Effect of Storage of Mueller-Hinton Agar Plates on Zone Sizes for Antimicrobial Testing

The length of time Mueller-Hinton agar plates can be stored at 4 C without affecting the size of zones of inhibition in susceptibility testing by the Bauer-Kirby method was studied. It was found that these plates can be stored for 3 weeks at 4 C without an appreciable affect on zone sizes. Storage of plates in sealed plastic bags did not alter the results significantly. The findings indicate that commercially prepared Mueller-Hinton agar plates, which may be several days old when received at the laboratory, are suitable for use in routine susceptibility tests by the Bauer-Kirby method.     

Fruit-body production of test cultures ofFlammulina velutipes preserved for seven years by freezing at three different temperatures

Two strains ofFlammulina velutipes were cultured on PDA plates, and mycelial disks punched out using a cork borer were used for preservation. Five disks of a strain were put into a vial containing one of three cryoprotectants, 10% glycerol, 5% DMSO or 10% polyethylene glycol. Vials were then stored for 7 yr at −20°C, −85°C or liquid nitrogen temperature. The mycelial growth on PDA plates of the cryopreserved mycelial disks, as well as the usual subcultures, were tested two times. After the second test, spawns were prepared for fruit-body production tests by bottle cultivation from selected plates of the second growth tests. The yields of fruit-bodies varied among the cultures derived from the mycelial disks of the same strain preserved under different conditions. Variation in yields was observed even among the mycelial disks preserved at liquid nitrogen temperature, although the range of yield variation was narrower. The yield variation was obvious for the cultures which showed large retardation in the growth test. Four mycelial disks out of the six preserved at −20°C showed higher yields than those preserved at other temperatures. Among the cultures derived from strain FMC224, the control cultures preserved by subculture showed the lowest yield.     

Sucrose and light effects on in vitro cultures of potato,chokecherry and saskatoon berry during low temperature storage

Cultures of potato (Solanum tuberosum) cv. Atlantic, chokecherry (Prunus virginiana L.) cv. Garrington and saskatoon berry (Amelancher alnifolia Nutt.) cv. Northline grown in vitro for 3 weeks at 24/22 °C, 16-h photoperiod, 150 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD) mixed fluorescent/incandescent light were stored for 6, 9 and 12 weeks at 4 °C under 0 (darkness) and 3 μmol m⁻² s⁻¹ PPFD (690 nm red light continuous illumination). Growth regulators free MSMO medium either with or without 30 g l⁻¹ sucrose was used to store the cultures. All cultures retained capacity to re-grow after storage. Tested factors, sucrose, light and the length of the storage period had an impact on shoot quality and re-growth capacity of the cultures. For either light treatment sucrose was essential for the low temperature maintenance of vigorous stock plants of potato, if stored for over 6 weeks. Chokecherry and saskatoon cultures stored well without sucrose; although chokecherry benefited from sucrose in the storage medium when the stock cultures were kept at the low temperature for 12 weeks. Low light significantly improved quality of the stored potato cultures, but had very little effect on both chokecherry and saskatoon berry cultures. The woody plant cultures grew during storage, and the longer the stock plants were stored, the more vigorous cultures they generated. The results indicate that growers can successfully use their existing facilities, small refrigerators and coolers with low light intensity, set at 4 °C, for short term storage of potato, chokecherry and saskatoon berry cultures. The potato cultures, which are known to be sensitive to prolonged low temperature storage, should be frequently monitored and subcultured as required. On the other hand, the woody plant stock cultures do not require any special attention when kept at 4 °C and re-grow the most vigorous shoots if stored for at least 12 weeks. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of storage and processing on the ascorbic acid content of concentrates prepared from Chaetoceros calcitrans

Microalgae concentrates, prepared by centrifuging axenic (bacteria-free) cultures of Chaetoceros calcitrans (Paulsen) Takano, were processed and stored under different experimental conditions. The content of ascorbic acid was examined in the concentrates, to assess potential changes in their nutritional properties. In algae pastes stored at 4 °C, it reduced by 29% after 4 weeks storage. As most of the ascorbic acid was retained intracellularly (92%) after resuspension, most of the cells had remained intact. In frozen and dried paste preparations, the losses of ascorbic acid ranged from minor (11% after liquid nitrogen storage for 4 weeks) to major (≥94% after drying at 100 °C for 2 h or at 60 °C overnight). However, most of the remaining ascorbic acid (>85%) in these preparations was rapidly leached from cells upon resuspension. Therefore, pastes stored at 4 °C may have the best potential as an ‘off-the-shelf’ microalgal food product for mariculture. Pastes should now be assessed in animal feeding trials, before being recommended for widespread use in the industry.     

