Towards microbiological quality assurance in radiation sterilization processing: the limiting case model applied to a microbial population having a distribution of radiation responses

A limiting case model, developed earlier to provide a conservative estimate of the proportion of non-sterile items ( P ) in an irradiated population of items contaminated with micro-organisms of a single type, has been extended to include items having organisms of different types. Estimates of P are compared with exact values derived on purely theoretical grounds and with values obtained by computer simulation of microbiological inactivation on items.     

Inactivation of Neurospora conidia by ultraviolet radiation

The inactivation of Neurospora conidia by ultraviolet radiation is a consequence of the inactivation of the nuclei. The nuclei exhibit a kinetics of inactivation which is first order with respect to the number of (viable) nuclei. Arguments based on the efficiency of inactivation indicate that only quanta absorbed within a nucleus can lead to its inactivation.There are at least two sets of inactivation processes. One involves the production of lethal mutations, the other is a non-genetic (not inheritable) process. The two processes are apparently intimately connected: they yield the same action spectrum and the effects of each are reduced by photo reactivation. The assumption that the two processes are independent leads to predictions which are at variance with the observed dependence of the frequency of lethal mutations on dose of ultraviolet radiation.     

Induced androgenesis in acipenserids may be obtained by ultraviolet radiation

The results are presented on haploid androgenesis in Siberian sturgeon and sterlet induced by UV irradiation of ovicells. During irradiation, the cells in Ringer solution were rotated around a UV lamp. The efficiency of genetic inactivation of ovicells was estimated by the following parameters: manifestation of Hertwig effect, the fraction of embryos demonstrating haploid syndrome at final developmental stages, by arrest of embryonic development in hybrids Siberian sturgeon × great sturgeon, and by absence of maternal alleles of microsatellite loci in embryos. The dose-effect curve suggests that, during UV irradiation of ovicells of Siberian sturgeon, the complete genetic inactivation is attained at exposition of 120 s, while that in sterlet is 90 or 105 s. The results show a principal possibility of inactivation of ovicells by UV irradiation and use of such cells for producing androgenetic progeny of acipenserids.     

Towards microbiological quality assurance in radiation sterilization processing: a limiting case model

A Limiting Case Model has been developed which describes the dependence on radiation dose of the proportion of items, in a population of items subjected to irradiation, which are contaminated by one or more organisms. This model is independent of the initial distribution of numbers of micro-organisms on items and represents a conservative approach to estimation of the proportions of non-sterile items in an irradiated population of items.     

Inactivation of macromolecules by ionizing radiation. Deterministic single-hit or stochastic multievent process?

A stochastic theory concerning the radiation inactivation of macromolecules such as enzymes or receptors is elaborated. In contrast with the single-hit theory, which assumes a complete inactivation of the target as the result of one hit, the stochastic theory postulates that the degree of inactivation by one hit is a random variable. This distinguishing feature has been considered in order to give a possible interpretation to the observed effect of temperature on the radiation-sensitivity of enzymes. As a consequence of the progressive inactivation during irradiation, the binding affinity of a ligand for the macromolecule is impaired by irradiation. Although this property might discriminate the stochastic theory from the classical single-hit theory on the basis of a statistical analysis of experimentally obtained data, it is shown that the commonly obtained degree of inaccuracy may render the statistical test non-conclusive.     

Molecular weight estimation of protoheme ferro-lyase by radiation inactivation.

The molecular weight of protoheme ferro-lyase [EC 4.99.1.1], a mitochondrial enzyme, was estimated by the radiation inactivation method. Irradiation in vacuo caused less inactivation than that in air. The approximate molecular weight determined by this method was in the range from 250,000 to 320,000. The significance of the location of the enzyme in connection with its role in heme synthesis is discussed on the basis of Singer's membrane theory.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

The substructure of phosphodiesterase as established by radiation inactivation: A reinterpretation of results

A model for the activation of phosphodiesterase by calmoduling based on a conversion of inactive dimers to active monomers, derived from radiation inactivation studies J. Biol. Chem. (1981) 256, 11351–11355 has been re-examined using a simple probability argument. We conclude that the original model is not supported by the radiation inactivation studies, since our analysis of this model would predict that the rate of radiation inactivation of calmodulin-dependent phosphodiesterase activity be exactly twice that for the decay in total activity in marked contrast with the results obtained.     

