Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores.

The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C. Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures. Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied. More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl. The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors. At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C). However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C. Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism. If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair. If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C. Thus, repair appeared to be associated with outgrowth. Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.     

Effect of prior refrigeration on botulinal outgrowth in perishable canned cured meat when temperature abused.

Perishable canned cured meat inoculated with Clostridium botulinum spores was placed at 4.4 or 10 degrees C after manufacture. Spore germination occurred at 10 degrees C. The germinated cell count remained stable over a period of 16 to 18 weeks. During that time period the inhibitory system and residual nitrite descreased. These factors combine to make perishable canned cured meats more prone to spoilage and potential hazard if they are temperature abused after a period of refrigerated storage.     

Effect of prior refrigeration on botulinal outgrowth in perishable canned cured meat when temperature abused.

Perishable canned cured meat inoculated with Clostridium botulinum spores was placed at 4.4 or 10 degrees C after manufacture. Spore germination occurred at 10 degrees C. The germinated cell count remained stable over a period of 16 to 18 weeks. During that time period the inhibitory system and residual nitrite descreased. These factors combine to make perishable canned cured meats more prone to spoilage and potential hazard if they are temperature abused after a period of refrigerated storage.     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse.

Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated.     

Sodium nitrite and sorbic acid effects on Clostridium botulinum spore germination and total microbial growth in chicken frankfurter emulsions during temperature abuse.

Samples of (i) a control or of (ii) sodium nitrite-containing or (iii) sorbic acid-containing, mechanically deboned chicken meat frankfurter-type emulsions inoculated with Clostridium botulinum spores, or a combination of ii and iii, were temperature abuse at 27 degrees C. Spore germination and total microbial growth were followed and examined at specified times and until toxic samples were detected. The spores germinated within 3 days in both control and nitrite (20, 40 and 156 micrograms/g) treatments. Sorbic acid (0.2%) alone or in combination with nitrite (20, 40, and 156 micrograms/g) significantly (P less than 0.05) inhibited spore germinations. No significant germination was recorded until toxic samples were detected. A much longer incubation period was necessary for toxin to be formed in nitrite-sorbic acid combination treatments as contrasted with controls or nitrite and sorbic acid used individually. Total growth was not affected by the presence of nitrite, whereas sorbic acid appeared to depress it. Possible mechanisms explaining the effects of nitrite and sorbic acid on spore germination and growth are postulated.     

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.     

Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation.

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.     

Inhibition by potassium ion of the pregerminative response to L-alanine of unactivated spores of Bacillus cereus T.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur. The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination.     

Nitrite-induced germination of putefactive anaerobe 3679h spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins.

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.     

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Glutamic Acid Decarboxylase in Spores of Bacillus megaterium and Its Possible Involvement in Spore Germination

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

Influence of NaCl, NaNO2 and oxygen on the germination and growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat

The thermal resistance of Bacillus licheniformis spores was increased from a D70-value of 590 min to one of 900 min by the addition of 4% NaCl to the heating medium [tryptone-yeast extract-glucose (TYG) broth, pH 6.8], but was decreased to 470 min in TYG broth acidified to pH 4.4. Sodium nitrite (0.02%) enhanced spore destruction at 80 degrees C but not at 70 degrees C; addition of 4% NaCl eliminated this effect. Less than half the number of spores surviving heat comparable to commercial cooking were heat-damaged to the extent of being unable to grow aerobically in the presence of 4% NaCl. No growth occurred during anaerobic incubation even when the media contained no added NaCl. Oxygen was not required to trigger spore germination, but trace amounts were needed for the successful outgrowth of germinated spores. Spore germination was accelerated and enhanced by the presence of at least 2% NaCl. Therefore under anaerobic conditions NaCl promotes microbiological stability because the germinated spores cannot develop further and become moribund. It is concluded that the plastic casing of luncheon-meat chubs is not sufficiently oxygen-impermeable to allow the product a long shelf-life other than at chill temperatures unless the chubs are stored in an oxygen-free atmosphere.     

Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25 degrees C

A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated. After the first pasteurization, potatoes are vacuum-packed and stored at 25 degrees-35 degrees C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again. When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5-9 d at 25 degrees C. To provide an appropriate reduction in the risk o survival and growth of Cl. botulinum, peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F(0)3 process. If they are not given such a heat treatment they should be stored at a temperature below 4 degrees C.     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5·5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5·5, 225 mg/1 of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6·5, 225 mg/1 of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5·5, but the addition of sorbate (225 mg/1 undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.