Effects of cycling temperatures on fiber metabolism in cultured cotton ovules

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [¹⁴C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.     

Ecology, Inhibitory Activity, and Morphogenesis of a Marine Antagonistic Bacterium Belonging to the Roseobacter Clade

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30°C but died rapidly at 37°C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37°C in less than 2 days and at 25°C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.     

Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28°C up to 40°C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45°C followed by incubation at 28°C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45°C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40°C or a brief (10-minute) pulse treatment at 45°C followed by a 28°C incubation provide protection (thermal tolerance) to a subsequent exposure at 45°C. Maximum thermoprotection is achieved by a 2-hour 40°C pretreatment or after 2 hours at 28°C with a prior 10-minute 45°C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45°C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.

During 40°C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28°C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40°C chase period. If the seedlings are subjected to a second HS after a 28°C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40°C to 45°C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28°C but become organelle-associated during a subsequent HS at 40°C.

     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.     

Role of Growth Temperature in Freeze-Thaw Tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37°C but are strongly repressed below 30°C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37°C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at −20°C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37°C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25°C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37°C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.Listeria monocytogenes remains a leading cause of deaths due to food-borne illness in the United States and other industrialized nations. Neonates, pregnant women, and immunocompromised people are at high risk for infection (15, 28, 37). Outbreaks of listeriosis tend to involve a relatively small number of closely related strains (“epidemic clones”), primarily of serotype 4b. Several major outbreaks have been attributed to epidemic clone I (ECI) and epidemic clone II (ECII), both of serotype 4b (5, 21).Unlike most other human food-borne pathogens, L. monocytogenes grows over a wide temperature range (1 to 45°C), with optimal growth at approximately 37°C (33). It has been known for some time that expression of several key virulence genes, including hly, encoding the hemolysin listeriolysin O and actA, encoding a protein that mediates actin polymerization required for intracellular pathogenesis, is optimal at 37°C but severely repressed at temperatures below 30°C (22). In contrast, flagellin, motility, and chemotaxis genes are repressed at 37°C but optimally expressed at temperatures below 25°C (9, 10).For an organism such as L. monocytogenes, which is commonly found in the environment but can also colonize and infect warm-blooded animals, temperature is likely to serve as a major signal differentiating environmental from vertebrate host-associated habitats (19). However, with the exception of the thermoregulated phenotypes described above (virulence factor production, motility, and chemotaxis), the impact of growth at different temperatures on specific responses and adaptations of the pathogen remains poorly understood.L. monocytogenes may be exposed to freezing as well as thawing in the course of its existence in natural environments (e.g., soils and water in temperate or cold regions), as well as during the storage and preservation of foods. The organism has been repeatedly isolated from frozen foods (e.g., ice cream) (7). After freezing (−18°C) in laboratory media or in foods and a single thawing cycle, survival depended on strain, freezing medium, and the presence of glycerol as cryoprotectant (13, 14). The freezing and thawing of bacteria grown at 30°C, in combination with essential oil, has been explored as one means to reduce the pathogen in foods (8). The possible role of the general stress sigma factor (sigma B) in survival of bacteria grown at 30°C and exposed to repeated freezing and thawing was also investigated (43). However, there is a surprising dearth of information on the possible impact of growth temperature on tolerance of L. monocytogenes to repeated freezing and thawing (cryotolerance).Studies with another gram-positive psychrotrophic bacterium (Exiguobacterium sibiricum and other Exiguobacterium spp.) revealed that growth in the cold (4°C), or growth on solid media regardless of temperature, resulted in increased tolerance of the bacteria against repeated freezing and thawing (40). It is not known whether low temperature or surface-associated growth may exert similar impacts on the cryotolerance of L. monocytogenes. The objective of the present study was to investigate the impact of growth temperature (4, 25, and 37°C) and of planktonic versus agar growth of L. monocytogenes on protection of the bacteria against repeated freezing and thawing.     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

Effect of Low Temperatures on Growth, Structure, and Metabolism of Campylobacter coli SP10

The effect of low temperatures on the survival, structure, and metabolism of Campylobacter coli SP10, a virulent strain, was investigated. C. coli became nonculturable rapidly at 20 and 10°C and slightly later at 4°C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37°C but also was noticeable at 20 and 10°C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10°C) seemed to be a degenerative form. The flagella were shorter at 20 and 10°C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C_19:0 cyc at 20°C with a concomitant decrease in C_18:1 trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20°C and was only occasionally detected at 10 and 4°C. At the same time, fumarate concentrations, which are normally not detectable at 37°C, were highest at 20°C and reduced at 10 and 4°C. Inactivation of fumarate reductase was considered to be a possible explanation.     

Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.     

Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.     

Effect of Chilling Temperatures upon Cell Cultures of Tomato

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv `VF36,' and cv `VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No growth was observed at 8°C, indicating an abrupt limit to growth between 8 and 12°C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8°C for up to 2 weeks. When cultures kept at 8°C for up to 30 days were transferred to 28°C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0°C, and for cells of `VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9°C but additional growth at 8°C did not occur, nor could growth be maintained by subculture at 8 or 9°C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10°C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.     

Effects of Temperature on Resistance in Phaseolus vulgaris Genotypes and on Development of Meloidogyne Species

Phaseolus vulgaris lines with heat-stable resistance to Meloidogyne spp. may be needed to manage root-knot nematodes in tropical regions. Resistance expression before and during the process of nematode penetration and development in resistant genotypes were studied at pre- and postinoculation temperatures of 24 °C and 24 °C, 24 °C and 28 °C, 28 °C and 24 °C, and 28 °C and 28 °C. Resistance was effective at all temperature regimes examined, with fewer nematodes in roots of a resistant line compared with a susceptible line. Preinoculation temperature did not modify resistance expression to later infections by root-knot nematodes. However, postinoculation temperatures affected development of Meloidogyne spp. in both the resistant and susceptible bean lines tested. The more rapid development of nematodes to adults at the higher postinoculation temperature of 28 °C in both bean lines suggests direct temperature effects on nematode development instead of on resistance expression of either of two gene systems. Also, resistance was stable at 30 °C and 32 °C.     

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

Combinations of Intervention Treatments Resulting in 5-Log10-Unit Reductions in Numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 Organisms in Apple Cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log₁₀ units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log₁₀ units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log₁₀-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log₁₀-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log₁₀-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

TIME-TEMPERATURE RELATIONS IN THE INCUBATION OF THE WHITEFISH,COREGONUS CLUPEAFORMIS (MITCHILL)

1. Whitefish eggs incubated in aerated lake water at controlled tempera tures of 0°, 0.5°, 2°, 4°, 6°, 8°, 10°, and 12°C., failed to hatch at either 0° or 12°C. 0.6 per cent hatched alive at 10°C., 72.67 per cent hatched alive at 0.5°C., and an intermediate proportion hatched at intermediate temperatures. 2. The percentage of abnormal embryos which developed to the hatching stage varied directly with temperature between 4° and 12°, all embryos being abnormal at 12°C.; but none were abnormal at either 0.5°, or 2°C. Normal development predominated from 0.5 to 6°C. The highest proportion of embryos to hatch alive was 72.67 per cent at 0.5°C., which is, hence, the optimum temperature. 3. Total incubation time ranged from 29.6 days at 10°C. to 141 days at 0.5°C. 4. The time (T) required to attain any given stage of development is expressed in equations See PDF for Equation where temperature, t, is a negative exponent of the constant, A, whose value differs above or below 6°C., a critical temperature. Values of A above 6° fluctuate about 1.13; those of A below 6° fluctuate about 1.19 as a mean. 5. Applying Arrhenius'' equation µ values for the total incubation period are 27,500 below 6° and 27,100 above it. 6. The relative magnitude of A values of the exponential equation and µ values of Arrhenius'' equation show corresponding changes from one developmental period to another. 7. When plotted, thermal increments show cyclic variations, with maxima during periods of cleavage and of organogenesis. These may indicate the interaction of two separate sets of embryonic processes, which give a maximal response to temperature differences during these two separate periods. 8. Above 6°, µ values during the hatching process are distinct from those of developmental stages and are regarded as being due to the action of hatching enzymes.