Radiation Sterilization of Bacon for Military Feeding

Sliced, cured bacon, packed in cans and seeded with 6 × 10⁵ spores per can of Clostridium botulinum strains 33A or 41B, or with 3 × 10⁶ spores per can of strains 36A, 12885A, 9B, or 53B, was irradiated to various dose levels with γ radiation. Evidence provided by swelling, toxicity, and recoverable C. botulinum with 2,200 inoculated, irradiated cans demonstrated that: (i) 4.5 Mrad were more than adequate as a sterilization dose; (ii) the experimental minimal sterilizing dose was 2.0 Mrad, and the theoretical 12-log reduction dose was 2.65 or 2.87 Mrad depending on the method of calculation; (iii) some spoilage occurred at dose levels below 2.0 Mrad; (iv) all visible spoilage of irradiated bacon was due to strains 33A and 12885A only, whose D values were, respectively, 0.141 and 0.177 Mrad based on spoilage data, and 0.221 and 0.188 Mrad, respectively, when based on recovery data; (v) toxic cans did not always result in swelling, nor did swollen cans always produce toxic spoilage; and (vi) viable C. botulinum can exist for at least 8 months in storage at 30 C without producing visible or toxic spoilage at doses below 2.0 Mrad.     

Cryogenic gamma irradiation of prototype pork and chicken and antagonistic effect between Clostridium botulinum types A and B.

Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 10(7) spores. The cans received a series of increasing doses of gamma rays (60Co) at -30 +/- 10 degrees C; they were incubated for 6 months at 30 +/- 2 degrees C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 less than ESD less than or equal to 3.2 Mrad for pork and 4.0 less than ESD less than or equal to 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of Type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was a least one type B strain that was at least as resistant as type A strains.     

Cryogenic gamma irradiation of prototype pork and chicken and antagonistic effect between Clostridium botulinum types A and B.

Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 10(7) spores. The cans received a series of increasing doses of gamma rays (60Co) at -30 +/- 10 degrees C; they were incubated for 6 months at 30 +/- 2 degrees C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 less than ESD less than or equal to 3.2 Mrad for pork and 4.0 less than ESD less than or equal to 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of Type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was a least one type B strain that was at least as resistant as type A strains.     

Radiation Sterilization of Prototype Military Foods: Low-Temperature Irradiation of Codfish Cake, Corned Beef, and Pork Sausage

"Screening" packs comprising 10 lots each of codfish cake, corned beef, and pork sausage, each lot containing about 10(6) spores of a different strain (five type A and five type B) of Clostridium botulinum per can, were irradiated at -30 +/- 10 C with a series of increasing doses (20 replicate cans/dose) of (60)Co gamma rays. The cans were incubated for 3 months at 30 C and examined for swelling, toxin, and recoverable botulinal cells. Based on the latter criterion of spoilage, median lethal dose (LD(50)) and D values were estimated for each strain in each food. The most resistant strain in codfish cake, corned beef, and pork sausage was, respectively, 53B, 77A, and 41B. There was no clear-cut trend in the comparative order of resistance between the two antigenic types among the three foods. LD(50) values gave essentially the same order of resistances as the D values and may be used interchangeably with the latter for the 10 test organisms. "Clearance" packs consisting of the most resistant strain (about 10(7) spores/can) with its respective food were irradiated with a variety of doses at -30 +/- 10 C, using 100 replicate cans/dose (about 10(9) spores/dose). These packs were incubated for 6 months at 30 C and assayed for the three types of spoilage. Based on recoverable cells, the experimental sterilizing doses (ESD) for codfish cake, corned beef, and pork sausage were 2.5< ESD 相似文献   

