Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Bicarbonate is a stimulus in the inter-species induced sporulation of strict anaerobic Syntrophomonas erecta subsp. sporosyntropha

Previously Syntrophomonas species had been described as the bacteria those did not form spores, however, in our previous studies, a newly isolated S. erecta subsp. sporosyntropha JCM13344^T was found to form spores in the co-culture with methanogens, while not in mono-culture or in co-culture with sulfate reducer. In this study, we examined the sporulation stimulus conferred by methanogens in the co-culture. By reducing bicarbonate in mono-culture and sulfate-reducing co-culture, we could induce S. erecta subsp. sporosyntropha JCM13344^T to form spores, so that bicarbonate at lower concentration was determined as another stimulus for sporulation. Based on the substrate degradation by strain JCM13344^T in different concentration of bicarbonate vs at different pHs, it was suggested that bicarbonate could stimulate the sporulation by mediating a nutrient deprivation but not pH drop. To further confirm the sporulation potential of this group of bacteria, spo0A fragments were amplified from strain JCM13344^T as well as all the recognized Syntrophomonas species, confirming that they were members of the spore-forming group. Since sporulation is a kind of response of spore-forming bacteria to environmental stresses, the observation in this work implies that stresses can be created even between the mutual beneficial partners, in this case, inducing sporulation.     

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A,B and F

AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum. METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus. CONCLUSIONS: Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses.

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Hypochlorite injury of Clostridium botulinum spores alters germination responses

Clostridium botulinum spores were sublethally damaged by exposure to 12 or 28 micrograms of available chlorine per ml for 2 min at 25 degrees C and pH 7.0. The damaging dose was 2.7 x 10(-6) to 3.1 x 10(-6) micrograms of available chlorine per spore. Damage was manifested by a consistent 1.6 to 2.4 log difference between the most probable number enumeration of spores (modified peptone colloid medium) and the colony count (modified peptone yeast extract glucose agar); this did not occur with control spores. Damaged spores could be enumerated by the colony count procedure. Germination responses were measured in several defined and nondefined media. Hypochlorite treatment altered the rate and extent of germination in some of the media. Calcium lactate (9 mM) permitted L-alanine (4.5 mM) germination of hypochlorite-treated spores in a medium containing 12 or 55 mM sodium bicarbonate, 0.8 mM sodium thiosulfate, and 100 mM Tris-hydrochloride (pH 7.0) buffer. Tryptose inhibited L-alanine germination of the spores. Treatments with hypochlorite and with hydrogen peroxide (7%, 25 degrees C, 2 min) caused similar enumeration and germination responses, indicating that the effect was due to a general oxidation phenomenon.     

Possible mechanisms for the revival of glutaraldehyde-treated spores of Bacillus subtilis NCTC 8236

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.     

Possible mechanisms for the revival of glutaraldehydetreated spores of Bacillus subtilis NCTC 8236

Spores of Bacillus subtilis NCTC 8236 were exposed to 2% alkaline glutaraldehyde and subsequently subjected to various treatments in an attempt to revive injured spores. Treatment with alkali (sodium or potassium hydroxide or, to a lesser extent, sodium bicarbonate) proved to be most successful. Some revival was achieved after thermal treatment. No revival was obtained with lysozyme or with various types of coat-removing agents. Experiments designed to distinguish between germination and outgrowth in the revival process established that sodium hydroxide (optimum concentration, 20 mmol/l) added to glutaraldehyde-treated spores increased the potential for germination. In contrast, spores which had been allowed to germinate before exposure to low concentrations of glutaraldehyde and then to sodium hydroxide were inhibited at the outgrowth phase to a much greater extent than germinated spores treated with the dialdehyde without subsequent alkali exposure. The results overall are discussed in terms of the possible mechanism and site of action of glutaraldehyde and the practical implications and significance of its use as a sporicide.     

Metabolic Activities of Isolated Spores of Anabaena cylindrica

A method for the isolation of spores of Anabaena cylindricais described, and results concerning the metabolic activitiesof isolated spores are presented. Isolated spores fixed carbondioxide in the light at a lower rate and evolved carbon dioxidein the dark at a higher rate than the corresponding whole filaments.Nitrogen-fixing ability in isolated spores could be detectedneither with the isotopic method nor with the new acetylenetechnique. Isolated spores were able to germinate at the samerate as spores within the original cell population when transferredinto fresh culture medium.     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.     

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).     

Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces vulgaris. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of S. albus were 10 to 50 times lower than those of T. vulgaris, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

Aziqne letale e azione mutagena della metil β β′ dicloroetilammina su spore di Aspergillus niger

Summary

It is shown that the lethal action of nitrogen mustard on spores of Aspergillus niger can be described in terms of the target theory, if one assumes that the intensity of this action varies with time. A two-hit curve can be fitted on the experimental data, on the assumption that a decrease of lethality depends on the inactivation of the substance with time.

By diluting the suspension containing the spores with a solution of glycine and sodium bicarbonate, the action of nitrogen mustard is stopped. The lethal action of nitrogen mustard is much weaker at 0° C than at 27° C.

Even though exact mutation rates have not been evaluated, an outstanding number of morphological and physiological mutants among the colonies developing from irradiated spores have been observed.     

Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica

Nystatin was used to develop a new method to select spores of the yeast Saccharomycopsis lipolytica. At low concentrations nystatin killed preferently growing cells of this yeast. At high concentrations nongrowing cells were affected as well. In contrast, spores were not sensitive to nystatin action. This differential response to the antibiotic suggested its use to select spores from sporulated yeast cultures.     

“Spore Plate Method” for Transformation of Steroids by Fungal Spores Entrapped in Silica Gel G

A new technique for investigating steroid biotransformations involving the use of glucose-treated Silica Gel G thin-layer chromatography plates spotted with fungal spores and steroid substrates is described. The conversion is followed by the detection and identification of steroid metabolites and is carried out on single plates by using the spores of different fungi. During the entire process, the spores remain on the original spots and microscopical examination revealed no germination. The method was successfully applied to as little as 30 μg of substrates, and a single plate could be used to detect the steroid metabolizing activity of spores of as many as 15 different cultures.     

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.     

"Spore plate method" for transformation of steroids by fungal spores entrapped in silica gel g

A new technique for investigating steroid biotransformations involving the use of glucose-treated Silica Gel G thin-layer chromatography plates spotted with fungal spores and steroid substrates is described. The conversion is followed by the detection and identification of steroid metabolites and is carried out on single plates by using the spores of different fungi. During the entire process, the spores remain on the original spots and microscopical examination revealed no germination. The method was successfully applied to as little as 30 μg of substrates, and a single plate could be used to detect the steroid metabolizing activity of spores of as many as 15 different cultures.     

Determination of the heat resistance of spores using a solid heating block system

A simple and accurate technique for the determination of the heat resistance of spores is described. The technique combines a modified capillary tube method with a solid heating block. The come-up time of spore suspensions was found to be short and simple and accurate technique is suggested for the correction of the come-up times. Experimental results are presented for the destruction of spores of Bacillus stearothermophilus at 120 which indicates the accuracy and reproducibility of the new method.     

Determination of the heat resistance of spores using a solid heating block system

A simple and accurate technique for the determination of the heat resistance of spores is described. The technique combines a modified capillary tube method with a solid heating block. The come-up time of spore suspensions was found to be short and simple and accurate technique is suggested for the correction of the come-up times. Experimental results are presented for the destruction of spores of Bacillus stearothermophilus at 120 degrees which indicates the accuracy and reproducibility of the new method.