A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

Alcoholic Hydrochloric Acid-Carmine as a Stain for Chromosomes in Squash Preparations

A regressive bulk carmine staining schedule was adapted from a formula proposed by P. Mayer. The stain is made by boiling gently 4 gm of certified carmine in 15 ml of distilled water to which 1 ml of concentrated HC1 has been added. After cooling, 95 ml of 85% alcohol is added, and the solution filtered. Fixed tissue is soaked in the stain until thoroughly penetrated; squashes are then prepared as usual, but plain 45% acetic acid is used as the temporary mounting medium instead of aceto-carmine. The advantages of this method are: intense, precise staining of chromosomes coupled with a lightly stained cytoplasm; consistency and uniformity of results; simplicity of use.     

The Use of Haematoxylin for Squash Preparations of Chromosomes

A simplified propionic-iron alum-haematoxylin stain for rapid squash preparations of chromosomes requires only two stock solutions: (A) 2% haematoxylin and (B) 0.5% iron alum, both in 50% propionic acid. For use, suitable volumes of A and B are mixed. With unripened solution A, equal volumes should be used and the stain is ready for use 1 day after mixing. As the haematoxylin ripens, progressively smaller amounts of B are required and the mixture may be used immediately. The stain gives excellent results when used in the same way that orcein and carmine are currently employed, with a wide range of animal and plant (including fungal) chromosomes, and with good nucleolar staining. It may be used either following acetic alcohol (1:3) fixation or as joint fixative and stain on unfixed material. In fungal material, where Lu's BAC fixative is recommended, the centrioles are also stained.     

Handling Avian Chromosomes

The somatic and meiotic chromosomes of Galius gal-lus dom. (as well as of the rat, mouse and possibly other animals) may be adequately prepared for miscroscopic examination by the following smear technic: Pith the fowl of appropriate age through the palatal fissure (other killing technics may be substituted for other animals), remove testes, slice medianly into halves (or quarters if large) and fix in mixture of 2 parts absolute ethanol, 1 part propionic acid, 1 part chloroform. Pour off and replace fixative at the end of 12-24 hours, after which the tissue may be stained or stored under refrigeration. Fresh material can be stained in propionic-carmine (0.5 g. carmine boiled in 45% aqueous solution of propionic acid), but material stored for several months should be stained in a boiled mixture of 10 ml. vinyl acetate, 10 ml. tertiary butyl alcohol, 50 ml. propionic acid, 75 ml. H₂O, 0.25 g. carmine, 0.25 g. orcein. A method is described for preparing and making permanent such preparations.     

The Use of Lacto-Propionic Orcein in Rapid Squash Methods for Chromosome Preparations

A solution of 2 gm of natural orcein dissolved in 100 ml of a mixture of equal parts of lactic and propionic acids, and diluted to 45% with water, proved more effective than other stain fixatives for meiotic preparations from fresh pollen mother cells. When used after 5 min fixation in modified Carnoy's fixative (alcohol, acetic acid, chloroform, formalin; 10:2:2:1) and 5 min maceration in 1 N HC1 at 60° C, the same stain proved the most suitable for the rapid preparation of root-tip chromosomes for counting and for studying detailed morphology.     

Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

Z-DNA immunoreactivity of Drosophila polytene chromosomes : Effects of the fixatives 45% acetic acid and 95% ethanol and of DNase I nicking

Drosophila salivary chromosomes have been isolated at neutral pH and physiological ionic strength. They display only background level binding of antibodies against Z-DNA. Following exposure to the commonly used fixative 45% acetic acid all of the polytene chromosomes, X and autosomes, show a massive increase in anti-Z-DNA antibody binding. The enhancement from background to intense fluorescence occurs whether the chromosomes are stabilised by two orders of magnitude lower concentration of formaldehyde than that used to minimise protein extraction in classical acid squash preparations, or by physiological concentrations of spermine and spermidine. Nicking of acetic acid-treated chromosomes by DNase I dramatically reduces their Z-DNA immunoreactivity. The histones and non-histones extracted by 45% acetic acid from unfixed and formaldehyde-fixed Drosophila chromatin have been analysed. Exposure of isolated salivary chromosomes to the non-protein-extracting fixative 95% ethanol also enhances Z-DNA immunoreactivity. All of these phenomena must be taken into account in the search for the Z-DNA conformation in cells by cytological techniques.     

