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).     

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.     

Alternative medium for Clostridium perfringens sporulation.

A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains. Comparatively this medium gave a greater percentage of sporulation than five other media described previously.     

Effect of metal ions on growth and sporulation of Clostridium perfringens in a synthetic medium.

All cells, whatever their origin, function in an essentially inorganic environment. In this environment, metal cations play an important role. Attempts were made in the present study to determine if different amounts of five metal ions (MG++, Fe++, Mn++, Zn++, Ca++) are needed for secondary metabolism of Clostridium perfringens than are needed for primary metabolism. Both the vegetative growth stage (primary metabolic stage) and spore stage (secondary metabolic stage) of Clostridium perfringens were studied. Endeavors were made to detect the effects of metal ions, if any, on growth and sporulation of the organisms. Mg++ was required for growth of vegetative cells and maintenance of normal cellular morphology, Fe++ was needed for sporulation as well as for vegetative growth, but the amount needed for spore formation was higher than that needed for growth. Ca++ was essential to refractility and heat-resistance of spores. Zn++ inhibited both growth and sporulation, if the Mg++ concentration was low. Mn++ was required for neither growth nor sporulation. For maximum heat-resistance of the spores, Ca++ plus unidentified organic substances were required.     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium.

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Endogenous radiolabeling of enterotoxin from Clostridium perfringens type A on a defined medium.

Four enterotoxin-positive strains of Clostridium perfringens were tested for sporulation and enterotoxin production on defined media. The medium described by Sacks and Thompson (Appl. Environ. Microbiol. 35:405-409, 1978) gave the highest enterotoxin production and was selected for the production of endogenously labeled enterotoxin. The specific radioactivity of the enterotoxin was 16,000 dpm/microgram when the tritiated amino acids were added to the growth medium just before the inoculum. Addition of the radioactive amino acids during the growth period gave consistently lower specific radioactivity. When the enterotoxin was produced on the medium described by Duncan and Strong (Appl. Microbiol. 16:82-89, 1968), the highest specific radioactivity of the enterotoxin was found when the radioactive amino acids were added to the growth medium 4 h after inoculation. In this case, the specific activity of the enterotoxin was 10,000 dpm/microgram.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Endogenous radiolabeling of enterotoxin from Clostridium perfringens type A on a defined medium

Four enterotoxin-positive strains of Clostridium perfringens were tested for sporulation and enterotoxin production on defined media. The medium described by Sacks and Thompson (Appl. Environ. Microbiol. 35:405-409, 1978) gave the highest enterotoxin production and was selected for the production of endogenously labeled enterotoxin. The specific radioactivity of the enterotoxin was 16,000 dpm/microgram when the tritiated amino acids were added to the growth medium just before the inoculum. Addition of the radioactive amino acids during the growth period gave consistently lower specific radioactivity. When the enterotoxin was produced on the medium described by Duncan and Strong (Appl. Microbiol. 16:82-89, 1968), the highest specific radioactivity of the enterotoxin was found when the radioactive amino acids were added to the growth medium 4 h after inoculation. In this case, the specific activity of the enterotoxin was 10,000 dpm/microgram.     

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.     

Enterotoxic strains of Clostridium perfringens and sporulation

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.     

Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.     

Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.     

Stimulation of growth and sporulation of Clostridium perfringens by its homologous enterotoxin

C. perfringens enterotoxin shortened the lag phase and time of onset of sporulation of the same organism in a dose-dependent manner. The toxin stimulated macromolecular synthesis of pre-exponential phase cells.     

Comparison of sulfite-polymyxin-sulfadiazine medium and tryptose-sulfite-cycloserine medium without egg yolk for recovering Clostridium perfringens.

The overall recoveries of spores and of actively growing, heat-stressed, coldshocked, and frozen cells of five strains of Clostridium perfringens were significantly greater (95% confidence limits) on tryptose-sulfite-cycloserine medium without egg yolk than on sulfite-polymyxin-sulfadiazine medium.     

High yields of coatless spores of Clostridium perfringens strain 8--6 in a defined medium.

Coatless spores of mutant strain 8--6 of Clostridium perfringens were formed reproducibly at levels exceeding 10(7)/mL in a defined medium. Confirming previous observations, the spores are quite stable in water and resistant to heat, octanol, and ethanol. They also resist 5% phenol. Electron microscopy confirms, with further evidence, the probable absence of any structure exterior to the cortex. The levels of sporulation attained are sufficient to permit recovery and purification for future studies on physicochemical and germination properties. The defined medium makes possible studies on spore coat (and exterotoxin) biosynthesis and coat assembly.     

Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, l-alanine, and a combination of these two germinants.