Radiation Sterilization of Prototype Military Foods: II. Cured Ham

Ten lots of diced cured ham, packed in cans, were inoculated with approximately 10(6)Clostridium botulinum spores per can. Each lot was seeded with a different strain (five type A and five type B strains). All cans were irradiated to various dose levels with Co(60). Evidence provided by swelling, toxicity, and recoverable C. botulinum with 6,350 cans demonstrated that: (i) 4.5 Mrad was more than adequate as a sterilization dose; (ii) the minimal experimental sterilizing dose (ESD) based on nonswollen nontoxic endpoints was 2.0 < ESD 相似文献   

Effect of Temperature of Liquid Nitrogen on Radiation Resistance of Spores of Clostridium botulinum

An apparatus consisting of a Dewar flask and a relay system controlling the flow of liquid nitrogen permitted the irradiation of samples in tin cans or Pyrex tubes at temperatures ranging from 0 ± 1.5 C to -194 ± 2 C. An inoculated pack comprising 320 cans of ground beef containing 5 × 10⁴ spores of Clostridium botulinum 33A per can (10 cans per radiation dose) was irradiated with Co⁶⁰ at 0 and -196 C. Incubation was carried out at 30 C for 6 months. Approximately 0.9 Mrad more radiation was required to inactivate the spores at -196 C than at 0 C. Cans irradiated at -196 C showed partial spoilage at 3.6 Mrad and no spoilage at 3.9 Mrad; the corresponding spoilage-no spoilage doses at 0 C were 2.7 and 3.0, respectively. The majority of positive cans swelled in 2 to 14 days; occasional swelling occurred as late as 20 days. At progressively higher doses, swelling was delayed proportionally to the radiation dose received. The remaining nonswollen cans had no toxin after 6 months of storage, although occasional cans contained very low numbers of viable spores comprising on the average 0.1% of the original spore inoculum. The D₁₀ values in phosphate buffer were 0.290 Mrad for 0 C and 0.396 Mrad for -196 C; in ground beef, the corresponding D₁₀ values were 0.463 Mrad and 0.680 Mrad, respectively. These D₁₀ values indicate that the lethal effect of γ rays decreased at -196 C as compared with 0 C by 13.5% in phosphate buffer, and by 47% in ground beef.     

Radiation Sterilization of Prototype Military Foods. III. Pork Loin

Ten lots of pork loin, packed in cans, were inoculated with approximately 10(6)Clostridium botulinum spores per can. Each lot was seeded with a different strain; five type A and five type B strains were used. The pack comprised 5,690 cans, including controls, and contained about 10(9) spores per dose. The cans were irradiated with Co(60) in the range of 0 to 5.0 Mrad (0.5 Mrad increments) at 5 to 25 C, incubated for 6 months at 30 C, and examined for swelling, toxicity, and recoverable C. botulinum. The minimal experimental sterilizing dose (ESD) based on nonswollen, nontoxic, but nonsterile end points was 2.5 < ESD 相似文献   

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Comparative Resistance of Strains of Clostridium botulinum to Gamma Rays

A total of 102 strains of Clostridium botulinum (56 strains of type A, 43 type B, and 3 nontoxigenic strains which could not be typed) was examined for resistance to gamma rays. When these organisms were suspended in neutral phosphate buffer in concentrations of 10⁴ spores per tube, the threshold sterilizing dose appeared to be 1.4 Mrad. Partial survival to 1.4 Mrad was shown by 10.7% of the type A strains, 18.6% of the type B strains, and one of three nontoxigenic strains. Over-all, type A strains indicated higher radioresistance than type B strains, although there was overlapping. Representatives of the most resistant strains had D values of 0.317 to 0.336 Mrad; the D values of an intermediate group were 0.224 to 0.253 Mrad, and the most sensitive strain studied, 51B, had a D value of 0.129 Mrad. The radioresistance of Putrefactive Anaerobe 3679, strain S-2, was comparable to the intermediate C. botulinum group (D = 0.209).     