Effect of low-temperature stress on abscisic acid,jasmonates, and polyamines in apples

The effect of low temperatures on polyamines, jasmonates, abscisic acid (ABA), and antioxidant activities was investigated in apple fruitlets. Although endogenous ABA concentrations were not significantly different between untreated control fruit kept at −2°C and those kept at 20°C, endogenous jasmonic acid (JA), putrescine, and spermidin concentrations at −2°C were generally higher than those at 20°C. Endogenous ABA concentrations increased in n-propyl dihydrojasmonate (PDJ)—or spermine-treated fruit in comparison to the untreated control at 20 and −2°C. The applications of PDJ or spermine decreased low-temperature injuries such as splitting and spotting in fruit. Although the IC₅₀ of 1,1-diphenil-2-pycrylhydrazyl (DPPH)-radical scavenging activities was not significantly different among the treatments, the IC₅₀ of O₂ ⁻-scavenging activities in PDJ-treated or Spm-treated fruit at 5 days after the low-temperature treatment was lower than in the untreated control at 20 and −2°C. The expression of MdCHS increased in Spm-treated fruit. The concentrations of ascorbic acid, catechin, chlorogenic acid, epi-catechin, and phloridzin in Spm-treated fruit were higher than in the untreated control at −2 or 20°C. These facts suggest that ABA, jasmonates and polyamines may be associated with low-temperature stress tolerance in apple fruitlets.     

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle,Chelonia mydas

Summary A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle,Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30°C although replication occurred between 16 and 37°C. These cells may be held as monolayers at 8°C or stored frozen in growth medium containing 10% dimethylsulfoxide at −70 or −196°C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.     

Storage and conversion of Eclipta alba synseeds and RAPD analysis of the converted plantlets

The encapsulated shoot tips and nodal segments of Eclipta alba were stored at 4, 12 and 20 °C under irradiance of 1.5 gmmol m⁻² s⁻¹ and high conversion was observed in synseeds stored at 4 °C for 8 weeks. Duration of storage was extended up to 12 weeks by decreasing sucrose concentration in the alginate matrix from 3 to 1 or 2 % and conversion frequency was 71.2–76.1 %. Synseed-derived plantlets survived by 100 % in ex vitro conditions. RAPD analysis revealed uniform amplification profile in donor and synseed derived plantlets.     

Microorganism inactivation using high-pressure generation in sealed vessels under sub-zero temperature

In order to test the possibility of utilizing high pressure in bioscience and biotechnology, a simple method for high-pressure generation and its use for microbial inactivation have been studied. When a pressure vessel was filled with water, sealed tightly and cooled to sub-zero temperatures, high pressure was generated in the vessel. The pressure generation was 60 MPa at −5 °C, 103 MPa at −10 °C, and 140 MPa at −15 °C, −20 °C, and −22 °C. The high pressure generated inactivated microorganisms effectively: yeasts (Saccharomyces cerevisiae and Zygosaccharomyces rouxii), bacteria (Lactobacillus brevis and Eschericia coli), and fungi (Aspergillus niger and Aspergillus oryzae) were completely inactivated when stored in sealed vessels −20 °C for 24 h. However, Staphylococcus aureus was only partly inactivated under the same conditions. This method opens up a new application of high pressure for storing, transporting, and sterilizing of foods and biological materials. Received: 28 July 1997 / Received last revision: 12 June 1998 / Accepted: 19 June 1998     

Growth promotion of wheat seedlings by <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 μg of P ml⁻¹ day⁻¹), and produced IAA (14.9 μg ml⁻¹ day⁻¹) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of spray-drying and storage on astaxanthin content of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> biomass

