccharides. Sudan Black B test for lipids also gave poor reaction. The classic Millon’s reaction for tyrosine gave positive result and DMAB method for tryptophan developed weak blue colour in some areas of the cytoplasm. Protein bound sulfhydril or disulfide groups could be identified. The histochemical tests thus revealed that the cement material was generally poor in carbohydrates and lipid and that it had good proportion of protein.

Abnormally enlarged muscle fiber cells of ring form were incidentally detected in transverse sections of muscles of the common squirrel monkey during microscopic investigation of the composition of muscle fibers. Longitudinal sections showed slender capsule-like cysts in the sarcoplasm. Detailed examination revealed these cysts to be those ofSarcocystis. Among the three types of muscle fiber cells that compose the skeletal muscle, staining with Sudan black B revealed that this parasite selectively infested type II white fibers with a large diameter and a high glycogen content.

The histological and histochemical changes in the thyroid gland ofSarotherodon mossambicus after treatment for a period of 20 days with 0.001 ppm DDT (50% Tafarol, Dichlorodiphenyltrichloroethane) and the pattern of recovery were observed. The observations revealed a trend of increased follicular height, nuclear diameter andE/T ratio (ratio between the weight of total epithelial height and total follicular weight) significantly (p < 0.001) to that of the normal control. The gland turned considerably goitrous and nonfunctional. The colloid was vacuolated and mostly exhausted since most of the follicles were in acolloidal state with obliterated lumina. PAS, alcian blue, toluidine blue, Sudan black B and luxol fast blue tests also reveal negative reaction in thyroid of treated animals.

1. The subject of the study is the life of the mould belonging to the Trichophytone group in the nail apparatus, i.e. in the nail plate as well as in the sealing barriere. 2. Morphological properties of the mould elements, filaments and spores are described. 3. The filaments attain the length of 15–17 mi, the thickness of 1–4 mi. Their thinner anterior end possesses a strong dyeing capacity, while the conspicuously broader posterior end is less liable to dyeing. The surface is covered with minute protuberances, the membrane shows a strong light refraction along its circumference. 4. The joining of the individual segments occurs by fixing the convex surface of the anterior part to the concave surface of the posterior part of the cell. 5. The inner structure of the filaments is well apparent not only under direct observation, especially in a phase microscope, but also after dyeing with haematoxylin, Sudan black B as well as metachromatic dyes applied in the process. 6. The filaments penetrate the cornified cells while forming irregular ragged passages by means of their enzymes. 7. The dyeing capacity enables to distinquish young, old and degenerating filaments. 8. The spores have an inner structure which becomes apparent in dyeing with haematoxyline, Sudan black B as well as metachromatic dyes. The spores are endowed with a membrane possessing a strong light refraction, they are deposited or freely spread over the dissociated cornified cells. 9. The mould filaments thrive in their optimum milieu, mostly in their sealing barrieres, at other times rarely directly in the nail plate.

