Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination

Aims: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. Methods and Results: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10⁶ spores while the stainless steel carriers did not inhibit the growth of spores. Conclusions: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. Significance and Impact of the Study: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 m long, and 2.2 ± 0.3 m wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 m and width of 1.9 ± 0.5 m. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.     

PELAGIC AND BENTHIC ENVIRONMENTAL CONTROLS ON THE SPATIAL DISTRIBUTION OF A VIABLE DIATOM PROPAGULE BANK ON THE SWEDISH WEST COAST1

The distribution of viable diatom resting stages in sediments on the Swedish west coast was assessed by the most probable number (MPN) culture technique. Multivariate analyses correlated benthic and pelagic environmental factors to the observed spatial variations in the size and taxonomic composition of the propagule bank. Viable diatom resting stages were plentiful (0.2–4.8 million cells·g ^{? 1} 1 Received 11 September 2001. Accepted 12 June 2002.
dry weight) and were dominated by the genera Skeletonema, Detonula, Chaetoceros, and Thalassiosira. Size of the propagule bank was primarily related to planktonic biomass (measured as chl a) and was highest in the Orust‐Tjörn fjord system. Species composition in this fjord system was dominated by D. confervacea (Cleve) Gran and T. nordenskioeldii Cleve in contrast to stations on the outer coast, which contained more cells of T. minima Gaarder, Asterionellopsis glacialis (Castracane) Round, and Leptocylindrus danicus Cleve. These taxonomic variations were principally influenced by deep water oxygen concentrations and water column stability. Benthic resting cells of S. costatum (Greville) Cleve were abundant all along the coast but showed reduced viability in low oxygen environments. Calculations based on MPN values estimated that resuspension of sediment could provide a sizable inoculum to the plankton, although the development of planktonic blooms will also depend on forces of hydrography and weather. Although benthic resting stages may not be absolutely necessary for survival of all diatoms, these cells may be important in determining species cycles, succession, and the spatial distribution of diatoms.     

The relationship between airborne pollen and fungal spore concentrations and seasonal pollen allergy symptoms in Cracow in 1997–1999

The investigation of airborne pollen and fungalspore concentrations was carried out in Cracowbetween 1997–1999. For this study thevolumetric method has been employed (Burkard).At the same time the clinical diagnosis ofpollen allergy in 40 patients was obtained onthe basis of an interview, positive skin pricktests with pollen extracts and increasedspecific IgE level. An increase in seasonalallergy symptoms in all patients occurred fromthe middle of May to the middle of August.Eighty eight percent of the patients (35 out of40) were sensitive to Poaceae pollen and about50% of them were additionally sensitive totree and herb pollen excluding grasses. Forpatients with additional allergy to tree pollenthe seasonal symptoms started at the end ofMarch (Betula) while for patients withadditional allergy to herb pollen it wasextended to the middle of September (Artemisia).Five people out of 40 revealed positive skinreactions to Alternaria spores and anincrease in specific IgE level. Positive skinreaction to Cladosporium spores with noincrease in specific IgE level occurred in 2patients. The increase in seasonal allergysymptoms in people sensitive to Alternariaspores noted in July and August could becaused not only by these spores but also byPoaceae pollen.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

NAGase activity in airborne biomass dust and relationship between NAGase concentrations and fungal spores

Inhalation of airborne fungal spores or fungalenzymes may cause adverse pulmonary healtheffects. The enzyme NAGase(N-acetyl--D-glucosaminidase) is achitinase presumed to be secreted by all fungi. In this study, NAGase activities andconcentrations of fungi are estimated inairborne biomass dust to acquire knowledgeabout the level of NAGase activity and therelationship between NAGase activity andconcentrations of airborne fungal spores.NAGase was sampled on both teflon andpolycarbonate filters, and polycarbonatefilters proved to be better for extraction ofNAGase than teflon filters. NAGase was foundin airborne dust at a biofuel plant and in dustgenerated from biomass. At a biofuel plant, themedian level of exposure to NAGase was 21 pmols^–1 m^–3. Significant correlationswere found between NAGase activities, totalnumber of fungal spores and CFU of fungi, withthe highest degree of correlation being betweenthe total number of fungal spores and theNAGase activity (r = 0.802, n = 76). Furthermore,when dust was stored for different periods, theculturability of fungal spores was stronglyreduced and the NAGase activity was not or onlyslightly reduced after up to 40 days ofstorage. Accordingly, NAGase activity may beused as a rapid method to get an estimate ofthe exposure level to airborne fungal spores.Whether pure NAGase or the NAGaseconcentrations observed here cause any healtheffects is not known, although it has beenshown that other fungal enzymes can causerespiratory disorders and a chitinase isdescribed as an allergen.     

Diversity of arbuscular mycorrhizal fungi across a fragmented forest in Panama: insular spore communities differ from mainland communities

It is now understood that alterations in the species composition of soil organisms can lead to changes in aboveground communities. In this study, we assessed the importance of spatial scale and forest size on changes in arbuscular mycorrhizal fungal (AMF) spore communities by sampling AMF spores in soils of forested mainland and island sites in the vicinity of Gatun Lake, Republic of Panama. We encountered a total of 27 AMF species or morphospecies, with 17, 8, 1 and 1 from the genera Glomus, Acaulospora, Sclerosystis, and Scutellospora, respectively. At small scales (<100 m²), we found little evidence for spatial structuring of AMF communities (decay of Morisita-Horn community similarity with distance). However, at large spatial scales, we found that the AMF spore community of a mainland plot was more similar to other mainland plots several kilometers (>5) away than to nearby island plots (within 0.7 km). Likewise, most island plots were more similar to other island plots regardless of geographic separation. There was no decay in AMF species richness (number of species), or Shannon diversity (number of species and their spore numbers) either with decreasing forest-fragment size, or with decreasing plant species richness. Of the six most common species that composed almost 70% of the total spore volume, spores of Glomus tsh and G. clavisporum were more common in soils of mainland plots, while spores of Glomus small brown and Acaulospora mellea were more abundant in soils of island plots. None of these common AMF species showed significant associations with soil chemistry or plant diversity. We suggest that the convergence of common species found in AMF spore communities in soils of similar forest sizes was a result of forest fragmentation. Habitat-dependent convergence of AMF spore communities may result in differential survival of tree seedlings regenerating on islands versus mainland.