Two-Phase Slug Flow Heat Exchanger for Microbial Thermal Inactivation Research

A continuous two-phase (air-liquid), slug flow, tubular heat exchanger was developed for microbial thermal inactivation research to give exposure times and temperatures in the range of high-temperature, short-time milk pasteurization as well as heat-treated sample volumes of at least 2 ml. The use of air to compartmentalize the liquid in the capillary tubing prevented the development of laminar flow, which enabled precise identification of the residence time of the fastest flowing particles in the heating, holding, and cooling sections of the instrument. Residence time distributions were quantitated by measuring the degree of spreading of radioactive tracers for water, whole milk, chocolate milk, cream, and ice-cream mix with holding temperatures from 50 to 72 C, holding times from 2 to 60 sec, and heating and cooling times of 1.7 sec each. For a holding time of 60 sec and a fastest particle velocity of 10.2 cm/sec, the velocity ratios of the fastest moving particle to the median particle were 1.05, 1.05, 1.10, and 1.13 for whole milk, chocolate milk, cream, and ice-cream mix, respectively. With shorter holding times, these velocity ratios were even closer to unity. These velocity ratios indicated that the instrument would be an effective tool for thermal inactivation research on microorganisms suspended in homogeneous fluids with a viscosity of 15 centipoises or less at the exposure temperature.     

Thermal Inactivation of Aerosolized Bacillus subtilis var. niger Spores

A hot-air sterilizer capable of exposing airborne microorganisms to elevated temperatures with an almost instantaneous heating time was developed and evaluated. With this apparatus, aerosolized Bacillus subtilis var. niger spores were killed in about 0.02 sec when exposed to temperatures above 260 C. This is about 500 times faster than killing times reported by others. Extrapolation and comparison of data on the time and temperature required to klll B. subtilis var. niger spores on surfaces show that approximately the same killing time is required as is necessary for spores in air, if corrections are made for the heating time of the surface.     

Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature

The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO(2) required long residence time and high temperatures, such as 60 degrees C. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO(2) for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO(2) not higher than 40 degrees C and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO(2) treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO(2) lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO(2) and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO(2) was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media.     

Procedure for Evaluating the Effects of 2,450- Megahertz Microwaves upon Streptococcus faecalis and Saccharomyces cerevisiae

Modifications of a commercial 2,450-megahertz microwave oven were made so that 6 ml of microbial suspension could be exposed to the microwave field for various periods of time. The microorganisms were contained in the central tube of a modified Liebig condenser positioned in the approximate geometric center of the oven cavity. Kerosene at -25 C was circulated through the jacket of the condenser during microwave exposure permitting microwaves to reach the microbial suspension. Flow rates of the kerosene were varied to permit the temperature of the suspension to range from 25 to 55 C during microwave exposure. Conductive heating experiments using similar temperatures were also conducted. A thermocouple-relay system was employed to measure the suspension temperature immediately after the magnetron shutoff. Continuous application of microwaves to suspensions of 10(8) to 10(9)Streptococcus faecalis or Saccharomyces cerevisiae per ml appeared to produce no lethal effects other than those produced by heat. Respiration rates of microwave-exposed Scerevisiae were directly related to decreases in viable count produced by increased microwave exposure times.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Development and validation of a model for radiofrequency ablation]

Since 1986, cardiac arrhythmias have been successfully treated by destroying the underlying arrhythmogenic substrate with radiofrequency energy (radiofrequency ablation). The aim of this study was to develop a model for radiofrequency ablation enabling evaluation of the temperature distribution within cardiac tissue and the influence of electrode tissue contact. The model describes a 7F electrode, 4 mm in length and positioned perpendicular to the tissue. Heat convection within the tissue and heat lost via the blood was taken into account. Simulation of constant tissue exposure to 4 W resulted in a temperature increase of 35 degrees C after 10 sec. The temperature increase in the depths was less steep, but constant, and exceeded the electrode temperature at depths of 1 mm after 40 sec, 2 mm after 100 sec, and 3 mm after 200 sec. Electrode tissue contact proved to have a great influence on tissue temperature. Poor contact resulted in a temperature rise of only 0.68 degree C with a maximum of 50 W, whereas with ideal contact, 4 W sufficed to achieve a chosen setpoint of 70 degrees C. The model was validated in an in vitro setup using ventricular tissue from the pig. A strong correlation was found between simulated heating efficiency during temperature-controlled ablations under different contact conditions, and the respective measured values in the in vitro setup with a correlation coefficient of 0.97.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

Dry-heat destruction of lipopolysaccharide: design and construction of dry-heat destruction apparatus.