Inactivation of single-celled Ascaris suum eggs by low-pressure UV radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m2 for intact eggs and from 0 to 500 J/m2 for decorticated eggs. With a UV fluence of 500 J/m2, 0.44-+/-0.20-log inactivation (mean+/-95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m2 resulted in 2.23-+/-0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m2), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m2), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80-+/-0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m2, suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-microm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (approximately 20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

Optimizing thermal and radiation effects for bacterial inactivation

The temperatures required for dry-heat spacecraft sterilization have been known to degrade heat-sensitive components. Thermoradiation, the simultaneous application of dry heat and gamma radiation, can provide the same degree of microbial inactivation as dry heat alone while substantially reducing component degradation. This is made possible by the synergistic effects produced when relatively low levels of these agents (e.g., 90 to 350 krads and 60° to 105°C) are applied simultaneously, thus permitting the use of lower temperatures and a reduced duration of heat exposure. The effects of temperature, radiation dose rate, and relative humidity on microbial inactivation during thermoradiation exposure have been established.This experimentation was supported by NASA Contract No. W-12853.     

Microbiological Quality Control of Sterilized Products: Evaluation of a Model Relating Frequency of Contaminated Items with Increasing Radiation Treatment

A model describing the dependence of the proportion of items contaminated ( P ) in a population of items on radiation dose ( D ) has been developed. The model was evaluated for a range of doses assuming a Poissonian distribution of micro-organisms on items prior to and during irradiation and radiation inactivation according to 'multiple-hit' kinetics; curves relating P and D were constructed. The influence on curve shape of changing the initial average number of micro-organisms on items and of varying parameters of microbial sensitivity to radiation was assessed. Consideration was given to the value of using sub-process radiation doses in microbiological quality control testing.     

Predicted inactivation of viruses of relevance to biodefense by solar radiation

UV radiation from the sun is the primary germicide in the environment. The goal of this study was to estimate inactivation of viruses by solar exposure. We reviewed published reports on 254-nm UV inactivation and tabulated the sensitivities of a wide variety of viruses, including those with double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA genomes. We calculated D(37) values (fluence producing on average one lethal hit per virion and reducing viable virus to 37%) from all available data. We defined "size-normalized sensitivity" (SnS) by multiplying UV(254) sensitivities (D(37) values) by the genome size, and SnS values were relatively constant for viruses with similar genetic composition. In addition, SnS values were similar for complete virions and their defective particles, even when the corresponding D(37) values were significantly different. We used SnS to estimate the UV(254) sensitivities of viruses for which the genome composition and size were known but no UV inactivation data were available, including smallpox virus, Ebola, Marburg, Crimean-Congo, Junin, and other hemorrhagic viruses, and Venezuelan equine encephalitis and other encephalitis viruses. We compiled available data on virus inactivation as a function of wavelength and calculated a composite action spectrum that allowed extrapolation from the 254-nm data to solar UV. We combined our estimates of virus sensitivity with solar measurements at different geographical locations to predict virus inactivation. Our predictions agreed with the available experimental data. This work should be a useful step to understanding and eventually predicting the survival of viruses after their release in the environment.     

Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus.

Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H2O2 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation. Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H2O2. Genetic analysis via transduction suggests that the nur mutation allele of the katF locus. As previously reported for broad-spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H2O2 inactivation is also independent of the recA and uvrA alleles. Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results.     