Morphological and histochemical changes occurring in nerve cells after experimental occlusion of the left middle cerebral artery have been studied. Material consisted of fifty-four dogs with ligation of that artery and ten controls with either only retraction of the cortex or ligation of the anterior or posterior cerebral arteries. Techniques employed were cresyl violet, PAS, Sudan black B and Gomori methods for alkaline and acid phosphatases. In twenty-two animals which survived 39 h to fourteen days, the distribution of chronic lesions and their histological type have been examined, infarcts being nearly always localized within the Sylvian region and the striated nuclei. Thirty-two other dogs had survivals inferior to or of 24 h and in these acute experiments nerve cell changes were carefully studied. Histologically, it was possible to detect three types of acute nerve cell change: (1) shrinkage and (2) hydropic change, the first appearing after 3 h, the second after 6 h, both with focal localization, thus easily distinguishable from similar bilateral artefactual images as the common chromophilic cells; (3) ischemic cell change, encountered for the first time after 6 h and having its peak at 24 h, whereas (1) and (2) became less frequent at this survival period. PAS and Sudan black B stained the three types of acutely damaged nerve cells faintly but were strongly positive in the chromophilic ones. Alkaline and acid phosphatase activities were absent both from changed and chromophilic neurones. However, nerve fibers existing throughout acutely lesioned areas as well as around chronic infarcts had strong acid phosphatase positivity and showed morphological changes such as interruptions, varicosities and terminal swellings. Owing to the rapid appearance of that increased activity, the A. considered it directly due to the ischemia of the nerve fibers. Die in Nervenzellen nach experimentellem Verschlus der A. cerebri media auftretenden morphlogischen und histochemischen Veranderungen wurden studiert. Das Untersuchungsmaterial bestand aus 54 Hunden mit Unterindung der A. cerebri media und 10 Kontrolltieren entweder mit Rindenentfernung oder Unterbindung der A. cerebri anterior oder posterior. Kresylviolett, PAS, Sudanschward B-Farbung sowie die Gomori-Methode auf alkalische und saure Phosphatase wurden angewendet. Bei 22 Tieren mit einer Uberlebenszeit von 39 Std bis zu 14 Tagen wurde die Verteilung chronischer Lasionen sowie ihr histologisches Bild untersucht. Infarkte waren fast immer in der Sylvischen Region sowie im Striatum lokalisiert. 32 Hunde hatten Uberlebenszeiten von weniger oder bis zu 24 Std. Die Nervenzellveranderungen dieser akuten Experimente wurden sorgfaltig untersucht. Histologische konnten drei Arten von akuten Nervenzellveranderungen festgestellt werden: 1. Schrumpfung, fruhestens nach 3 Std; 2. Wasserveranderungen nach 6 std, wobei beide deutliche Herdlokalisation zeigten, und daher leicht von ahnlichen bilateralen Artefakten — wie den ublichen chromohilen Zellen — zu unterscheiden waren; 3. ischamische Zellveranderungen, wobei 1. und 2. zu diesem Uberlebenszeitpunkt abnehmen. Mit PAS und sudanschwarz B werden diese drei Arten von akut geschadigten Nervenzellen schwach gefarbt, die Chromohilen hingegen reagieren stark positiv. Alkalische und saure Phosphataseaktivitat konnte weder in den veranderten noch in den chromohilen Nervenzellen festgestellt werden. Jedoch zeigten Nervenfasern in akut geschadigten Arealen als auch um chronische Infarkte stark positive saure Phosphataseaktivitat und liesen morphologische Veranderungen, wie Unterbrechungen, Unterbrechungen, Schwellungen und Endauftreibungen erkennen. Der Autor vermutet, das die rasch auftretende Aktivitatssteigerung in direkter Beziehung zu der Ischamie der Nervenfasern steht.

Paraffin sections of tissues fixed in absolute alcohol or Carnoy's fluid were mordanted in a 1% aqueous solution of phosphomolybdic acid, stained in saturated solutions of Sudan black B, acetylated Sudan black, various solvent and basic dyes in 70% ethyl alcohol for 5 min at room temperature, dehydrated in alcohol and covered in Permount. Sudan black B and other dyes with basic groups stained basement membranes, reticulum and collagen fibers intensely. Acetylated Sudan black, Sudan IV and oil red 0 did not color any tissue structures. Control sections, without pretreatment, did not bind Sudan black B. These findings indicate interaction between basic groups of the dye and free acid groups of phosphomolybdic acid.

Adrenocortical cells of the zona fasciculata of adult Syrian hamsters given 250 mg/kg triparanol daily by stomach tube were compared with cortical cells of untreated control animals. Dark cytoplasmic bodies which stained with toluidine blue, Nile blue sulfate, Sudan black B and acid hematein were observed with the light microscope. The bodies disappeared following the pyridine extraction-acid hematein test and control animals as well as animals treated with triparanol failed to give a positive reaction for cholesterol. These findings offer evidence that the cytoplasmic bodies are phospholipids. With the electron microscope the cytoplasmic granules exhibited rounded edges and were occasionally surrounded by a membrane. They displayed a crystalline latticework with opaque lines approximately 40–60 A separated by clear spaces 50–70 A. These observations coupled with the biochemical findings of other investigators suggest that the crystals may represent either the accumulations of enzymes or the matrix for a steroid precursor such as desmosterol.

