Chromosome Preparations from Mouse Embryos During Early Organogenesis: Dissociation After Fixation, Followed by Air Drying

Embryos are put into 1% sodium citrate at 37 C; 7- and 8-day specimens requiring about 20 min. With increasing age, the duration of treatment is increased up to 50 min. Handling is facilitated by keeping specimens in a small glass vessel for observation under a binocular microscope, and by changing fluids with a fine-tipped pipette. Fixation in ethanol-acetic acid 3:l for 2-3 hr is uncritical, as material may be stored in the fixative overnight at 4 C. Staining in toto with 2% orcein in 50% acetic acid follows, requiring 0.5-1 hr (storage in this solution up to 2 wk at 4 C is permssible). After staining, specimens are subjected to cellular dissociation in a mixture of glacial acetic and 50% lactic acid, the action of which is controlled by the duration of treatment and by increasing the ratio of lactic to acetic from 1:Z (younger embryos) to 3:2 (older embryos). Only 1-3 drops of the dissociating fluid is used for each embryo, to favor concentration of the free-floating cells. Since the time required varies from several minutes to nearly an hour, the most favorable degree of dissociation can best be judged by the cloudiness produced in the dissociating fluid. A small drop not exceeding 2 mm in diameter, of the cell suspension, is placed on a slide and followed immediately by a normal-sized drop of fresh 3:1 ethanol-acetic. After drying, the chromosomes are stained with lactic-acetic-orcein or other suitable stain. The method gives satisfactory results with embryos from the 7th to 11th day of pregnancy.     

Production of vigorous,desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor

Summary This report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20–50 M abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.Plant Research Centre contribution No. 1523     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in Whole-Mount Skeletons in Vitro

Cartilage and bone of the developing skeleton can be reliably differentiated in whole-mount preparations with toluidine blue-alizarin red S staining after FAA fixation. The recommended staining procedure is based chiefly on the use of newborn white and Swiss-Webster mice, 4-9 days postnatal, but was tested also on mice and rats 3-8 wk of age. Procedure: Sacrifice, skin, eviscerate, remove body fat, and place specimens in FAA (formalin, 1; acetic acid, 1; 70% alcohol, 8) for approximately 40 min. Stain in 0.06% toluidine blue made in 70% ethyl alcohol for 48 hr at room temperature. Use 20 volumes of stain solution to the estimated volume of the specimen. Destain soft tissues in 35% ethyl alcohol, 20 hr; 50%, 28 hr; and 70%, 8 hr. Counterstain in a freshly prepared 1% aqueous solution of KOH to which is added 2-3 drops of 0.1% alizarin red S per 100 ml of solution. Each day for 3 days, transfer the specimen to a fresh 1% KOH-alizarin mixture, or until the bones have reached the desired intensity of red and soft tissues have cleared. Rinse in water, and place in a 1:1 mixture of glycerol and ethyl alcohol for 1-2 hr, then transfer the specimen to fresh glycerol-alcohol for final clearing and storage. Older mice and rats require procedural modifications: (1) fixation for 2 hr, (2) 0.12% toluidine blue, (3) maceration for 4 days in 3% KOH-alizarin, and (4) preliminary clearing for 24 hr in a mixture of glycerol, 2; 70% ethyl alcohol, 2; and benzyl alcohol, 1 (v/v) before placing in a 1:1 alcohol-glycerol mixture.     

Enhanced microbial safety of channel catfish (Ictalurus punctatus) fillet using recently invented medium molecular weight water-soluble chitosan coating

Chitosan with higher molecular weight exhibited higher antimicrobial efficacy against foodborne pathogens. However, the poor water solubility of higher or medium molecular weight chitosan limits its applications. To overcome the challenge, our research team searched for simple preparation procedure for fast-dissolving medium molecular weight chitosan in water. Throughout the process, we were able to obtain a higher concentration of medium molecular weight water-soluble (MMWWS) chitosan (400 kDa). The MMWWS chitosan showed physicochemical properties that are suitable for edible coating. Antibacterial activities of 400-kDa chitosan coating prepared in acetic acid (1% v/v) or aspartic acid (1% or 3% w/v) were examined. The surface of catfish cubes was inoculated with six foodborne pathogens and then coated with chitosan solutions. The survival of each pathogen was evaluated during shelf life storage. Compared with the control, 3% w/v chitosan coating in aspartic acid solution exhibited the most effective antibacterial activities among other coating treatments, completely inhibiting Vibrio parahaemolyticus on the surface of catfish. The study suggested that chitosan dissolved in aspartic acid has the potential for use as an alternative antimicrobial coating for catfish fillet.     