The main objective of this study was to evaluate the stability of astaxanthin after drying and storage at different conditions during a 9-week period. Recovery of astaxanthin was evaluated by extracting pigments from the dried powders and analysing extracts by HPLC. The powders obtained were stored under different conditions of temperature and oxygen level and the effects on the degradation of astaxanthin were examined. Under the experimental conditions conducted in this study, the drying temperature that yielded the highest content of astaxanthin was 220°C, as the inlet, and 120°C, as the outlet temperature of the drying chamber. The best results were obtained for biomass dried at 180/110°C and stored at −21°C under nitrogen, with astaxanthin degradation lower than 10% after 9 weeks of storage. A reasonable preservation of astaxanthin can be achieved by conditions 180/80°C, −21°C nitrogen, 180/110°C, 21°C nitrogen, and 220/80°C, 21°C vacuum: the ratio of astaxanthin degradation is equal or inferior to 40%. In order to prevent astaxanthin degradation of Haematococcus pluvialis biomass, it is recommended the storage of the spray dried carotenized cells (180/110oC) under nitrogen and −21°C.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Production of haploid plants from in vitro culture of unpollinated ovules of Cucurbita pepo

Ovaries from squash plants (cv. Eskandarani) were picked one day before anthesis, and exposed to cold temperature (4 °C) for 0, 2, 4 and 8 days. The ovules were cultured on MS medium with 30 g l⁻¹sucrose, 8 g l⁻¹agar and supplemented with four concentrations of 2,4-D, i.e., 0.1, 1.0, 5 and 10 mg l⁻¹. Then the dishes were incubated at 25 ± 1 °C under 16-h photoperiod for 4 weeks. After that ovules were transferred to growth regulator free MS medium for 4 weeks. Data indicated that the most plantlets per 100 cultured ovules resulted from the ovule of ovaries without cold treatment, when cultured in MS medium supplemented with 1 or 5 mg l⁻¹2,4-D. The cytological study revealed that one third of examined plants were haploid (2n = x = 20) and the others were double haploid (2n = 2x = 40). This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

Changes in fungi and mycotoxins in pearl millet under controlled storage conditions

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25°C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25°C in either air (aerobic) or N₂ (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25°C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), “Helminthosporium”-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25°C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20–22%. Aflatoxin contamination averaged 174 ng g⁻¹ over all treatments, and increased up to 798 ng g⁻¹ in high-moisture grain at stored at 25°C.     

Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents

Six crude oil-degrading bacterial strains isolated from different soil and water environments were combined to create a defined consortium for use in standardized efficacy testing of commercial oil spill bioremediation agents (OSBA). The isolates were cryopreserved in individual aliquots at pre-determined cell densities, stored at −70°C, and thawed for use as standardized inocula as needed. Aliquots were prepared with precision (typically within 10% of the mean) ensuring reproducible inoculation. Five of the six strains displayed no appreciable loss of viability during cryopreservation exceeding 2.5 years, and five isolates demonstrated stable hydrocarbon-degrading phenotypes during inoculum preparation and storage. When resuscitated, the defined consortium reproducibly biodegraded Alberta Sweet Mixed Blend crude oil (typically ± 7% of the mean of triplicate cultures), as determined by quantitative gas chromatography–mass spectrometry of various analyte classes. Reproducible biodegradation was observed within a batch of inoculum in trials spanning 2.5 years, and among three batches of inoculum prepared more than 2 years apart. Biodegradation was comparable after incubation for 28 days at 10°C or 14 days at 22°C, illustrating the temperature tolerance of the bacterial consortium. The results support the use of the synthetic consortium as a reproducible, predictable inoculum to achieve standardized efficacy tests for evaluating commercial OSBA. Received 31 August 1998/ Accepted in revised form 30 November 1998     

Effect of storage temperature on prokaryotic cell counts and community composition analysis from fixed and filtered seawater samples