An Air-Drying Procedure for Mammalian Male Meiotic Chromosomes, Following Softening in Gluconic Acid and Cell Separation by An Ethanol-Acetic Mixture

A I cm³ sample of tubules from testes is placed in 5 ml of 0.7% Na-citrate for 20-30 min, then 5 ml of glacial acetic acid is added, mixed well, and allowed to stand for 30 min. The mixture is centrifuged, the supernatant removed, and 3 ml of 3 M gluconic acid is mixed with the tissue and allowed to act for 3 hr. The gluconic acid is removed with a pipette and the tissue is suspended in 5 ml of a freshly made 1:1 absolute ethanol-glacial acetic acid mixture. The tissue is drawn into and discharged from a syringe several times through an 18, a 20, and finally a 22 gauge needle to separate and suspend the cells. The cells are centrifuged and resuspended several times in fresh fixative to remove the gluconic acid. Finally, the cells are suspended in sufficient fixative to give a smear of suitable density, and air-dried preparations are made, or the suspension may be stored at 0-5 C for several days. The cells can be stained by any of the usual stains for chromosomes. This technique results in the improved spreading produced by the air-drying technique and permits recovery of all stages of meiosis and mitosis present.     

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A₃ R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.Abbreviations PI propidium iodide - NOR(s) nucleolus organizer region(s) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Deceased, April 23, 1988     

DNA loss during C-banding of chromosomes of Lilium longiflorum

Root tip chromosomes were uniformly labelled with ³H-thymidine and replicate squashes were made. One set was untreated, one incubated in Ba(OH)₂ solution, and a further set treated sequentially in Ba(OH)₂ and hot saline-citrate (2 × SSC) to reveal C-bands. All replicates were autoradiographed and comparative grain counts made. Differences in grain numbers per metaphase cell showed that Ba(OH)₂ extracted 40% of label, and that a further 23% was lost in the subsequent SSC incubation. The distribution of grains was mapped along a sample of each of five individually-recognisable chromosomes at the three treatment stages. Within each chromosome, the number of grains per segment did not differ significantly from a random distribution. This was true for all five chromosomes at all three stages of treatment, whether or not the regions were C-banded. — We conclude that DNA extraction occurs progressively during C-banding in Lilium, but that C-bands are not dark because of their relatively high retention of DNA.     

Staining Algal Pyrenoids with Carmine After Fixation in an Acidified Hypochlorite Solution

Pyrenoids but not nucleoli are clearly stained with propionocarmine after a special fixation. The pyrenoid is distinguished as the largest dark purple, intracellular body that stains homogeneously. The general procedure is as follows: unicells and filaments are collected by centrifugation and any liquid discarded; cells are fixed 5 rain in a mixture of 10 volumes of 50% ethanol, 10 of concentrated HC1, and 5 of Clorox; washed in 95% ethanol 3 times or more to remove all chlorophyll and fixative; mordanted for 5 min with a suitably adjusted concentration of ferric propionate in 50% propionic acid (reddish orange in color); stained 5 min in 0.5% carmine in 50% propionic acid heated in a boiling water bath. The material is mounted in 50% propionic acid. The results are reproducible when material is processed in centrifuge tubes and when the optimum time in the mordant and stain is determined for a particular species and then repeated accurately. This technique was successful in staining pyrenoids of all of the 8 species tested, representing 6 algal divisions.     

Degradation of acrylic acid by fungi from petrochemical activated sludge

Summary Geotrichum sp. and Trichoderma sp. isolated from petrochemical effluent treatment plant activated sludge completely degraded 2 g acrylic acid/l as sole carbon source in 4 d. However, after neutralization with Ca(OH)₂, concentration as high as 10 g/l was degraded in 6 d. During the degradation acetic acid and -hydroxy propionic acid transiently accumulated.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Modification of Fertility of Soil Materials for Restoration of Acid Grassland Habitat

