Heat inactivation of Mycobacterium avium-Mycobacterium intracellulare complex organisms in aqueous suspension.

Isolants from swine and from humans representing serotypes 1, 2, 4, 8, and 10 of the Mycobacterium avium-Mycobacterium intracellulare complex were compared for heat tolerance in aqueous suspension. The most heat-resistant isolant found was a serovar 10 isolated from a human. This isolant was examined further to determine the rate of kill at various temperatures and pH's, the effect of meat protein and fat, and the effect of nitrite. Kill rates were not significant at 60 degrees C or below. Decimal reduction values were 4 min or less at 65 degrees C and 1.5 min or less at 70 degrees C. Kill rates were slightly higher at pH values of 6.5 and 7.0 than at 5.5 or 6.0. the water-soluble fraction of wiener emulsion did not alter kill rates, but the saline-soluble fraction protected the organism somewhat. Fat did not affect the survival of the organisms except to eliminate the protective effect of saline extract when the suspension contained 50% fat. The addition of sodium nitrite to the suspension did not alter the heat sensitivity of the organisms.     

Causes of variation in botulinal inhibition in perishable canned cured meat.

Final internal processing temperatures within the range of 63 to 74 degrees C did not alter the degree of botulinal inhibition in inoculated perishable canned comminuted cured pork abused at 27 degrees C. Adding hemoglobin to the formulation reduced residual nitrite after processing and decreased botulinal inhibition. Different meats yielded different rates of botulinal outgrowth when substituted for fresh pork ham. Pork or beef heart meat showed no inhibition of the Clostridium botulinum inoculum even with a 156-microgram/g amount of sodium nitrite added to the product. This effect appears to be one of stimulating outgrowth, since residual nitrite depletion was not measurably altered.     

Causes of variation in botulinal inhibition in perishable canned cured meat.

Final internal processing temperatures within the range of 63 to 74 degrees C did not alter the degree of botulinal inhibition in inoculated perishable canned comminuted cured pork abused at 27 degrees C. Adding hemoglobin to the formulation reduced residual nitrite after processing and decreased botulinal inhibition. Different meats yielded different rates of botulinal outgrowth when substituted for fresh pork ham. Pork or beef heart meat showed no inhibition of the Clostridium botulinum inoculum even with a 156-microgram/g amount of sodium nitrite added to the product. This effect appears to be one of stimulating outgrowth, since residual nitrite depletion was not measurably altered.     

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Pasteurization of salted whole egg inoculated with Arizona or Salmonella.

Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.     

Pasteurization of salted whole egg inoculated with Arizona or Salmonella.

Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.     

Generic vapor heat treatments to control Maconellicoccus hirsutus (Homoptera: Pseudococcidae)

Vapor heat treatments were developed against life stages of the mealybug Maconellicoccus hirsutus (Green) (Homoptera: Pseudococcidae). Treatments tested were 47 degrees C for 5-50 min in 5-min increments and 49 degrees C for 3, 5, 8, 10, and 12 min. All tests were conducted with mixed age M. hirsutus on Chinese pea, Pisum sativum L. Treatment at 47 degrees C required 45 min to kill all M. hirsutus, whereas treatment at 49 degrees C required 10 min. The adult female and nymphal stages were the most heat tolerant at 47 degrees C, but the egg stage was the most heat tolerant at 49 degrees C. Use of the vapor heat treatments on other commodities will require achieving or exceeding the proper temperature and duration at all locations on the host where M. hirsutus may reside.     

A laboratory study of survival of selected microorganisms after heat treatment of biowaste used in biogas plants

The aim of the study was to assess the effect of pasteurisation, as set by the European regulation EC 1774/2002, on selected pathogens and indicator organisms. Unpasteurised substrate (biowaste), including animal by-products from a full-scale biogas plant was heat treated under laboratory conditions at 70 degrees C and 55 degrees C for 30 min and 60 min. Heat treatment at 55 degrees C for 60 min was not sufficient to achieve a hygienically acceptable product. Heat treatment at 70 degrees C for 30 min and 60 min was effective in reducing pathogenic bacteria, Ascaris suum eggs, Swine vesicular disease virus and indicator organisms. However, this level of pasteurisation will still not reduce the quantity of Clostridia spores, or completely inactivate heat-resistant viruses such as Porcine parvovirus or Salmonella phage 28B. The results still give cause for some concern regarding the use of digested residue from biogasplants in agriculture.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat.

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

Effects of Ingredients Used in Condensed and Frozen Dairy Products on Thermal Resistance of Potentially Pathogenic Staphylococci

A cell suspension of Staphylococcus aureus (196E) was injected into raw skim milk which contained different concentrations of sugar, serum solids, fat, stabilizers, and emulsifiers. The ingredient samples were exposed for the desired length of time in a constant-temperature water bath (60 C). Standard plate counts were made, and the number of surviving organisms was determined. Regression coefficients for each ingredient concentration were calculated and plotted against the per cent ingredient concentration to give an indication of protective action. Analyses of variance were conducted on bacterial counts to test the protective action of each ingredient. A comparison of the number of survivors in different sugar concentrations showed that with up to 14% sugar all the organisms were killed within 30 min. In sugar concentrations above 14%, the number of survivors increased regularly with each increase in sugar concentration up to 57%, which was the maximum used. In concentrations of serum solids above 9%, some organisms survived 35 min of heat treatment. Butter fat, stabilizer, and emulsifier did not offer any protective action in the concentrations observed.     