Marine, pelagic prokaryotes commonly are visualized and enumerated by epifluorescence microscopy after staining with fluorescent, DNA-binding dyes and sample preparation and storage has a major influence on obtaining reliable estimates. However, sampling often takes place in remote locations and the recommended continuous sample storage at −20°C until further sample evaluation is often logistically challenging or infeasible. We investigated the effect of storage temperature on fixed and filtered seawater samples for subsequent enumeration of total prokaryotic cells and community composition analysis by fluorescence in situ hybridization (FISH). Prokaryotic abundance in surface seawater was not significantly different after 99 days when filters were stored either at room temperature (RT) or at −20°C. Furthermore, there was no loss in detection rates of phylotypes by FISH from filters stored at RT or −20°C for 28–30 days. We conclude that fixed and filtered seawater samples intended for total prokaryote counts or for FISH may be maintained long-term at room temperature, and this should logistically facilitate diverse studies of prokaryote ecology, biogeography, and the occurrence of human and fish/shellfish pathogens.     

Thermal tolerance and acclimation response of larvae of the sub-Antarctic beetle Hydromedion sparsutum (Coleoptera: Perimylopidae)

Hydromedion sparsutum is a locally abundant herbivorous beetle on the sub-Antarctic island of South Georgia, often living in close association with the tussock grass Parodiochloa flabellata. Over a 4-day period in mid-summer when the air temperature varied from 0 to 20°C, the temperature in the leaf litter 5–10 cm deep at the base of tussock plants (the microhabitat of H. sparsutum) was consistently within the range of 5–7.5°C. Experiments were carried out to assess the ability of H. sparsutum larvae collected from this thermally stable environment to acclimate when maintained at lower (0°C) and higher (15°C) temperatures. The mean supercooling points (freezing temperature) of larvae collected in January and acclimated at 0°C for 3 and 6 weeks and 15°C for 3 weeks were all within the range of −2.6 to −4.6°C. Larvae in all treatment groups were freeze tolerant. Acclimation at 0°C significantly increased survival in a 15-min exposure at −8°C (from 27 to 96%) and −10°C (from 0 to 63%) compared with the field-fresh and 15°C-treated larvae. Similarly, survival of 0°C-acclimated larvae in a 72-h exposure at −6°C increased from 20 to 83%. Extending the acclimation period at 0°C to 6 weeks did not produce any further increase in cold tolerance. The concentrations of glucose and trehalose in larval body fluids increased significantly with low temperature acclimation. Larvae maintained at 15°C for 3 weeks (none survived for 6 weeks) were less able to survive 1-h exposures between 30 and 35°C than the 0°C-treated samples. Whilst vegetation and snow cover are an effective buffer against low winter temperatures in many polar insects, the inability of H. sparsutum larvae to acclimate or survive at 15°C suggests that protection against high summer temperatures is equally important for this species. Accepted: 2 August 1999     

Electrophysiological and ultrastructural correlates of cryoinjury in sciatic nerve of the freeze-tolerant wood frog, Rana sylvatica

We investigated function and ultrastructure of sciatic nerves isolated from wood frogs (Rana sylvatica) endemic to the Northwest Territories, Canada, following freezing at −2.5 °C, −5.0 °C, or −7.5 °C. All frogs frozen at −2.5 °C, and most frogs (71%) frozen at −5.0 °C, recovered within 14 h after thawing began; however, frogs did not survive exposure to −7.5 °C. Sciatic nerves isolated from frogs frozen at −7.5 °C were refractory to electrical stimulation, whereas those obtained from frogs surviving exposure to −2.5 °C or −5.0 °C generally exhibited normal characteristics of compound action potentials. Frogs responded to freezing by mobilizing hepatic glycogen reserves to synthesize the cryoprotectant glucose, which increased 20-fold in the liver and 40-fold in the blood. Ultrastructural analyses of nerves harvested from frogs in each treatment group revealed that freezing at −2.5 °C or −5.0 °C had little or no effect on tissue and cellular organization, but that (lethal) exposure to −7.5 °C resulted in marked shrinkage of the axon, degeneration of mitochondria within the axoplasm, and extensive delamination of myelin sheaths of the surrounding Schwann cells. Accepted: 28 April 1999