Use of composts for habitat restoration offers advantages in terms of efficient use of resources. Chemical amendment of compost to reduce its pH and P availability was investigated in order to improve suitability for use in reclamation of blocky quarry waste to acidic grassy heathland. The effect of these amendments was observed on competition between two grass species: Agrostis capillaris and Festuca ovina. A factorial, pot‐scale greenhouse experiment was set up using two composts (one a mixture of green waste and catering waste, and the other a mixture of green waste and sewage sludge). In addition, two soils were collected from upland acidic grassland to provide a natural comparison. S⁰ was applied to reduce soil pH, and Fe(OH)₃ from a coal waste treatment plant was applied to counteract the expected increase in P availability due to acidification of the composts by S⁰. Addition of S⁰ significantly reduced soil solution pH and addition of Fe(OH)₃ significantly reduced soil solution P concentration. In one compost S⁰ reduced the biomass of F. ovina while increasing that of A. capillaris, whereas Fe(OH)₃ had no significant effect on the biomass of either species. Although S⁰ and Fe(OH)₃ did adjust the chemical properties of the soil solution, Fe(OH)₃ did not bind P strongly enough to make it unavailable to plants. Further work is required, however, the use of chemically amended composts provides a sustainable sink for organic wastes and we conclude from this study that they have great potential for large‐scale restoration of blocky waste tips.     

The influence of LBG addition to carrageenan on the mechanical stability of the gel and the fermentative activity of immobilized propionic acid bacteria

In the first part of the experiments, the mechanical properties of 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/locust bean gum (LBG) gels stored in various concentrations of propionic and acetic acids and their mixtures were examined. The stability of these materials was measured by uniaxial compression between two parallel plates using the Instron Universal Testing Machine. A mathematical model explaining the dependence of the destroying force on the storage time was chosen for data analysis. Using this model, the average rate of gel deterioration was calculated. The structural properties of the examined gels were most influenced by the highest concentration of propionic and acetic acids and their mixtures (1% acetic acid and 2% propionic acid). The addition of LBG to carrageenan decreased the gel destroying force and increased its resistance to acids. In the second part of the experiments, the Propionibacterium freudenreichii subsp. shermanii NCFB 1081 and NCFB 566 were immobilized in a living state in 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/LBG gels. The ammonia consumption, glucose utilization, production of propionic and acetic acids and the biosynthesis of vitamin B₁₂ were examined. An increase in the productivity of propionic acid and a significant decrease in the vitamin B₁₂ produced in the biosynthesis were observed when immobilized cells were used. The immobilization of cells enhanced the productivity of propionic acid by up to 40% compared to free cells. The best results were obtained for the second and third applications of immobilized cells in all concentrations of carrageenan gels and 2% and 3% carrageenan/LBG gels The results showed that carrageenan/LBG is a better support material for the immobilization of propionic acid bacteria than the pure carrageenan.     

Aceto-Carmine-Lactophenol Preparations Of Paramecium Aurelia

Paramecia were killed and stained by adding a sat. soln. of carmine in acetic acid to a small drop of culture, cleared with 45% acetic acid as soon as the nuclei became darkly colored, and mounted in lactophenol (phenol crystals, 20 g.; lactic acid, 20 ml.; glycerol, 40 ml.; distilled water, 20 ml.). The mounting mixture was prepared by warming to dissolve the phenol and 20 drops of aceto-carmine added after cooling. Cover glasses were ringed with colorless nail polish or with asphaltum after slides had stood several days.     

The end products of the metabolism of aromatic amino acids by clostridia

The end products of the metabolism of phenylalanine, tyrosine and tryptophan by growing cultures of clostridia have been identified. The species used were Clostridium aminovalericum; C. bifermentans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. cochlearium; C. difficile; C. ghoni; C. histolyticum; C. lentoputrescens; C. limosum; C. lituseburense; C. malenomenatum; C. mangenoti; C. propionicum; C. putrefaciens; C. sordellii; C. sporogenes; C. sporosphaeroides; C. sticklandii; C. subterminale; C. tetani; C. tetanomorphum. The mixture of aromatic compounds formed, which depended upon the species, included phenyl acetic acid, phenyl propionic acid, phenyl lactic acid, phenol, p-cresol, p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, indole, indole acetic acid and indole propionic acid.Abbreviation GLC gas liquid chromatography     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Stains recently certified

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.