A series of chemocytological observations were made on two distinctly different epithelial cell types, named barrel-shaped and rod-shaped cells, in the gall bladders of the mouse, golden hamster and guinea pig. Barrel-shaped cells contain small amounts of tetrazonium-reactive protein, Sudan black B stainable lipid, PAS-reactive polysaccharides and nucleic acids. The cells are further found to be weakly positive for α-naphthyl acetate esterase. On the other hand, rodshaped cells contain large amounts of the substances named and reveal a rather pronounced esterase activity. These results were discussed in reference to cytological and chemocytological data previously reported on the two cell types. Earlier concepts regarding the barrel-shaped cells to be specifically liquid absorptive and the rod-shaped cells to be physiologically inactive, are not supported by present evidence. Though further extensive studies are necessary, it can be presumed that barrel-shaped cells are functionally declining and rod-shaped cells highly active.

Les effets de l'acetylation reversible et de l'esterification sulfurique sur la coloration de polysaccharides par le noir Soudan B sont etudies apres inclusion a la paraffine; les faits suivants sont a noter. 1° Une affinite nette pour le noir Soudan B en solution alcoolique existe, dans le cas des polysaccharides tres acides, sans aucun pretraitement des coupes. 2° Tous les polysaccharides presentent, apres acetylation ou esterification sulfurique, une forte affinite pour le noir Soudan B, le bleu alcian et le bleu de toluidine; la teinte est souvent metachromatique avec ce dernier colorant. 3° L'affinite des polysaccharides pour les trois colorants disparait apres saponification des coupes acetylees. 4° Les teintes les plus intenses sont obtenues avec les solutions vieillies ou acetifiees de noir Soudan B. — Ces faits plaident en faveur de l'hypothese suivant laquelle le noir Soudan B possede certaines proprietes des colorants basiques.

The method of Sheehan and Storey, used for demonstrating stable Sudanophilia of neutrophils in smears, was modified by substituting other alcohols (from methanol to amyl alcohol) for ethanol, and the effect of this on the resulting staining reactions was investigated. The intensity of the staining of the neutrophil granules decreases with increasing number of carbon atoms in the alcohol, with a gradation of staining from black to yellow, while, at the same time the staining of the erythrocytes increases. The best solvent for Sudan black B for haematological purposes is methanol. This solvent was further used for investigations on various procedures for detecting so-called masked sudanophilia. The most constant and interesting results were obtained after the action of mercuric ions on smears fixed in formalin vapour. Under certain conditions, a characteristic binding of different fractions of Sudan black B on different blood cells can be attained, so that neutrophil granules are stained brown, monocytes mostly grey, lymphocytes, platelets, megakaryocytes, plasmocytes and perinuclear areas of normoblasts blue. The present experiments with various substrates indicate that demasked blue staining in the cytoplasm may be conditioned by phospholipids, although a reaction with other components of the cytoplasm cannot be excluded. The method described may be useful in haematology for studying cytogenetic relations in the monocytic and granulocytic series.

Rabbits were immunized with a low-density lipoprotein (LDL) preparation isolated from rabbit serum by ultracentrifugation. This elicited precipitin isoantibodies which distinguished two antigenically different genetic variants, i.e., allotypes of serum LDL. Both allotypes were identified as LDL by the following criteria: (1) the precipitin lines stained intensely with the lipid stain Sudan black B; (2) the antigens were found in the low-density but not the high-density lipoprotein fraction; (3) the antigens migrated electrophoretically on Agarose in the α2to β region. That the inheritance of these two allotypes is controlled by a pair of allelic genes at an autosomal locus is based on allotypes present in 323 progeny from six possible mating combinations. This LDL locus designated Lpqwas shown not to be linked to the light-chain or heavy-chain loci of immunoglobulins.

In Fortfuhrung fruherer Untersuchungen kann gezeigt werden, das sich nach Invertseifenbehandlung neben Proteinen auch saure Mukopolysaccharide mit Sudanschwarz B darstellen lassen. Nach geeigneter Differenzierung kann die C 4-SSB-Methode zur Darstellung von sauren Mukopolysacchariden in Mastzellen und im Knorpel verwendet werden.

Auf Filterpapier aufgetragene Reinsubstanzen (Lipide, Eiweissubstanzen und Polysaccharide) wurden mit Sudan III, Sudan IV, Sudanrot B, Sudanrot VII B, Scharlach R med und Sudanschwarz B (SSB) gefarbt.