Chemical Sensitization of Clostridium botulinum Spores to Radiation in Meat

Beef ground round inoculated with 1,000,000 spores of Clostridium botulinum 33-A per gram and containing various additives was exposed to gamma radiation. Spores were inactivated in samples (irradiated at 2.0, 2.5, and 3.0 Mrad) which contained sodium nitrate (1,000 ppm) plus sodium chloride (2.5%). Similar results were obtained when sodium nitrite (200 ppm) was substituted for sodium nitrate, except that there was evidence of spore survival in 1 of 120 cans irradiated at 2.0 Mrad. Spore destruction was based upon the absence of spores and mouse-lethal toxin in meat subcultures made from cans incubated at 35 C for 120 days. Spores were not destroyed when exposed to 2.5 or 3.0 Mrad in the absence of sodium nitrate, sodium nitrite, or sodium chloride. Furthermore, the use of these chemicals individually, together with radiation, was ineffective. The additives alone in the absence of radiation also did not cause spore destruction. Radiation levels of 2.0, 2.5, and 3.0 Mrad, when used with sodium chloride at 1.5 or 2.0% and sodium nitrate at 500 ppm or sodium nitrite at 100 ppm, were ineffective.     

Resistance of Clostridium perfringens Type A Spores to γ-Radiation

The radiation resistance of the spores of a classical strain and of an atypical, heat-resistant strain of Clostridium perfringens was determined. Spores were produced in Ellner's and in a Trypticase broth medium. Approximately 10⁶ viable spores per milliliter were suspended in 0.06 m phosphate buffer and irradiated with γ rays from cobalt-60; the survivors were counted in Tryptone-yeast extract-agar by the Prickett-tube technique. Radiation D values for spores of the atypical strain in phosphate buffer and in cooked-meat broth were 0.23 and 0.30 Mrad, respectively, and the D value of the classical strain was 0.25 Mrad in phosphate buffer. Spores of the classical and atypical strains of C. perfringens type A are characterized by differences in heat resistance; yet, all strains tested demonstrated similar radiation resistance. Also, the spores were more resistant to ionizing radiation in cooked-meat broth than in phosphate buffer.     

Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA⁺ and rodA⁻ strains. Tapping-mode AFM images of wild-type and rodA⁺ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA⁻ spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA⁺ spores. Rodlets were removed from rodA⁺ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA⁺ and rodA⁻ spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10² and 10⁶ spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor''s desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA''s efficacy standard for a biocide, although they were found to be as effective for concentrations of 10² spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.Developing a decontamination approach that can be safely and effectively applied to civilian water resources and facilities following a terrorist or catastrophic release of Bacillus anthracis spores poses many challenges. For example, if a municipal drinking water system were contaminated directly or indirectly during or after such an incident, it would be essential to assess the potential health risks posed by water consumption or other water uses (e.g., recreational and bathing) and then to apply one or more proven technologies, if deemed necessary, to decontaminate the water supply quickly and cost-effectively. Treatment of drinking water implies the use of a decontamination approach that would not pose adverse health risks to humans or result in unacceptable damage to the environment. A major obstacle in killing spores of Bacillus spp. on or in virtually any matrix is their high level of resistance to treatments such as harsh chemicals, heat, desiccation, and UV light (14, 20). Because of the substantial and widely reported resistance of Bacillus spores to inactivation, a decontaminant proven to be efficacious in killing such spores for site-specific applications is likely to be effective against all other biological warfare agents as well.Whereas nearly all biological warfare agents are intended for aerosol application, many have strong potential as waterborne threats and could inflict heavy casualties when ingested (2). B. anthracis in particular has been identified as a “probable” (12) or an actual (24) water threat. Even though the principal risk associated with the consumption of water containing B. anthracis spores would likely arise from an ingestion hazard, water used for bathing, showering, or recreational purposes might also pose cutaneous as well as aerosol exposure hazards. There is controversy regarding the long-term viability of B. anthracis in water, and experimental evidence is limited. However, according to a review of nonkinetic studies on survival of virulent strains in the environment (21), B. anthracis spores can survive from 2 to 18 years in pond water and 20 months in seawater or distilled water. B. anthracis spores have been reported by others to be stable in water for 2 years (24).Various decontamination approaches have been evaluated for efficacy against biological warfare agents, including Bacillus spores, on hard, nonporous surfaces. Recommendations by the U.S. Environmental Protection Agency (EPA) include the use of sodium hypochlorite (1:9 dilution of bleach to 5,250 to 6,000 ppm, corrected to pH 7, with a 60-min contact time at 20°C [6, 17]), and liquid chlorine dioxide with a 30-min wet contact time at 20°C (7). Liquid hydrogen peroxide/peroxyacetic acid (known as peroxy compounds and marketed as ready-to-use solutions), generally with a 15- to 20-min wet contact time and concentration as specified by the manufacturer, has also been recommended (13). Other products, such as hydrogen peroxide solution (3 to 25%) and potassium peroxymonosulfate, have been evaluated for efficacy against Bacillus spores as well (27). Although disinfectants at various concentrations have been tested previously against the spores of B. anthracis and their surrogates, wide variations in test protocols make meaningful comparisons among studies virtually impossible (9, 11, 17).In contrast to surface cleanup of spores, fewer assessments of efficacy utilizing suspension tests with the aforementioned chemicals or other methods have been reported for the decontamination of Bacillus species spores in water, and much of the published work has assessed only relatively high concentrations of spores in water. For example, one previous investigation commenced evaluations with 0.2-ml suspensions of approximately 10⁹ spores/ml of various Bacillus spp. to which 20 ml of aqueous ozone or 20 ml of hydrogen peroxide solution was added to assess sporicidal action (10), and others have reported mechanisms of deactivating B. subtilis spores prepared in concentrations of up to approximately 10⁸ spores/ml (26) and approximately 10⁹ spores/ml (17). Inactivation by chlorination of various Bacillus spp. with initial concentrations of approximately 1 × 10⁴ CFU/ml has also been tested (16). However, relatively low spore concentrations would be expected to result from dilutions following contamination of a large public water system. Therefore, it is reasonable to evaluate the effectiveness of decontaminants or other methods against even lower spore concentrations in water than have been previously assessed. In addition to assessing the parameter of Bacillus spore concentration in water, it is essential to identify the most effective commercially available chemical that will kill all the spores or minimize population growth, while considering the effects of the chemical on the environment and in humans.Several objectives served to focus our investigation. First, five potential candidate decontaminants were selected because of their relative safety and ultimate degradation in the environment without substantive adverse consequences. The five chemicals were also chosen as a way of comparing the effectiveness of available free chlorine content, pH, and oxidation potential on spore inactivation. From an evaluation of those chemical parameters, we sought to determine the most effective option for inactivating Bacillus spore surrogates suspended in water. As a second objective, we attempted to identify the lowest concentration of the selected chemicals necessary to achieve the EPA''s biocide standard of a >6-log kill. As a third objective, we wanted to assess the effect of reduced spore concentration on chemical biocide efficacy. As an important step in ascertaining an efficient, safe, and cost-effective water treatment method that could potentially provide safe water to the general population in the event of B. anthracis contamination—and limit the potential risk of contracting gastrointestinal or cutaneous anthrax as well—the following parameters were evaluated: chemical decontaminant type, chemical decontaminant concentration (0.01% to 5%), contact time of spores with chemical decontaminant (10 min to 1 h), spore type (Bacillus atrophaeus or Bacillus thuringiensis), and low versus relatively high spore concentrations (approximately 10² and 10⁶ spores/ml, respectively).Use of B. atrophaeus and B. thuringiensis spores as surrogates for B. anthracis is widely reported in the literature. For example, Szabo et al. (23) used B. atrophaeus subsp. globigii spores as a surrogate for B. anthracis to investigate the persistence and decontamination of those surrogates on corroded iron in a model drinking water system, and Rice et al. (16) used spores of B. thuringiensis as an “appropriate surrogate for spores of B. anthracis” for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Furthermore, the EPA (5) concluded that “B. globigii can serve as a conservative surrogate for B. anthracis during studies of inactivation by chlorination.”     

