Mechanism of microwave sterilization in the dry state

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Mechanism of microwave sterilization in the dry state.

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Effect of Temperature and Gas Velocity on the Dry-Heat Destruction Rate of Bacterial Spores

Spores of Bacillus subtilis were dried in vacuo for use in dry-heat thermal destruction tests. Survivor curve tests were conducted in a specifically designed dry-heat oven. This oven provided accurate temperature control and permitted air or nitrogen to be passed over the spores during the lethal treatment. Experiments were carried out at various flow rates of the two gases (air and nitrogen) and various temperatures, and the data were expressed as survivor curves from which the decimal reduction time (D value) was obtained. Linear regression analysis methods were used to compute the slope of the survivor curves. The results indicated that as the flow rate of gas is increased, the effect of temperature on the destruction rate of the spores is lessened, the z value becoming very large. It is believed that the higher flow rates of dry gas cause greater dehydration of the spores and that spore moisture loss is one of the major factors in determining the dry-heat thermal destruction rate of bacterial spores.     

Dry-heat destruction of lipopolysaccharide: mathematical approach to process evaluation.

A mathematical model was developed to estimate the number of logarithmic cycles (LDec) of lipopolysaccharide concentration destroyed by a dry-heat sterilization process. The LDec values calculated from the mathematical model agreed well with those obtained from the destruction of lipopolysaccharide by a dry-heat treatment. A discussion of how the mathematical model may be used to evaluate a dry-heat sterilization cycle is presented. This mathematical model and the dry-heat destruction curves indicated existence of a maximum LDec value at each temperature. The implications of this finding are discussed.     

Dry-heat destruction of lipopolysaccharide: mathematical approach to process evaluation.

A mathematical model was developed to estimate the number of logarithmic cycles (LDec) of lipopolysaccharide concentration destroyed by a dry-heat sterilization process. The LDec values calculated from the mathematical model agreed well with those obtained from the destruction of lipopolysaccharide by a dry-heat treatment. A discussion of how the mathematical model may be used to evaluate a dry-heat sterilization cycle is presented. This mathematical model and the dry-heat destruction curves indicated existence of a maximum LDec value at each temperature. The implications of this finding are discussed.     

Dry-heat resistance of selected psychrophiles.

The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.     

Effect of temperature on emergence, survival and infectivity of cercariae of the marine trematode Renicola roscovita (Digenea: Renicolidae)

Marine bivalves harbour a diversity of trematode parasites affecting population and community dynamics of their hosts. Although ecologically and economically important, factors influencing transmission between first (snail) and second (bivalve) intermediate hosts have rarely been studied in marine systems. In laboratory experiments, the effect of temperature (10, 15, 20, 25 degrees C) was investigated on (1) emergence from snails, (2) survival outside hosts and (3) infectivity in second intermediate hosts of cercariae of the trematode Renicola roscovita (Digenea: Renicolidae), a major parasite in North Sea bivalves. Emergence of cercariae peaked at 20 degrees C (2609 +/- 478 cercariae snail(-1) 120 h(-1)) and was considerably lower at 10 degrees C (80 +/- 79), 15 degrees C (747 +/- 384) and 25 degrees C (1141 +/- 334). Survival time decreased with increasing temperature, resulting in 50% mortality of the cercariae after 32.8 +/- 0.6 h (10 degrees C), 26.8 +/- 0.8 h (15 degrees C), 20.2 +/- 0.5 h (20 degrees C) and 16.6 +/- 0.3 h (25 degrees C ). Infectivity of R. roscovita cercariae in cockles Cerastoderma edule increased with increasing temperature and was highest at 25 degrees C (42.6 +/- 3.9%). However, mesocosm experiments with infected snails and cockle hosts in small aquaria, integrating cercarial emergence, survival and infectivity, showed highest infection of cockles at 20 degrees C (415 +/- 115 metacercariae host(-1)), indicating 20 degrees C to be the optimum temperature for transmission of this species. A field experiment showed metacercariae of R. roscovita to appear in C. edule with rising water temperature in April; highest infection rates were in August, when the water temperature reached 20 degrees C. Since another trematode species (Himasthla elongata; Digenea: Echinostomatidae) occurring at the experimental site showed a similar temporal pattern, trematode transmission to second intermediate bivalve hosts may peak during especially warm (> or = 20 degrees C) summers in the variable climate regime of the North Sea.     