A dry-heat oven with automatic, multiple-sample introduction and withdrawal has been constructed to achieve instantaneous heating and cooling of samples. The oven temperature fluctuation at set points of 170 to 250 degrees C was +/- 0.1 degrees C, with temperature variation between the replicate samples of +/- 0.2 degrees C. Correction required for a sample come-up time was minimal, i.e., less than 0.25 min of the dry-heat destruction time.     

Enhancement of hyperthermic killing cultured mammalian cells by treatment with anisotonic NaCl or medium solutions

The exposure of cultured Chinese hamster cells (CHO) to anisotonic medium increased the cellular sensitivity to heat treatment at 42.3°C. A greater potentiation of heat killing is observed when the anisotonic solution consists of pure NaCl in water compared to growth medium made anisoltonic by dilution or by addition of NaCl. Hypertonic treatment caused greater heat sensitization than hypotonic treatment. Thermal tolerance observed in the control cells after 4–6 hours of heating in medium was also observed for cells exposed to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic NaCl solutions during heating removed the shoulder from the heat survival curve, while the curves for cells heated in medium made anisotonic retained their shoulders. These studies suggest: (1) that either the plasma membrane is a primary target for heat inactivation of mammalian cells, or (2) that changes in intracellular ion concentrations enhance thermal damage occurring in critical intracellular structures.     

Thermal inactivation and injury of Moraxella-Acinetobacter cells in ground beef.

The thermal inactivation and injury (sensitivity to 0.8% NaCl) of a radiation-resistant culture of Moraxella-Acinetobacter mixed in minced beef were determined. Survival curves for Moraxella-Acinetobacter cells in beef had an initial shoulder preceding a logarithmic decline when the cells were heated at 65, 70, and 75 degrees C, but not at 80 degrees C. In all cases, the experimental points not included in the shoulder were linearized by means of a least-squares straight line, and the latter was used to determine D values. Shoulder values of 12.2, 4.1, and 0.6 min at temperatures of 65, 70, and 75 degrees C were added to the respective D values of 35.4, 6.6, and 1.4 min to determine the time required to destroy one log cycle. The Z value was 7.3 degrees C. Moraxella-Acinetobacter cells in meat were more rapidly injured than inactivated, on initial exposure to heat. The number of cells injured by this initial exposure increased as the temperature was increased. At 65 degrees C the percentage of injured cells increased more rapidly with exposure time than did the inactivated cells. As the temperature was increased, the rates of inactivation and injury became more and more similar.     

Thermal adjustments to nonexertional heat stress in mature and senescent Fischer 344 rats

The purpose of this study was to test the hypothesis that the rise in colonic temperature (Tc) during nonexertional heat stress is exaggerated in senescent (SEN, 24 mo, n = 12) vs. mature (MAT, 12 mo, n = 15) conscious unrestrained Fischer 344 rats. On 2 separate days (48 h apart) each SEN and MAT animal was exposed to an ambient temperature (Ta) of 42 degrees C (relative humidity 20%) until a Tc of 41 degrees C was attained and then cooled at a Ta of 26 degrees C until Tc returned to the initial control level. Control Tc was similar in the two groups for both trials. The rate of Tc change during heating was 63% greater (0.070 +/- 0.005 vs. 0.043 +/- 0.004 degrees C/min, P less than 0.05) and the time to 41 degrees C reduced by 36% (54 +/- 6 vs. 85 +/- 10 min, P less than 0.05) in MAT vs. SEN animals during the first exposure, although the cooling rate was slower in the MAT (0.048 +/- 0.004 degrees C/min) vs. SEN (0.062 +/- 0.006 degrees C/min) animals (P less than 0.05). The heating rate was unchanged in MAT animals between trials 1 and 2. However, SEN animals had a 95% increase in heating rate in trial 2 compared with trial 1 (P less than 0.05), and the corresponding time to 41 degrees C was decreased by 44% (P less than 0.05). As a result, rate of heating and time to 41 degrees C were similar in the two groups during trial 2. The cooling rate was similar between trials within each group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nanoscale Structural and Mechanical Analysis of Bacillus anthracis Spores Inactivated with Rapid Dry Heating