Resistivity of Spores to Ultraviolet and γ Radiation while Exposed to Ultrahigh Vacuum or at Atmospheric Pressure

Viability studies were conducted on microbial spores subjected to ultrahigh vacuum (UHV) in the 10^-9 to 10^-10 torr range. After 5 to 7 days in vacuum, they were exposed to ultraviolet (UV) or to γ radiation either while still under vacuum or in the presence of dried air. Among the four test organisms subjected to UHV and ultraviolet radiation, Aspergillus niger was the most resistant; Bacillus megaterium, B. subtilis var. niger, and B. stearothermophilus were about equally less resistant. All four spores were more sensitive to ultraviolet radiation when UHV-dried than when desiccant-dried. Of the four test organisms subjected to UHV and γ radiation, B. megaterium proved to be the most resistant; A. niger was the least resistant; and the remaining two organisms were of intermediate resistivity. All four organisms were less radiation resistant when UHV-dried than when irradiated in their normally hydrated state, and all showed an increased radiosensitivity after vacuum drying when oxygen was present. In addition, spores of B. subtilis var. niger and A. niger were less radiosensitive when UHV-dried and irradiated in vacuum than when “wet” and irradiated in air, whereas the reverse relationship was observed for the remaining two organisms. Based on the fact that microbial contaminants can be readily shielded from UV light by soils, metal particles, etc., and considering that the levels of ionizing radiations reported to be present in interstellar space are generally lower than those used in these experiments, the decrease in radioresistivity imparted by UHV drying is not of a sufficient magnitude to sterilize dependably portions of a spacecraft while on a mission.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Recovery of Spores from Thermophilic Dairy Bacilli and Effects of Their Surface Characteristics on Attachment to Different Surfaces

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of ~10⁷ spores ml⁻¹; spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from −10 to −20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 × 10⁴ spores cm⁻²), with fewer spores attaching to glass (3 × 10³ spores cm⁻²). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (~1 × 10³ spores cm⁻²), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.     

Radiation Injury of Clostridium botulinum Spores in Cured Meat

Cans of chopped ham, inoculated with spores of Clostridium botulinum strains 33A and 41B at levels of 2,500 and 250 per gram, were subjected to an enzyme-inactivating heat treatment and irradiation with 0.5, 1.5, 2.5, or 3.5 Mrad of Co(60). A portion of the pack was not irradiated, and received a commercial thermal process (F(0) = 0.2). Viable spores were enumerated after treatment and after 6 months of incubation at 30 to 37.7 C. Toxic spoilage occurred at 0 and 0.5, but not at 1.5, 2.5, or 3.5 Mrad. More spoilage and toxin formation occurred in the product irradiated at 0.5 Mrad than in identical product receiving no radiation treatment. Confirmed botulinal spores were isolated from all of the radiation variables of 2,500 per gram-inoculated product and from all but the 3.5 Mrad low-inoculum cans. However, neither growth nor toxin was observed in unspoiled product. The "injury" phenomenon previously described in thermally processed cured meats (survival of botulinal spores without capacity for multiplication or toxigenesis) apparently occurs also in irradiated cured meats.     

Sporicidal properties of hydrogen peroxide against food spoilage organisms

The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H₂O₂ were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with „D” values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H₂O₂ concentration and the temperature reduced the lag period.     

Identification by Quantitative Carrier Test of Surrogate Spore-Forming Bacteria To Assess Sporicidal Chemicals for Use against Bacillus anthracis

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H₂O₂, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37°C for 72 h. The spore suspensions, heat treated at 80°C for 10 min to rid them of any viable vegetative cells, contained 1 × 10⁸ to 3 × 10⁸ CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 μl (≥10⁶ CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 μl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of ≥10⁶ in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580^T) and Bacillus subtilis (ATCC 6051^T) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.