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.     

Ultrafast microwave energy fixation for electron microscopy

We demonstrate that microwave (MW) energy can be used in conjunction with chemical cross-linking agents to fix tissue blocks rapidly for electron microscopy in as brief a time as 26 msec. The optimal ultrafast MW fixation methodology involved immersing tissue blocks up to 2 mm3 in dilute aldehyde fixative and immediately irradiating the specimens in a 7.3 kW MW oven for 26-90 msec, reaching a fixation temperature range of 32-42 degrees C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hr at 25 degrees C. Potential applications for this new fixation technology include investigation of rapid intracellular processes (e.g., vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (e.g., antigens and enzymes).     

Optimal collection of blood samples for the measurement of tumor necrosis factor alpha

We have examined how delayed separation of plasma from cells affects the recovery of recombinant human tumor necrosis factor alpha (rhTNF alpha) from whole blood. Storage of heparinized whole blood samples at room temperature for 1 hr results in a significant (p = 0.036) fall in recovery of plasma TNF alpha from 788 +/- 119 pg/mL to 472 +/- 77 pg/mL, measured by specific enzyme-linked immunosorbent assay (ELISA). Storage of whole blood samples at 4 degrees C for 1 hr reduces but does not prevent the fall in recovery of plasma TNF alpha: 725 +/- 82 pg/mL at time 0, 472 +/- 81 pg/mL after 1 hr, p = 0.038. Recovery of bioactive TNF alpha (cytotoxocity for L929 cells) after 1 hr at room temperature is also significantly reduced from 576 +/- 139 pg/mL to 450 +/- 154 pg/mL, p = 0.036. Studies with 125I-rhTNF alpha confirmed the fall in plasma activity and revealed a rapid commensurate increase in 125I-rhTNF alpha activity in the cell fractions. We recommend that clinical samples for the measurement of cytokines should be kept at 4 degrees C and separated rapidly (within half an hour) before storing the plasma at -70 degrees C.     

Influence of body temperature on the development of fatigue during prolonged exercise in the heat.

We investigated whether fatigue during prolonged exercise in uncompensable hot environments occurred at the same critical level of hyperthermia when the initial value and the rate of increase in body temperature are altered. To examine the effect of initial body temperature [esophageal temperature (Tes) = 35.9 +/- 0.2, 37.4 +/- 0. 1, or 38.2 +/- 0.1 (SE) degrees C induced by 30 min of water immersion], seven cyclists (maximal O2 uptake = 5.1 +/- 0.1 l/min) performed three randomly assigned bouts of cycle ergometer exercise (60% maximal O2 uptake) in the heat (40 degrees C) until volitional exhaustion. To determine the influence of rate of heat storage (0.10 vs. 0.05 degrees C/min induced by a water-perfused jacket), four cyclists performed two additional exercise bouts, starting with Tes of 37.0 degrees C. Despite different initial temperatures, all subjects fatigued at an identical level of hyperthermia (Tes = 40. 1-40.2 degrees C, muscle temperature = 40.7-40.9 degrees C, skin temperature = 37.0-37.2 degrees C) and cardiovascular strain (heart rate = 196-198 beats/min, cardiac output = 19.9-20.8 l/min). Time to exhaustion was inversely related to the initial body temperature: 63 +/- 3, 46 +/- 3, and 28 +/- 2 min with initial Tes of approximately 36, 37, and 38 degrees C, respectively (all P < 0.05). Similarly, with different rates of heat storage, all subjects reached exhaustion at similar Tes and muscle temperature (40.1-40.3 and 40. 7-40.9 degrees C, respectively), but with significantly different skin temperature (38.4 +/- 0.4 vs. 35.6 +/- 0.2 degrees C during high vs. low rate of heat storage, respectively, P < 0.05). Time to exhaustion was significantly shorter at the high than at the lower rate of heat storage (31 +/- 4 vs. 56 +/- 11 min, respectively, P < 0.05). Increases in heart rate and reductions in stroke volume paralleled the rise in core temperature (36-40 degrees C), with skin blood flow plateauing at Tes of approximately 38 degrees C. These results demonstrate that high internal body temperature per se causes fatigue in trained subjects during prolonged exercise in uncompensable hot environments. Furthermore, time to exhaustion in hot environments is inversely related to the initial temperature and directly related to the rate of heat storage.     