Induced lactic acid fermentation during the preservation stage of ripe olives from Hojiblanca cultivar

The influence of initial sodium chloride concentration (6 and 0%, w/v), acetic acid concentration (0.6, 0.3 and 0.0%, v/v), type of process (natural and inoculated), and storage system (anaerobic and aerobic) on the inducement of a lactic fermentation for the preservation stage of Hojiblanca cultivar ripe olives was investigated. The addition of 6% NaCl prevented colonization by lactic acid bacteria in all cases. A high level of acetic acid (0.6%) was effective in preserving olives for 2 months, although yeast growth was not inhibited for longer periods of storage. Natural growth of Lactobacillus plantarum did not occur. Inoculation with this micro-organism was effective only in the two treatments with tap water (with no NaCl) as the initial covering solution, although survival was reduced to a half of the added organisms when the initial pH was corrected with 0.3% acetic acid. In these two treatments pH quickly reached appropriate values (<4.0) for olive stabilization. Aerobic conditions led to low concentrations of carbon dioxide, without disturbing growth of lactic acid bacteria. Thus, the aerobic lactic acid fermentation, with tap water initially, was the most adequate preservation procedure for the storage of ripe olives prior to their oxidation treatment. Results of trials conducted on an industrial scale showed the same pattern and confirmed the viability of the new procedure.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

A simple method for preparing meiotic chromosomes from mammalian testis

Freshly removed testis tubules are treated with hypotonic citrate solution and fixed in 31 acetic alcohol without undue rupture. The material is transferred to 60% acetic acid and the resulting suspension is air-dried onto warm slides using a micropipette. Finally the slides are stained with lacto-acetic orcein. — Provided suitable storage is made, slides need not be prepared in the same week as fixation and this may facilitate the collection of material during field study.     

Automated differential staining for cartilage and bone in whole mount preparations of vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

Automated Differential Staining for Cartilage and Bone in Whole Mount Preparations of Vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

Rheological and Physicochemical Studies on Emulsions Formulated with Chitosan Previously Dispersed in Aqueous Solutions of Lactic Acid

Chitosan, a natural, cationic polysaccharide, may be a hydrocolloid strategic to formulate acidic food products, as it can act as both bio-functional and technofunctional constituent. Typically, acetic acid is used to disperse chitosan in aqueous media, but the use of this acid is limited in food formulations due to its flavor. In this study, chitosan was firstly dispersed (0.1% m/V) in lactic acid aqueous solutions (pH 3.0, 3.5 or 4.0), and then evaluated regarding its thickener and emulsion stabilizer properties. O/W emulsions were prepared and characterized in terms of rheological properties, droplets average diameters and droplets ζ-potential. Emulsions containing chitosan were 3 times more viscous than controls without chitosan, and presented storage modulus (G’) higher than loss modulus (G”). Furthermore, they displayed two different populations of droplets (average diameters of 44 and 365 nm) and positive ζ-potential values (+50 mV). Droplets average diameters and ζ-potential did not present significant changes (p > 0.05) after storage at 25 °C during 7 days. This study showed that i) food organic acids other than acetic acetic acid can be used to disperse chitosan for technological purposes, and ii) chitosan dispersed at very low concentrations (0.1 m/V %) had relevant effects on rheological and physicochemical aspects of food-grade emulsions.     

In vitro response of encapsulated somatic embryos of Lagerstroemia indica L

A method to produce encapsulatable units for synthetic seeds was developed in L. indica. Somatic embryos were harvested from leaf derived embryogenic callus on Murashige and Skoog's basal medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 0.5 mg/l), 6-benzyl amino purine (BAP, 1 mg/l) and ascorbic acid (AA, 50 mg/l). The embryos were encapsulated in alginate beads and dehydrated. Germination ability of the artificial seeds were investigated. The frequency of regeneration from the encapsulated embryos was significantly affected by (i) the concentration of alginate (ii) the duration of storage, and (iii) the effect of different types of media. A 2% sodium alginate concentration on MS salts resulted in significantly higher germination frequencies than at other concentrations. L. indica showed maximum germination on MS medium (93.84%) after 6 weeks of culture. The germinated synthetic seeds with well developed roots and shoots were transferred successfully to green house. This is the first report on artificial seeds in Lagerstroemnia indica.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Effects of ethanol and acetic acid on the transport of malic acid and glucose in the yeast Schizosaccharomyces pombe: implications in wine deacidification

Abstract Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of l-malic acid in Schizosaccharomyces pombe . The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics. Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v). The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport. Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects. Accordingly, deacidification by Ss. pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae .     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

Fractionation of wheat straw by atmospheric acetic acid process

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.