Microbiological Evaluation of a Large-Volume Air Incinerator

Two semiportable metal air incinerators, each with a capacity of 1,000 to 2,200 standard ft(3) of air per min, were constructed to sterilize infectious aerosols created for investigative work in a microbiological laboratory. Each unit has about the same air-handling capacity as a conventional air incinerator with a brick stack but costs only about one-third as much. The units are unique in that the burner housing and combustion chamber are air-tight and utilize a portion of the contaminated air stream to support combustion of fuel oil. Operation is continuous. Aerosols of liquid and dry suspensions of Bacillus subtilis var. niger spores and dry vegetative cells of Serratia marcescens were disseminated into the two incinerators to determine the conditions required for sterilization of contaminated air. With the latter organisms (concentration 2.03 x 10(7) cells/ft(3) of air), a temperature of 525 F (274 C), measured at the firebox in front of the heat exchanger, was sufficient for sterilization. To sterilize 1.74 x 10(7) and 1.74 x 10(9) wet spores of B. subtilis per ft(3), the required temperature ranged from 525 to 675 F (274 to 357 C) and 625 to 700 F (329 to 371 C), respectively. Air-sterilization temperature varied with each incinerator. This was because of innate differences of fabrication, different spore concentrations, and use of one or two burners With dry B. subtilis spores (1.86 x 10(8)/ft(3)), a temperature of 700 F was required for sterilization. With dry spores, no difference was noted in the sterilization temperature for the two incinerators.     

Effect of Various Gas Atmospheres on Destruction of Microorganisms in Dry Heat

The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.     

Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transitions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic (P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observable ranged from 0.06 (P. vittata) to 0.12 (P. setiferum)gH(2)Og(-1)dry weight, and were highly correlated with water affinity parameters. In spores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum)gH(2)Og(-1)dry weight, some water partitioned into a near pure water fraction that melted at about 0 degrees C. These sharp peaks near 0 degrees C were associated with lethal freezing treatments. The enthalpy of water melting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores compared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fern spores, water content can be precisely manipulated to avoid both desiccation and freezing damage.     

Effects of temperature and desiccation on ex situ conservation of nongreen fern spores

? Premise of the study: Fern spores are unicellular and haploid, making them a potential model system to study factors that regulate lifespan and mechanisms of aging. Aging rates of nongreen spores were measured to compare longevity characteristics among diverse fern species and test for orthodox response to storage temperature and moisture. ? Methods: Aging of spores from 10 fern species was quantified by changes in germination and growth parameters. Storage temperature ranged from ambient room to -196°C (liquid nitrogen); spores were dried to ambient relative humidity (RH) or using silica gel. ? Key results: Survival of spores varied under ambient storage conditions, with one species dying within a year and two species having greater than 50% survival after 3 years. Few changes in germination or growth were observed in spores stored at either -80°C or -196°C over the same 3-yr study period. Spores stored at -25°C aged anomalously quickly, especially those dried to ambient RH or subjected to repeated freeze-thaw cycles. ? Conclusions: Spore longevity is comparable to orthodox seed longevity under ambient storage conditions, with wide variation among species and shelflife extended by drying or cooling. However, faster aging during freezer storage may indicate a similar syndrome of damage experienced by seeds categorized as "intermediate". The damage is avoided by storage at -80°C or liquid nitrogen temperatures, making cryoconservation an effective and broadly applicable tool to extend fern spore longevity. The study demonstrates that spore banks are a feasible approach for ex situ conservation of this important plant group.     

Moist-heat resistance, spore aging, and superdormancy in Clostridium difficile

Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log(10) within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log(10) within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log(10), 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.     

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.     

Life from the ashes: survival of dry bacterial spores after very high temperature exposure

We found that spores of Bacillus amyloliquefaciens rank amongst the most resistant to high temperatures with a maximum dry heat tolerance determined at 420 °C. We found that this extreme heat resistance was also maintained after several generations suggesting that the DNA was able to replicate after exposure to these temperatures. Nonetheless, amplifying the bacterial DNA using BOXA1R and (GTG)₅ primers was unsuccessful immediately after extreme heating, but was successful after incubation of the heated then cooled spores. Moreover, enzymes such as amylases and proteases were active directly after heating and spore regeneration, indicating that DNA coding for these enzymes were not degraded at these temperatures. Our results suggest that extensive DNA damage may occur in spores of B. amyloliquefaciens directly after an extreme heat shock. However, the successful germination of spores after inoculation and incubation indicates that these spores could have a very effective DNA repair mechanism, most likely protein-based, able to function after exposure to temperatures up to 420 °C. Therefore, we propose that B. amyloliquefaciens is one of the most heat resistant life forms known to science and can be used as a model organism for studying heat resistance and DNA repair. Furthermore, the extremely high temperature resistivity of these spores has exceptional consequences for general methodology, such as the use of dry heat sterilization and, therefore, virtually all studies in the broad area of high temperature biology.

     

Resistance of Spores of Bacillus subtilis var. niger on Kapton and Teflon Film to High Temperature and Dry Heat

To determine parameters that would assure sterility of a sealed seam of film for application in "split-seam entry," spores of Bacillus subtilis var. niger were sprayed onto pieces of Kapton and Teflon film. Short-time, high-temperature (200 to 270 C) exposures were made with film pieces between aluminum blocks in a hot-air oven, and the D and z values were determined after subculture of surviving spores. The use of Kapton film allowed the study of high temperatures, since it is not heat sealable and could be used to make thin packages for heat treatment. Spores on Teflon were dry-heat treated in a package designed to simulate an actual seam to be sealed. The z values of 29.1 C (52.4 F) for spores on Kapton and 139 C (250.4 F) for spores on Teflon were calculated.     