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Susceptibility of Tribolium castaneum life stages exposed to elevated temperatures during heat treatments of a pilot flour mill: influence of sanitation, temperatures attained among mills floors, and costs

The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), was investigated in a pilot flour mill subjected to three, 24-h heat treatments by using forced-air gas heaters fueled by propane. Two sanitation levels, dusting of wheat flour and 2-cm-deep flour, were created in 25 plastic bioassay boxes, each holding eggs, young larvae, old larvae, pupae, and adults of T. castaneum plus two temperature sensors. Data loggers (48) were placed on the five mill floors to record air temperatures. The time required to reach 50 degrees C, time above 50 degrees C, and the maximum temperature among mill floors and in bioassay boxes were measured. The maximum temperature in bioassay boxes and in the mill was lower on the first floor than on other floors. This trend was apparent in time required to reach 50 degrees C and time above 50 degrees C, especially in compartments with 2-cm-deep flour. The mean +/- SE mortality of T. castaneum life stages on the first floor was 55.5 +/- 12.9-98.6 +/- 0.8%; it was 93.2 +/- 6.7-100 +/- 0.0% on other floors. Adults were the least susceptible stage. Mortality of T. castaneum stages in compartments with 2-cm-deep flour was generally lower than those with flour dust. Costs for the three heat treatments ranged from US$27,438 to $28,838. An effective heat treatment can be conducted within 24 h, provided temperatures on mill floors reach 50 degrees C in 8-12 h and are held above 50 degrees C for at least 10-14 h, with maximum temperatures held between 50 and 60 degrees C.     

Muscle and femoral vein temperatures during short-term maximal exercise in heart failure

Core temperature decreases throughout short-term maximal exercise in heart-failure patients. To investigate possible causes for this unusual response to exercise, we studied core (pulmonary arterial blood), femoral vein, muscle, and skin temperatures in eight patients with severe heart failure who performed maximal upright incremental bicycle exercise to 50 W. A normal group (n = 4) was exercised for comparison. In the heart-failure patients, core temperature was 36.95 +/- 0.37 degrees C at rest, significantly (P less than 0.05) decreased at 25 W of exercise to 36.59 +/- 0.40 degrees C, and at 50 W remained decreased to 36.57 +/- 0.40 degrees C. In comparison, we found that the resting core temperature in the normal subjects was 37.28 +/- 0.34 degrees C, was the same at 25 W (37.29 +/- 0.41 degrees C), and increased significantly (P less than 0.05) to 37.50 +/- 0.32 degrees C at 50 W of exercise. Femoral vein temperature in heart-failure patients (n = 6) was below core temperature throughout exercise to 25 and 50 W (36.22 +/- 0.62 and 36.34 +/- 0.65 degrees C, respectively). Muscle temperature (n = 7) was significantly (P less than 0.05) lower in the heart-failure patients (34.8 +/- 1.1 degrees C) at rest compared with the normal subjects (36.2 +/- 1.0 degrees C). During exercise, muscle temperature increased above core temperature in only four of the heart-failure patients and was significantly (P less than 0.05) lower (36.5 +/- 1.3 degrees C) compared with the normal subjects (38.0 +/- 0.2 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of long-term exposure to an air-conditioned environment on the diurnal cortisol rhythm