Na,K-ATPase: radiation inactivation studies

Na,K-ATPase from duck salt gland and ox brain in the membrane-bound or solubilized form was studied by the radiation inactivation technique using ATP, CTP, GTP or p-NPP as substrates. The values of radiation inactivation size (RIS) were compared with the target size (TS) for the alpha-subunit of the enzyme obtained by an independent method as well as with analytical centrifugation data obtained for C12E8-solubilized enzyme. It was concluded that during ATP (CTP) hydrolysis the enzyme operates as an oligomeric structure; the complex formation requires the presence of K+ and adenosine triphosphate binding to the sites with a low affinity for the nucleotide. Specially designed experiments revealed that the degree of enzyme oligomerization increases with an increase in the microviscosity of the membrane lipid environment.     

Differential effects of growth temperatures on inactivation and mutation of Candida albicans by ultraviolet radiation

Summary Candida albicans exhibits greater susceptibility to inactivation by ultraviolet (uv) radiation if grown before or after irradiation at 37° C rather than 25° C. Caffeine, acriflavin or amino acid analogues potentiate inactivation during postirradiation growth at 37° C but have little effect at 25° C. In contrast to inactivation, mutation induction by uv is unaffected by pre- or postirradiation growth temperatures or by metabolic antagonists. These findings are not explicable in terms of possible effects of growth temperatures on known mechanisms for repair of uv damaged DNA. They are consistent, however, with a previous proposal that a temperature dependent mechanism for dark recovery exists in C. albicans which involves synthesis of protein essential for repair of lethal, non-genetic uv damage.     

Solar UV radiation does not inactivate marine birnavirus in coastal seawater

We examined the inactivation kinetics of marine birnavirus (MABV) in a coastal sea, in seawater samples collected from 50 cm depth. MABV was added to both natural and autoclaved seawater at a concentration of 6 x 10(6.43) TCID50 (50% tissue culture infectious dose) ml(-1), put in dialysis tubes and incubated at the original depth. The inactivation of MABV by solar UV radiation was examined using light and dark tubes. The infectivity titer of MABV was measured by the TCID50 method using CHSE-214 cells. Virus infectivity in natural seawater decreased quickly and was below the detection limit by 270 min in both light and dark conditions; however, virus infectivity was maintained in the autoclaved seawater until 420 min. These results suggest that the loss of virus infectivity is not caused by sunlight UV radiation.     

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.     

Effects of ionizing radiation on artificial (planar) lipid membranes. I. Radiation inactivation of the ion channel gramicidin A

The ion channel formed by the pentadecapeptide gramicidin A in planar lipid membranes is extremely sensitive to ionizing radiation. The membrane conductance may drop by several orders of magnitude under appropriate experimental conditions (low pH and presence of oxygen). The radiation sensitivity is strongly reduced for gramicidin M-. This analogue has the four tryptophan residues replaced by phenylalanines. Experiments performed in the presence of various radical scavengers suggest that the inactivation of the channel is due to a combined action of OH and of HO2 radicals at the tryptophan residues. The shape of the inactivation curves following continuous radiolysis or pulse radiolysis were found to be in fair agreement with a simple model which assumes that the damage of a single tryptophan residue is sufficient for channel inactivation. The conductance of inactivated channels could not be resolved within the experimental accuracy. This is contrary to photolysis of gramicidin channels found by Busath and Waldbilling (1983), where a broad distribution of low conductance states was observed. The inactivation by radiolysis seems to represent an 'all-or-none-process' of the channel conductance.     

The inactivation of bacteriophage by ionizing radiation

The loss of energy by a fast, charged particle in dry material of atomic constitution approximating a protein is described and the important features tabulated. The inactivation of T-1, T-3, and T-7 bacteriophages in the dry state by deuterons is described and follows a simple logarithmic survival curve. The cross section per deuteron is much less than the electron microscope cross section in all three cases and indicates some kind of structure to the viruses. The variation of cross section with ionization density has been measured for T-1. It is linear and the kind of internal-structure which could account for this is discussed.