Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.     

Visible light inactivation of bacteria and fungi by modified titanium dioxide.

Visible light induced photocatalytic inactivation of bacteria (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis) and fungi (Candida albicans, Aspergillus niger) was tested. Carbon-doped titanium dioxide and TiO2 modified with platinum(IV) chloride complexes were used as suspension or immobilised at the surface of plastic plates. A biocidal effect was observed under visible light irradiation in the case of E. coli in the presence of both photocatalysts. The platinum(IV) modified titania exhibited a higher inactivation effect, also in the absence of light. The mechanism of visible light induced photoinactivation is briefly discussed. The observed detrimental effect of photocatalysts on various microorganism groups decreases in the order: E. coli > S. aureus approximately E. faecalis>C. albicans approximately A. niger. This sequence results most probably from differences in cell wall or cell membrane structures in these microorganisms and is not related to the ability of catalase production.     

Rate of inactivation of adenyl cyclase and phosphodiesterase: determinants of brain cyclic AMP

In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.     

Different effects of microwave energy and conventional heat on the activity of a thermophilic beta-galactosidase from Bacillus acidocaldarius

The effect of microwave (f = 10.4 GHz) irradiation on a thermostable enzyme was experimentally tested, showing that irreversible inactivation is obtained. Enzymatic solutions (500 microliters, with concentrations between 10-100 micrograms/ml) were exposed at 70 degrees C, at SAR levels of 1.1 and 1.7 W/g for 15, 30, 45, or 60 min, and their activity was compared to that of a sample heated in a water bath at the same temperature. The residual activity of the exposed samples depends on enzyme concentration, microwave power level, and exposure time; activity was reduced to 10% in 10 micrograms/ml solutions treated at 1.7 W/g for 60 min. Microwave effects disappeared at concentrations above 50 micrograms/ml. These results were not found following water bath heating at the same temperature and durations.     

Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation.

Raw ground beef patties inoculated with stationary-phase cells of Escherichia coli O157:H7, salmonellae, or Campylobacter jejuni were subjected to gamma irradiation (60Co) treatment, with doses ranging from 0 to 2.52 kGy. The influence of two levels of fat (8 to 14% [low fat] and 27 to 28% [high fat]) and temperature (frozen [-17 to -15 degrees C] and refrigerated [3 to 5 degrees C]) on the inactivation of each pathogen by irradiation was investigated. In ascending order of irradiation resistance, the D10 values ranged from 0.175 to 0.235 kGy (C. jejuni), from 0.241 to 0.307 kGy (E. coli O157:H7), and from 0.618 to 0.800 kGy (salmonellae). Statistical analysis revealed that E. coli O157:H7 had a significantly (P < 0.05) higher D10 value when irradiated at -17 to -15 degrees C than when irradiated at 3 to 5 degrees C. Regardless of the temperature during irradiation, the level of fat did not have a significant effect on the D10 value. Salmonellae behaved like E. coli O157:H7 in low-fat beef, but temperature did not have a significant effect when the pathogen was irradiated in high-fat ground beef. Significantly higher D10 values were calculated for C. jejuni irradiated in frozen than in refrigerated low-fat beef. C. jejuni was more resistant to irradiation in low-fat beef than in high-fat beef when treatment was at -17 to -15 degrees C. Regardless of the fat level and temperature during inactivation, these pathogens were highly sensitive to gamma irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Thymineless Death in Escherichia coli: Inactivation and Recovery

The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, B(s-1), B(s-3), B(s-12), and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 mug of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 mug/ml actually increased the sensitivity of E. coli B, B(s-1), B(s-3), and B(s-12) to inactivation by either TLD or NA; at 150 mug of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and B(s-12). Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli B(s-1) and B/r did not recover viability after any mode of inactivation, and E. coli B(s-3) and B(s-12) recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events.     

Evaluation of the necessity for rapid inactivation of brain enzymes prior to analysis of norepinephrine dopamine and serotonin in the mouse.

Norepinephrine, dopamine and serotonin concentrations were measured in mouse whole brain. Animals were killed either by decapitation or by exposure to 250 msec microwave irradiation which produces irreversible inactivation of brain enzymes. The biogenic amines were determined by mass fragmentometry, fluorometry and by a combination of high performance liquid chromatography and an electrochemical detector. No differences were found in the levels of these neurochemicals between the two methods of animal sacrifice. Therefore, rapid inactivation of brain enzymes is not necessary prior to analysis for catecholamines and serotonin in mouse whole brain.     

Assay of norepinephrine and dopamine in the rat brain after microwave irradiation

Rapid inactivation of enzymes prior to the assay of rat brain catecholamines was evaluated. Regional levels of norepinephrine and dopamine were measured by high performance liquid chromatography with electrochemical detection after enzyme inactivation by microwave irradiation at levels of 1.3 kw and 5 kw, and compared with decapitation. The differences found in regional levels of catecholamines between the two methods of euthanasia indicate that rapid inactivation of brain enzymes is necessary for accurate analysis of catecholamines in rat brain.     

Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity

The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C). Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature. When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed. These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity. At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures. Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E. coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity. The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable.     

Heat transfer analysis of Staphylococcus aureus on stainless steel with microwave radiation

Staphylococcus aureus (NCTC 6571; Oxford strain) on stainless steel discs was exposed to microwave radiation at 2450 MHz and up to 800 W. Cell viability was reduced as the exposure time increased, with complete bacterial inactivation at 110 s, attaining a temperature of 61.4 degrees C. The low rate of temperature rise, RT, of the bacterial suspension as compared with sterile distilled water or nutrient broth suggests a significant influence of the microwave sterilization efficacy on the thermal properties of the micro-organisms. The heat transfer kinetics of thermal microwave irradiation suggest that the micro-organism has a power density at least 51-fold more than its surrounding liquid suspension. When the inoculum on the stainless steel disc was subjected to microwave radiation, heat conduction from the stainless steel to the inoculum was the cause of bacteriostasis with power absorbed at 23.8 W for stainless steel and 0.16 W for the bacteria-liquid medium. This report shows that the microwave killing pattern of Staph. aureus on stainless steel was mainly due to heat transfer from the stainless steel substrate and very little direct energy was absorbed from the microwaves.     

Regional levels of cyclic AMP in rat brain: pitfalls of microwave inactivation.

Techniques of in-vivo microwave irradiation to inactivate brain enzymes in rats were varied as to exposure configuration and output power. The rate at which metabolism was stopped was studied in various regions of the rat brain, using changes in levels of cyclic AMP and phosphodiesterase activity. Exposure times required to obtain stabilized levels of cyclic AMP varied in different brain regions, i.e., hypothalamus, cortex and cerebellum. Levels of cyclic AMP in selective regions of the brain decreased as more rapid inactivation was achieved. The authors identify important sources of variability of present microwave inactivation systems and the need for improved control of signficant microwave parameters.     

A facile solid-state synthesis and in vitro antimicrobial activities of some 2,6-diarylpiperidin/tetrahydrothiopyran and tetrahydropyran-4-one oximes

Some 2,6-diarylpiperidin/tetrahydrothiopyran/tetrahydropyran-4-one oximes were synthesized in dry media under microwave irradiation and were evaluated for their in vitro antibacterial activity against clinically isolated bacterial strains i.e. S.aureus, beta-H.Streptococcus, E.coli, P.aeruginosa, S.typhii and in vitro antifungal activities against fungal strains i.e. C.albicans, Rhizopus, A.niger and A.flavus. Structure-activity relationships for the synthesized compounds showed that compounds 12 and 15 exerted excellent antibacterial activity against all the tested bacterial strains except 15 against S.aureus and beta-H.streptococcus. Against C.albicans and A.flavus, compound 15 exerted potent antifungal activities while against Rhizopus, compound 16 showed promising activity.     

Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay

Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.     

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.     

Kinetics of Escherichia coli destruction by microwave irradiation.

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.     

Kinetics of Escherichia coli destruction by microwave irradiation.

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.     

Preservation of structure and cytochemical reactivity at the ultrastructural level, using microwave irradiation

The application of microwave irradiation at various steps in 'normal' EM procedures has been investigated. 1. The temperatures in various aqueous volumes were measured during microwave irradiation. Increases were small and the final temperature could be controlled by cooling the glass base plate. 2. The influence of microwave irradiation on the various fixation schedules for the electron microscopy of single cell populations was studied and the results favoured the idea that by the application of microwave irradiation a more life-like ultrastructural preservation could be obtained. 3. Peroxidase-like activity in erythrocytes, acid phosphatase activity in resident macrophages and peroxidase activity in monocyte granules was apparently not influenced by microwave irradiation during aldehyde fixation and incubation. 4. The anticipated microwave-induced penetration enhancement of cerium ions in a cytochemical reaction procedure to detect acid phosphatase activity in resident macrophages was not observed.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Does microwave irradiation have other than thermal effects on histological staining of the mammalian CNS? A light microscopical study of microwave stimulated staining under isothermal conditions in man and rat.

The question whether or not microwave irradiation exerts other than thermal effects on histological staining is still a matter of controversy. The present study was undertaken to reveal or reject such a so far hypothetical non-thermal irradiation effect. A device was developed, which enables exposure of histological sections or tissue pieces to microwave irradiation under isothermal conditions, i.e. with synchronous removal of the internal heat produced. Three classical neuroanatomical staining methods were tested on human and rat CNS. As control, identical procedures were performed without simultaneous microwave irradiation. The experiments were performed at three different temperature levels ranging from 5 to 50 degrees C. In none of the cases studied was a light microscopically appreciable difference observed between the microwave and non-microwave versions of a stain at the same temperature. The hypothesis of a separate non-thermal effect of microwave irradiation on histological staining is therefore rejected.