In the present study, the influence of the long-term use of air-conditioning in summer on the cortisol rhythm was examined by measuring the rhythm in subjects who had been exposed to air-conditioning for a short [S] or long [L] time. Investigations were conducted twice in July and September. Atmospheric temperature and relative humidity near the subjects were measured for three days in each season. Saliva samples for cortisol analysis were collected every 2 hours during the daytime beginning at 8:00 h with subsequent sampling times at 10:00, 12:00, 14:00, 16:00, 18:00, 20:00 and 22:00 h. A questionnaire on sleep and duration of air-conditioning use was also undertaken. Ambient mean temperature was higher in the S group (mean+/-SD; 30.8+/-1.2 degrees C in July, 28.0+/-0.8 degrees C in September) than in the L group (28.0+/-1.2 degrees C in July, 27.3+/-1.0 degrees C in September) (p<0.01), while mean relative humidity did not differ. There were no differences in bedtime, waking time and sleeping hours either between groups or months. Diurnal patterns of salivary cortisol rhythm in July and September were similar in the S group, but the L group had a delayed rise of morning cortisol secretion in September compared with July. These results suggest that long-term exposure to an air-conditioned environment might adversely affect the human cortisol rhythm.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Effect of saline infusion on body temperature and endurance during heavy exercise

We tested the hypothesis that volume infusion during strenuous exercise, by expanding blood volume, would allow better skin blood flow and better temperature homeostasis and thereby improve endurance time. Nine males exercised to exhaustion at 84.0 +/- 3.14% (SE) of maximum O2 consumption on a cycle ergometer in a double-blind randomized protocol with either no infusion (control) or an infusion of 0.9% NaCl (mean vol 1,280.3 +/- 107.3 ml). Blood samples and expired gases (breath-by-breath), as well as core and skin temperatures, were analyzed. Plasma volume decreased less during exercise with the infusion at 15 min (-13.7 +/- 1.4% control vs. -5.3 +/- 1.7% infusion, P less than 0.05) and at exhaustion (-13.6 +/- 1.2% vs. -1.3 +/- 2.2%, P less than 0.01). The improved fluid homeostasis was associated with a lower core temperature during exercise (39.0 +/- 0.2 degrees C for control and 38.5 +/- 0.2 degrees C for infusion at exhaustion, P less than 0.01) and lower heart rate (194.1 +/- 3.9 beats/min for control and 186.0 +/- 5.1 beats/min for infusion at exhaustion, P less than 0.05). However, endurance time did not differ between control and infusion (21.96 +/- 3.56 and 20.82 +/- 2.63 min, respectively), and neither did [H+], peak O2 uptake, and CO2 production, end-tidal partial pressure of CO2, blood lactate, or blood pressure. In conclusion, saline infusion increases heat dissipation and lowers core temperature during strenuous exercise but does not influence endurance time.     

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.     

Assay of frozen boar semen with Sephadex filtration

The suitability of filtration of frozen boar semen through Sephadex G-15-120 as a viability assay was investigated. Semen thawed on a hot plate at 38 degrees C was counted with a Coulter Counter before and after filtering through Sephadex columns with 0.1 M sodium citrate as flushing medium. More spermatozoa passed through the column when the temperature of the flushing medium was elevated from room temperature to 37 degrees C and added with 5 mM caffeine (12.3 vs. 22.8% p<0.01). The use of caffeine and 37 degrees C flushings produced a filtrate containing spermatozoa with 89+/-4% motility and 97.6+/-1.6% normal acrosome ridges with the use of frozen semen from 5 different boars. The repeatability was +/-7.3%. The unfiltered samples were judged to contain 45 +/- 8% motile spermatozoa and 66.6 +/- 7.1% spermatozoa with normal acrosome ridges. Filtering of frozen boar semen through Sephadex is proposed as a rapid, objective assay combining the benefits of differential counting of normal acrosome ridges and motility determination.