Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production.

The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.     

Influence of Carbohydrates on Growth and Sporulation of Clostridium perfringens Type A

Growth and sporulation of Clostridium perfringens type A in Duncan and Strong (DS) sporulation medium was investigated. A biphasic growth response was found to be dependent on starch concentration. Maximal levels of heat-resistant spores were formed at a starch concentration of 0.40%. Addition of glucose, maltose, or maltotriose to a sporulating culture resulted in an immediate turbidity increase, indicating that biphasic growth in DS medium may be due to such starch degradation products. Amylose and, to a lesser extent, amylopectin resulted in biphasic growth when each replaced starch in the sporulation medium. A levels of heat-resistant spores approximately equal to the control was produced with amylopectin but not amylose as the added carbohydrate. Addition of glucose or maltose to a DS medium without starch at stage II or III of sporulation did not alter the level of heat-resistant spores as compared with the level obtained in DS medium with starch. Omission of starch or glucose or maltose resulted in an approximately 100-fold decrease in the number of heat-resistant spores, although the percentage of sporulation (90%) was unaffected. The role of starch and amylopectin in the formation of heat-resistant spores probably involves the amyloytic production of utilizable short-chain glucose polymers that provide an energy source for the completion of sporulation.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Clear, defined medium for the sporulation of Clostridium perfringens.

A new, defined medium for the sporulation of Clostridium perfringens is presented. Sporulation levels exceeding 10(6) to 10(7) heat-resistant spores per ml were obtained for seven strains: PS49, PS52, FD-1, T-65, NCTC strains 8798, 8238, and 10240. In the presence of theophylline, a methylxanthine, higher levels of heat-resistant spores were attained for strains PS49, PS52, FD-1, ant T-65; photomicrographs demonstrated a higher fraction of sporulating cells when these strains were grown in the presence of methylxanthines. Use of washed, highly diluted (less than 100 cells) inocula resulted in no reduction in spore yield. Strain KA3 grew well but sporulated poorly on this medium. The medium was clear and free of precipitate when small amounts (100 microgram/ml) of methylxanthine were incorporated.     

Effect of purine derivatives, papaverine hydrochloride, and imidazole on enterotoxin formation by Clostridium perfringens type A

The percentage sporulation and enterotoxin specific activity were improved for all of five Clostridium perfringens strains, and numbers of heat-resistant spores were improved for four of five strains by replacing proteose peptone with peptone in Duncan-Strong (DS) medium. When raffinose replaced starch in DS, peptone was superior to proteose peptone in increasing percentage sporulation, numbers of heat-resistant spores, and enterotoxin formation for four of five strains. Enterotoxin levels for a strain varied when different lots of the same peptone were used. Additional experiments were conducted with three C. perfringens strains grown in DS medium with peptone. Enterotoxin specific activity was increased for three strains by adding papaverine (hydrochloride crystalline), for two strains by adding each of caffeine and 3-isobutyl-l-methylxanthine, for one strain by adding each of theophylline, 6-mercaptopurine, and 2-amino-6-mercaptopurine, and for none of the strains by adding imidazole. When enterotoxin formation was improved for a strain by one of the compounds, percentage sporulation increased, but growth decreased. Effective compounds also increased numbers of heat-resistant spores for strains H6 and R42, but slightly or not at all for strain E13. The action of these compounds was concentration dependent, with the optimal concentration differing between compounds and between strains grown in the presence of the same compound.     

Sporulation of Penicillium camemberti in submerged batch culture.

Sporulation of Penicillium camemberti was studied in submerged batch fermentation. A defined medium was used with glucose and ammonium as C- and N-sources. Temperature was set to 25 degrees C at pH 5.6. Essential for submerged sporulation was the presence of calcium (14 mM) which was adsorbed to the cell walls in all sporulating strains and inhibited mycelial growth. Acetate led to highly branched conidiophores and was the second main factor for efficient sporulation. The chelating properties of citrate were necessary for keeping calcium and phosphate in solution. Fermentation conditions allowed high spore yields after 96 h (1.6 x 10(8) spores/ml). Cyclopiazonic acid, the mycotoxin common for P. camemberti was produced during fermentation. The levels observed (0.5-4 ppm at 96 h) were strain specific and not related to spore yield.     

Improved Medium for Sporulation of Clostridium perfringens

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.     

Sporangila autolysis in Clostridium perfringens type A

The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of Clostridium prefringens was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co²⁺ (0.3 · 10⁻³ M CoCl₂) and trypsin (48 μg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N-Acetylmuramyl-l-alanine amidase activity which released N-terminal alanine, and endopeptidase activity which hydrolysed the d-alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not known if one or two enzyme are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to that of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporilated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation medium or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.     

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.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).     

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Effect of glucose on sporulation and extracellular amylase production byClostridium perfringens type A in a defined medium

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine.

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.