Thin layer chromatography of commercial Sudan Black B on silica gel with chloroform-benzene (1∶1) as the developing solvent reveals two blue main fractions with Rf values of 0.49 and 0.19, SBB-I and SBB-II respectively. Furthermore at least eighteen secondary fractions or impurities have been found. SBB-I and SBB-II were isolated and purified by preparative thin layer chromatography. Commercial Sudan Black B consists of about 20 p.c. SBB-I, 60 p.c. SBB-II and 20 p.c. secondary fractions. From spectrophotometrical and histochemical investigations it appeared that SBB-I stains lipids more pronounced than SBB-II; moreover SBB-I is more specific for neutral lipids than SBB-II, which fraction may also stain some proteins and acid mucopolysaccharides. Contrary to SBB-II the staining with SBB-I is fairly independent of pH. Finally, the colour of SBB-II changes under the influence of light and air, while SBB-I is much more stable. A physico-chemical study of the nature of SBB-I and SBB-II, including spectrophotometry, chromatography, infrared spectroscopy and chemical analysis revealed, that SBB-II is a basic dye, while SBB-I in spite of the structural resemblance behaves as a neutral one, dissolving therefore better in neutral lipids. As yet the chemical composition of SBB-I and SBB-II, and the relation to the scheme of synthesis of Sudan Black B has not been solved. The unspecificity of lipid staining by Sudan Black B is due to the basicity of SBB-II, and to the instability of this dye toward light and air. Moreover the some eighteen impurities may have some influence on the staining properties. The question of solubility or adsorption processes in the case of lipid staining by Sudan dyes is at least partially answered by the proposition of a dissolving fraction SBB-I and an adsorbed fraction SBB-II. The changing absorption spectra by the corresponding solvatochromic and metachromatic effects may give information about the nature of the lipids stained.

In normal specimens of the bug,Dysdercus koenigii, the cells of the pars intercerebralis stain highly positively with Sudan black B and acid haematein, while the corpora cardiaca and allata stain lightly. After the administration of parathion, carbamate and endrin the situation is reversed. The increased level of lipids in the corpora cardiaca and allata coincides with the degree of loss of lipids from the cells of the pars intercerebralis.

LIPID metabolism in mammals has been widely investigated with reference to cold acclimation and hibernation. In this laboratory, work on lipid metabolism is hamsters during cold exposure and hibernation has been focused on rates of lipid synthesis or utilization in tissues in vitro. This communication is concerned with measurement of the circulating total lipid in the intact animal. Analyses were performed on blood from, cold-exposed and hibernating golden hamsters (Mesocricetus auratus). A similar series of analyses were performed on white Wistar rats to provide comparison with a non-hibernating mammal. Blood samples were taken from animals kept at room temperature as control, and kept in the cold (5° ± 1° C) for 48 h, 3 weeks or 6–8 weeks. Samples were also taken from hamsters in hibernation, arousing from hibernation (cheek pouch temperature 18°–20° C) and immediately after the animals regained full consciousness and normal temperature. The animals were stunned by a sharp blow on the head, except those in hibernation. Approximately 2 c.c. of blood were withdrawn by cardiac puncture. After removal of the needle the syringe was discharged into a centrifuge tube and the sample allowed to clot. The samples were centrifuged and the serum removed by suction. The serum was then analysed for total lipid by a colorimetric method employing Sudan black B, described by Swahn1. The samples were compared with a standard of corn oil in ethanol. The concentrations were calculated as mg total lipid/100 ml. of serum. The results are shown in Fig. 1.

DURING investigations in which sudan black B was used for studying the lipoid inclusions in cells of bone and teeth1, it was found that dentin, enamel, cartilage and bone did not stain significantly unless they were previously extracted with pyridine at 60° C., when characteristic staining reactions were obtained.

IN 19581 a method was described for staining sites of calcification at the ‘calcification front’—the edge of the preosseous matrix in bone, the cartilage around the hypertrophic cells of the epiphyseal cartilage and the junction of predentin arid dentin. The method consisted, after fixation, in extracting the tissue with hot pyridine, decalcifying, embedding in gelatin, and staining with an alcoholic solution of sudan black B after sectioning on a freezing microtome. The sections were mounted in glycerine jelly. A fuller account of the method was published later2.