Factors influencing the altered thermogenic response of rat brown adipose tissue in streptozotocin-diabetes.

1. Adipocytes were isolated from the interscapular brown fat of male rats maintained at 21 degrees C. These animals were controls, streptozotocin-diabetics or 2-day insulin-treated diabetics. 2. With adipocytes from diabetic animals, maximum rates of noradrenaline-stimulated O2 uptake were decreased by 58%, and the Bmax. of [3H]GDP binding to mitochondria was decreased by 55%. Insulin administration reversed both of these changes. 3. Streptozotocin-diabetes increased basal lipolysis in adipocytes incubated with adenosine deaminase (1 unit/ml), decreased the EC50 (concn. giving 50% of maximum effect) for noradrenaline, but did not change the maximum rate of noradrenaline-stimulated lipolysis. Except for some small differences at very low concentrations (10-100 pM), diabetes or insulin treatment did not alter the sensitivity of noradrenaline-stimulated lipolysis or O2 uptake to the inhibitory effect of N6-phenylisopropyladenosine. It is therefore concluded that the lesion(s) in thermogenesis in diabetes are not attributable to any changes in lipolysis. 4. Blood flow through interscapular brown fat, measured by accumulation of [14C]DDT [14C-labelled 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] was increased by 2.3-fold 70 min after a single administration of insulin to diabetic rats. This treatment decreased blood flow through epididymal white fat by 58%. 5. Propranolol treatment of diabetic rats muted the ability of insulin treatment to increase the maximum rate of noradrenaline-stimulated O2 uptake, suggesting that this action of insulin may be a secondary one rather than a direct effect of the hormone on the adipocytes.     

Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Influence of heat transmission mode on heating rates and on the selection of patches for heating in a mediterranean lizard

Heliothermy (heat gain by radiation) has been given a prominent role in basking lizards. However, thigmothermy (heat gain by conduction) could be relevant for heating in small lizards. To ascertain the importance of the different heat transmission modes to the thermoregulatory processes, we conducted an experimental study where we analyzed the role of heat transmission modes on heating rates and on the selection of sites for heating in the Mediterranean lizard Acanthodactylus erythrurus (Lacertidae). The study was conducted under laboratory conditions, where two situations of different operative temperatures (38 degrees and 50 degrees C) were simulated in a terrarium. In a first experiment, individuals were allowed to heat up during 2 min at both temperatures and under both heat transmission modes. In a second experiment, individuals were allowed to select between patches differing in the main transmission mode, at both temperatures, to heat up. Experiences were conducted with live, nontethered lizards with a starting body temperature of 27 degrees C. Temperature had a significant effect on the heating rate, with heat gain per unit of time being faster at the higher operative temperature (50 degrees C). The effect of the mode of heat transmission on the heating rate was also significant: at 50 degrees C, heating rate was greater when the main heat transmission mode was conduction from the substrate (thigmothermy) than when heating was mainly due to heat gain by radiation (heliothermy); at 38 degrees C, heating rates did not significantly differ between transmission modes. At 38 degrees C, selection of the site for heating was not significantly different from that expected by chance. However, at 50 degrees C, the heating site offering the slowest heating rate (heliothermic patch) was selected. These results show that heating rates vary not only with environmental temperature but also with different predominant heat transmission modes. Lizards are able to identify and exploit this heterogeneity, selecting the source of heat gain (radiation) that minimizes the risk of overheating when temperature is high.     

Growth factors and hyperthermia. I. The relationship between hyperthermic cell killing and the mitogenic response to serum and growth factors

Chinese hamster ovary HA-1 cells were tested for their ability to respond to mitogenic stimulation after hyperthermia at 45 degrees C. Cells were arrested by 24 h incubation in serum-free Eagle's MEM. Heating of arrested cells in serum-free medium did not alter heat sensitivity compared to exponentially growing cells heated in serum-containing medium. After hyperthermia cells exhibited a delay in the ability to undergo mitogenesis. Recovery of the capacity for mitogenesis occurred during the 24 h following heating and was able to take place in the absence of serum. After recovery in serum-free medium, cells were simultaneously assayed for survival and mitogenesis as measured by [3H]Thy uptake. With increasing heating time, surviving fraction and mitogenesis decreased. The reduction in survival was similar to the reduction in [3H]Thy incorporation. The relationship between mitogenesis and cell death was studied in more detail with flow cytometry. At a relatively mild heat dose of 30 min at 45 degrees C (survival = 30%), a small population of cells (9%) was found to be clonogenically dead yet capable of being stimulated to progress from G1 to G2-M. At a more severe heat dose of 40 min at 45 degrees C (survival = 3%), stimulation of dead cells could not be detected. Therefore, hyperthermia impairs mitogenic ability, but at low heat doses, a subpopulation of killed cells can still be stimulated to progress through the cell cycle.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.