Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Thermal inactivation and injury of Bacillus stearothermophilus spores

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.     

Efflux of chloride from lactating rat mammary tissue slices

1. The efflux of chloride (using 36Cl) from lactating rat mammary tissue slices has been investigated. 2. Chloride efflux was found to be temperature dependent; lowering the temperature of the incubation medium reduced the fractional efflux. 3. The stilbene derivatives DIDS was without effect on the fractional release of Cl when studied at 20 degrees C. However, DIDS was found to attenuate the increase in efflux found upon transferring the tissue from a medium maintained at 4 degrees C to one at 20 degrees C. 4. The loop-diuretic furosemide, also reduced the temperature-sensitive portion of Cl efflux. 5. Chloride efflux was transiently increased when tissue slices were transferred from a medium containing gluconate as the principal anion to one containing Cl. 6. The results appear to confirm that mammary Cl transport is mediated via anion exchange and via (Na + K + Cl) cotransport.     

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.     

Increase of enzyme activities in Neurospora crassa during incubation at low temperatures.

The effect of lowering the incubation temperature of sucrose-grown cultures of Neurospora crassa on the level of various enzyme activities was investigated. Of twelve inducible/derepressible activities studied, three, in addition to glycerol kinase, were found to increase during 48 h of incubation at 4-6 degrees C: trehalase (increase in specific activity of 3-10-fold), beta-glucosidase (6-12-fold) and beta-N-acetylglucosaminidase (4 to 6-fold). The maximum increases occurred at 6 degrees C and no increases took place in mycelia incubated at 0 degrees C. The kinetics of the changes in activity were markedly different from those observed previously with glycerol kinase. The increases were inhibited by cycloheximide. Trehalase, beta-glucosidase and beta-N-acetylglucosaminidase activities were not rapidly lost when cultures incubated at 6 degrees C were returned to 26 degrees C.     

Cytochemical discrimination between catalases and peroxidases using diaminobenzidine.

The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.     

Increased mesquite gum formation in nodal explants cultures after treatment with a microbial biomass preparation.

Prosopis laevigata nodal explants cultures were established in Murashige and Skoog medium. Simultaneously these cultures were subjected to stress with biotic elicitors and an environmental factor (temperature increase to promote heat stress) in order to promote and increase exuded mesquite gum production. The biotic elicitors were: Aspergillus nidulans and Pseudomonas pseudoalcaligenes both used in concentrations of 10, 20 and 30 mg, whereas the environmental condition was different incubation temperatures (25, 35 and 40 degrees C). The greatest gum production (approximately 13 mg of pooled gum from 100 explants after 14 days incubation) took place when the culture medium was added 10, 20 and 30 mg of autoclaved fungal mycelium of A. nidulans or 30 mg of autoclaved bacterial biomass of P. pseudoalcaligenes in combination with an incubation temperature of 35 degrees C. These treatments were non-significantly different among themselves (P < 0.05), but were significantly different to the rest of the treatments (P > 0.05).     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3 degrees C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4 degree C for the three strains. Lag times of 1-3 d and 3 to greater than 34 d were observed with incubation at 5 and 0 degrees C respectively. Corresponding generation times ranged from 13-24 h at 5 degrees C and 62-131 h at 0 degree C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4 degrees C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30 degrees C.     

Effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Lepidoptera: Noctuidae)

The ability to store different insect stadia for prolonged periods provides considerable flexibility and ability to conduct experiments properly. Therefore, studies were undertaken to determine the effect of storage temperature and duration on viability of eggs of Helicoverpa armigera (Hübner). The percentage egg hatch and incubation period were significantly (P=0.01) influenced by egg age, storage temperature, and storage duration. Egg hatch ranged from 0.0 to 96.8% across temperatures and storage durations. None of the eggs hatched when stored at -20 and 0 degrees C. The regression model with the optimum Mallow Cp statistic for any of the identified linear and quadratic terms did not improve the precision of prediction in egg hatch beyond 67.0%. Forecasting of incubation period based on egg age, storage duration, and durationxtemperature was quite effective (R2=84.2%). Day degrees required for egg hatching decreased with an increase in temperature from 10 to 27 degrees C, and egg age from 0 to 3 days. The day degree requirements were highest for 0-day-old eggs at 10 degrees C, and lowest at 27 degrees C. Although the incubation period was higher, the hatchability was lower for 0- and 1-day-old eggs stored at constant 10 degrees C, these eggs can be stored for 10 days at 10 degrees C, with a hatchability of >75.0%. It was safer to store the H. armigera eggs for 10 days at 10 degrees C, which will hatch within 1.6 to 2.0 days after restoration at 27 degrees C with a hatchability of >75.0%. This information will be useful in planning and execution of experiments involving H. armigera on various aspects of research in entomology.     