Effective killing of Bacillus anthracis spores is of paramount importance to antibioterrorism, food safety, environmental protection, and the medical device industry. Thus, a deeper understanding of the mechanisms of spore resistance and inactivation is highly desired for developing new strategies or improving the known methods for spore destruction. Previous studies have shown that spore inactivation mechanisms differ considerably depending upon the killing agents, such as heat (wet heat, dry heat), UV, ionizing radiation, and chemicals. It is believed that wet heat kills spores by inactivating critical enzymes, while dry heat kills spores by damaging their DNA. Many studies have focused on the biochemical aspects of spore inactivation by dry heat; few have investigated structural damages and changes in spore mechanical properties. In this study, we have inactivated Bacillus anthracis spores with rapid dry heating and performed nanoscale topographical and mechanical analysis of inactivated spores using atomic force microscopy (AFM). Our results revealed significant changes in spore morphology and nanomechanical properties after heat inactivation. In addition, we also found that these changes were different under different heating conditions that produced similar inactivation probabilities (high temperature for short exposure time versus low temperature for long exposure time). We attributed the differences to the differential thermal and mechanical stresses in the spore. The buildup of internal thermal and mechanical stresses may become prominent only in ultrafast, high-temperature heat inactivation when the experimental timescale is too short for heat-generated vapor to efficiently escape from the spore. Our results thus provide direct, visual evidences of the importance of thermal stresses and heat and mass transfer to spore inactivation by very rapid dry heating.     

High-throughput PCR in silicon based microchamber array

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 °C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).     

Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios

Aerobic composting is a common management practice to inactivate pathogens in manure; however, additional research on the role of compost composition in pathogen inactivation is needed. The objective of this study was therefore to determine the effect of the carbon:nitrogen (C:N) ratio and the presence of ammonium sulfate on inactivation of Salmonella spp. in cow manure-based mixtures composted in a bioreactor under controlled conditions. Compost preparations with an initial C:N ratio of 20:1 required a maximum of 4 days of storage before Salmonellae were inactivated by 7 log₁₀, whereas preparations with C:N ratios of 30:1 and 40:1 C:N required more than 5 and 7 days of storage, respectively. The pH values of both the 20:1 and 30:1 C:N preparations decreased during the onset of composting before increasing to >8. In contrast, pH values of 40:1 C:N preparations increased immediately to >8, generally within the first day of storage. Maximum temperatures observed in 20:1 C:N preparations for inactivation of pathogens were less than 50 °C, and the cumulative heat exposure required for pathogen inactivation in 20:1 C:N preparations was 15-fold less than in 40:1 C:N preparations. Supplementation of compost mixtures with 0.08% ammonium sulfate resulted in slightly higher temperatures; however, these higher temperatures did not translate into more rapid rates of pathogen inactivation.     

Heat stability and species range of purified staphylococcal alpha-toxin

Cooper, Louis Z. (New England Medical Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Heat stability and species range of purified staphylococcal alpha-toxin. J. Bacteriol. 91:1686-1692. 1966.-Heating of high-titer purified staphylococcal alpha-toxin at 60 and 80 C resulted in a double-sloped curve of inactivation of the hemolytic effect on rabbit erythrocytes. Early inactivation was less at the lower temperature, but activity persisted for a longer time at 80 C. Toxin inactivated at 60 C showed renewed activity when heated briefly at 80 C. A precipitate which formed during heating of alpha-toxin at 60 or 80 C yielded hemolytic activity when resuspended and heated at 80 but not at 60 C. Supernatant fluid of heat-precipitated toxin was heat-labile and did not regain activity when heated at 80 C. The results indicate that the "paradoxical effect" of heating of staphylococcal alpha-toxin is not due to a thermolabile inhibitor, but results from alteration of the toxin molecule to a heat-stable active form. Demonstration of renewed activity by 80 C heating of purified toxin requires potent toxin preparations and brief heating periods. Hemolysis of erythrocytes of several animal species by purified alpha-toxin was generally similar to that produced by impure toxin. Rabbit cells were most susceptible. Human and horse erythrocytes hemolyzed to less than 0.1% of the extent of rabbit cells. Blood cells of other species were intermediate in their response to the lytic effect of alpha-toxin.     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae.

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 degrees C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 degrees C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 degrees C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage.     

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.     

Heat inactivation of Salmonella typhimurium DT104 in beef as affected by fat content

The heat resistance of an eight-strain cocktail of Salmonella typhimurium DT104 was determined at 58-65 degrees C in beef containing 7, 12, 18 or 24% fat. Inoculated beef was packaged in bags completely immersed in a circulating water bath and held at 58, 60, 62.5 and 65 degrees C for a predetermined length of time. The surviving cell population was enumerated by spiral plating heat-treated samples onto tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. Preliminary studies on thermal inactivation of the Salmonellae isolates in chicken broth indicated no correlation between heat resistance and origin of the isolates. While linear survival curves were observed in chicken broth, inactivation kinetics in beef showed deviations from the first order kinetics, represented by an initial lag period or shoulder before any death occurred with time. Overall, increased fat levels in beef resulted in longer lag periods and lower D-values, suggesting that the lag periods must be taken into account and added to the D-values for calculating the time required at a specific temperature for achieving a specific lethality for Salm. typhimurium DT104 in beef. Thermal death times from this study will assist the retail food industry to design cooking regimes that ensure safety of beef contaminated with Salm. typhimurium DT104.