Effects of water activity and temperature on the survival of Aspergillus carbonarius spores in vitro

AIMS: To determine the effect of water activity (a(w)) and temperature on the survival of Aspergillus carbonarius spores. METHODS AND RESULTS: Spores of A. carbonarius were dried onto filter membranes. These filters were held at 1.0, 0.9, 0.8, 0.6 and 0.4 a(w) and at 1, 15, 25 and 37 degrees C for up to 618 d. At intervals, spores were recovered from filters and assessed for viability by enumeration on dichloran rose bengal chloramphenicol agar. Survival and subsequent growth of spores was prolonged at low temperatures and at a(w) below 0.6. Above 15 degrees C, 0.6-0.9 a(w) were often more deleterious than 1.0. However, at 1 degrees C and 1.0 a(w), spores lost viability more rapidly than at lower a(w). CONCLUSIONS: Increased incidence of black Aspergillus spp. in dry soils and from grapes in dry conditions may result partly from prolonged survival of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Delineating the direct effect of a(w) and temperature on survival of A. carbonarius spores may aid in understanding the incidence of this ochratoxin A-producing species in vineyard soils and on grapes.     

Heat Resistance of Spores of Marine and Terrestrial Strains of Clostridium botulinum Type C

Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 10(6) spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D(220) (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C).     

Inactivation of bacillus spores in dry systems at low and high temperatures.

A plot of the thermal resistance of Bacillus subtilis var. niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level. The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value). However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment. The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C). The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process. By extrapolation of the thermal resistance plot obtained under these conditions for B. subtilis var. niger spores, the D value at 0 degrees C would be about 4 years.     

Destruction of Bacillus anthracis strain Sterne 34F2 spores in postal envelopes by exposure to electron beam irradiation

AIMS: To determine the irradiation dose necessary to reduce the populations of Bacillus anthracis spores in a dry medium in postal envelopes. METHODS AND RESULTS: Bacillus anthracis Sterne 34F2 spores were dispersed in non-fat dry milk and then placed into standard business postal envelopes. The spores were treated with a sequence of irradiation doses to determine the decimal reduction value (D10) in kiloGrays (kGy). The average D10 value was 3.35 +/- 0.02 kGy. CONCLUSIONS: An irradiation dose of 40.2 kGy would be required to result in a process equivalent to the thermal canning process (12 D10 reduction) to eliminate Clostridium botulinum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Irradiation is an effective means of reducing or eliminating B. anthracis spores in a dry medium in postal envelopes.     

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures

Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.     

Laboratory bioassays of entomopathogenic fungi for control of Delia radicum (L.) larvae

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 x 10(6)spores/g dry soil) and broadcast (3.85 x 10(5)spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 x 10(6) and 1.8 x 10(8)spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on the rust,Maravalia cryptostegiae,a potential biological control agent of rubber-vine weed,Cryptostegia grandiflora (Asclepiadaceae: Periplocoideae), in Australia,II: Infection

The parameters which govern infection of rubber-vine weed by the rustMaravalia cryptostegiae were investigated. The infection process, from appressorial formation to sporulation, is described and illustrated. Uredinioid teliospores have an optimum temperature range for germination at 22–27 °C, both in vitro and in vivo. However, germination on the rubber-vine leaf was more than double (81–92%) that in the absence of the host, and appressoria were formed only in vivo. An optimum temperature of 20–22°C and a dew period of 12 hours or more gave the highest level of infection as measured by sporulation density. The latent period from inoculation to pustule formation decreased with increasing temperature; the shortest period (8–11 days) being recorded at 25–27°C. At the lower temperatures (18°C), this was significantly extended (19–21 days). Four successive inoculations significantly reduced plant height and dry weight, although a compensatory growth flush occurred after the third inoculation. The addition of cryoprotectants had a negative affect on spore viability and subsequent infectivity. Cooling dry spores to –196°C at the rate of 10°C min^–1 gave the best results, with high germination (93–65%) up to 8 days after thawing.     

Analysis of the properties of spores of Bacillus subtilis prepared at different temperatures

AIMS: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. METHODS AND RESULTS: Bacillus subtilis spores prepared at temperatures from 22 to 48 degrees C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. CONCLUSIONS: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.     

Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.     

Effect of storage method on spore viability in five globally threatened fern species

Spore germination of five globally threatened fern species [Culcita macrocarpa C. Presl, Dryopteris aemula (Aiton) O. Kuntze, D. corleyi Fraser-Jenkins, D. guanchica Gibby and Jermy and Woodwardia radicans (L.) Sm.] was determined after 1, 6 or 12 months of storage in glass vials (dry storage) or on agar (wet storage) at -20, 5 or 20 degrees C. In all species, storage technique, storage temperature and the technique-temperature interaction all had a significant effect on germination percentage. In most cases, the germination percentage was best maintained by wet storage at 5 or 20 degrees C. In the case of the hygrophilous species C. macrocarpa and W. radicans, 6 or 12 months' dry storage killed most spores. Only Woodwardia radicans germinated in the dark during wet storage at 20 degrees C. Wet storage at 5 degrees C prevented dark germination, and reduced bacterial and fungal contamination. Wet storage at -20 degrees C killed all or most spores in all species. In the three Dryopteris species, the differences among the storage conditions tested were smaller than in C. macrocarpa and W. radicans, and the decline in spore viability during storage was less marked, with high germination percentages being observed after 12 months of dry storage at all three temperatures. Dry storage, which has lower preparation time and space requirements than wet storage, was generally more effective at the lower temperatures (-20 or 5 degrees C).