Effect of ambient temperature on serum prolactin and luteinizing hormone levels during the reproductive life cycle of the female turkey (Meleagris gallopavo)

An ambient temperature of 30 degrees C compared to 18 degrees C accelerated the increase in serum prolactin (Prl) level induced by photostimulation of female turkeys. The contribution of reproductive stage and nesting behavior to this serum Prl elevation was assessed by housing adult female turkeys in individual wire cages while allowing other females free access to nests on the floor. Birds of both groups were exposed to 10 degrees C, 24 degrees C or 30 degrees C beginning 4 wk prior to photostimulation and continuing throughout the reproductive phase. Lapsed time between the onset of photostimulation and onset of sexual maturity, and between the onset of sexual maturity and onset of incubation behavior was shorter in birds housed at 30 degrees C with access to nests than in corresponding birds housed at 24 degrees C and 10 degrees C. The increases in serum Prl associated with sexual maturity or incubation behavior occurred at a greater rate in the birds maintained at 30 degrees C. Cage-reared birds had the same lapsed time between onset of photostimulation and onset of sexual maturity and the same sustained low Prl level regardless of ambient temperature exposure. All groups exhibited similar luteinizing hormone profiles. These findings indicate that the accelerated increase in Prl under elevated temperature in floor-reared turkeys is related to accelerated development of reproductive function, and not the direct effect of ambient temperature on mechanisms controlling Prl.     

Effects of hypertonic and sodium-free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells

Incubation of cultured cells in hypertonic medium and sodium-free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV-G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV-G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5 degrees C. In addition, VSV-G accumulates in the post-ER pre-Golgi compartment when cells are incubated at 15 degrees C and in the trans-Golgi network (TGN) when cells are incubated at 18 degrees C. Upon transfer of cells to 32 degrees C in control medium, VSV-G exits each of these compartments and is transported to the cell surface. Incubation in sodium-free medium at 32 degrees C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre-Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps; the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro

The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39 degrees C in 5% CO2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37 degrees C or 39 degrees C) in 5% CO2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37 degrees C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes.     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia.

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of γ-linolenic acid by Mortierella isabellina grown on hexadecanol

AIMS: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol. METHODS AND RESULTS: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied. The production of gamma-linolenic acid reached 2.44 mg ml-1 (271 mg g-1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23 degrees C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5 degrees C. CONCLUSION: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.     

The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes.

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.     

Tissue culture of the deep-sea eel<Emphasis Type="Italic"> Simenchelys parasiticus</Emphasis> collected at 1,162 m

We successfully cultivated fin cells of the deep-sea eel Simenchelys parasiticus (collected at 1,162 m) in L-15 medium supplemented with fetal bovine serum (FBS) and additional NaCl. We found that the pectoral fin cells proliferated in L-15 medium enriched with 4 g/l of NaCl salt (pH 7.3) containing 10% FBS at 10 degrees C and 15 degrees C. No cells were attached to the plastic culture plates when Dulbecco's modified Eagle's medium (pH 7.8) or 0-2 g/l of NaCl was added to the medium or when incubation was carried out at 4 degrees C. The majority of the explant outgrowth cells were detached when temperature increased to higher than 15 degrees C. The rate of proliferation of the fin cells was extremely slow and was dependent on the FBS concentration. Cell growth was enhanced by approximately 2.2-fold, and doubling time decreased from 170 h to 77 h when the FBS concentration was increased from 10% to 20% (v/v). Our established deep-sea eel cells were passaged 16 times over a 1-year period under atmospheric pressure conditions.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.