Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

The formation of spores in biofilms of Anoxybacillus flavithermus

Aims:  To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable – temperature – on spore formation.
Methods and Results:  A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8·5 h period at three different temperatures of 48, 55 and 60°C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6–8 h. Spores formed at 55 and 60°C and amounted to approx. 10–50% of the biofilm. No spores formed at 48°C.
Conclusions:  The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores.
Significance and Impact of the Study:  This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus .     

Properties and attempted culture of Pasteuria penetrans, a bacterial parasite of root-knot nematode (Meloidogyne javanica)

When they were subjected to a range of physical and chemical treatments, spores of Pasteuria penetrans showed properties similar to those of other endospore-forming bacteria. The spores did not take up some stains, were resistant to desiccation and sonication and showed extrusion of spore contents ('spore popping') on prolonged exposure to 0.1% KMnO₄ in 0.3 n HNO₃. Calcium and dipicolinic acid (DPA) were present at concentrations of 0.28% and 0.96% of the spore dry weight respectively, giving a Ca: DPA molar ratio of 1.2. The infectivity of P. penetrans spores was reduced to a low level after heating at 100°C for 5 min, but spore attachment was not markedly affected by heating at 100°C for 15 min. Evidence for the presence of catalase in P. penetrans spores was equivocal because the low levels of catalase activity observed in spore suspensions may have been due to contamination from catalase-positive nematode tissue. When P. penetrans spores were exposed to a range of substances known to act as germinants for spores of Bacillus spp., germination or loss of refractility was not observed by phase microscopy. In vitro culture of P. penetrans was attempted by inoculating either spores or vegetative mycelial bodies onto a diverse range of simple and complex media and incubating them in aerobic, reduced oxygen, anaerobic and increased CO₂ environments. Signs of spore germination or growth of vegetative stages were never observed.     

The Behaviour of a Food Poisoning Strain of Clostridium welchii in Beef

S ummary : An inoculum of 10⁵ spores of Clostridium welchii F2985/50 in meat survived steaming at 100° for 5 h, the number being reduced sevenfold for every hour of steaming. They also survived for at least 6 months in frozen meat stored at -5° and -20°, whereas vegetative cells died more rapidly at -5° than at -20°. In beef stored for 13 days at 1°, 5°, 10° and 15° there was no multiplication but a slow destruction of vegetative cells, but there was little change in the spore count. Slow multiplication occurred at 20° but at 25° and 37° growth was rapid. Only about 3% of the spores germinated without prior heat shock, so the majority failed to germinate in raw meat stored at any temperature, but did so once the meat had been heated. In meat which had been heated and allowed to cool almost all of the spores had lost their heat resistance.
It was found that the minimal growth temperature was related to pH and medium, so that meat with a pH higher than that used in these experiments (pH 5°7–5°8) would probably have a lower minimal growth temperature for these organisms and would thus be more susceptible to spoilage.     

Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates

The effect of continuous (689 MPa with holding times of 5, 15 or 25 min) and oscillatory (one, three or five cycles at 689 MPa with holding times of 1 s) high hydrostatic pressure treatments on the viability of Byssochlamys nivea ascospores suspended in apple and cranberry juice concentrates adjusted by dilution to water activities (a_w) of 0·98 and 0·94 was evaluated at 21 and 60 °C. Inactivation of the initial spore inocula was achieved after three or five cycles of oscillatory pressurization at 60 °C when the a_w was 0·98 in both fruit juices. With a_w 0·94, the initial inocula were reduced by less than 1 log-cycle after five pressure cycles. Inactivation was not observed within 25 min with continuous pressurization at 60 °C. In treatments at 21 °C, no effect on spore viability was observed with continuous or oscillatory treatments.     

A cotyledon screen for resistance to scab (Cladosporium cucumerinum) in cucumber (Cucumis sativus) seedlings

An efficient protocol for differentiating cucumber (Cucumis sativus) seedlings resistant and susceptible to scab (Cladosporium cucumerinum) has been developed by examining the interacting factors of spore density and cotyledon growth stage. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 1 day after emergence by placing a 5 μl inoculum droplet (2 × 10⁶ spores/ml) on the newly expanded cotyledons. Seedlings were incubated in a darkened dew chamber for 48 h at 20°C and 100% r.h., and then grown for 6 days at 20°C. Plants could be rated as resistant or susceptible 8 days after inoculation.     

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 D ₇₀-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°C but not at 70°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.     

Tailing of Survival Curves of Bacillus licheniformis Spores Treated with Hydrogen Peroxide

An attempt has been made to rule out possible causes of artefacts in establishing survival curves of Bacillus licheniformis spores heated (30–80 °C) in 4.4 mol/l hydrogen peroxide (pH 2.0). A tailing phenomenon apparent as that of a suspension of spores produced by routine subculture was obtained with those grown-up from a single spore selected by micromanipulation. No spore fraction differing in size or density could be separated from the whole population. The tail was not due to decomposition of hydrogen peroxide, protective effect by other spores, release of protective factors, or temperature heterogeneity during treatment. Changing from an open vessel to a closed tube did not influence the tailing. The only apparent artefact was therefore the formation of clumps under the conditions of the treatment. Since the spore catalase was demonstrated to be highly resistant, it was concluded that a spore could be protected against hydrogen peroxide by the catalase of the other spores in the clump. Conditions resembling those arising in spore suspensions could occur under industrial conditions, for example in sterilizing surfaces contaminated with aggregates of Bacillus spores.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

A note on the effect of storage on the chemical resistance of spores of Bacillus subtilis SA22 and Bacillus subtilis globigii B17

Spores of Bacillus subtilis SA22, harvested from nutrient agar after 9 d at 30°C and stored in distilled water at 4°C, were unaltered in their resistance to 17.7% hydrogen peroxide or 0.04% peracetic acid after storage for up to 134 weeks. Three spore crops of B. subtilis globigii were unaffected by storage for up to 134 weeks with respect to 17.7% hydrogen peroxide resistance but were significantly more resistant to 0.04% peracetic acid following storage.     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

THE INCIDENCE AND THERMAL RESISTANCE OF MESOPHILIC SPORES FOUND IN MILK AND RELATED ENVIRONMENTS

SUMMARY: A spore 'spectrum' is described of aerobic mesophiles capable of resisting different heat treatments. It is shown that B. licheniformis is the most common spore former found in bulk milk but since its spores are rapidly destroyed at 100°, the more heat resistant B. subtilis is the dominant surviving spore former in commercial sterilized milk. The thermal resistance of strains of B. subtilis and B. licheniformis isolated from different sources has been investigated and the strains of B. subtilis typed according to the behaviour of their spores when heated at 100°. All strains of B. licheniformis were destroyed more rapidly by boiling for 2 min than strains of B. subtilis but only those strains of the latter which showed some degree of heat activation were more resistant than B. licheniformis . The 'resistant' and heat activated strains of B. subtilis appear to be sparsely distributed in nature and were only isolated from sterilized milk where the heat treatment applied would tend to eliminate other strains. The spore content of bovine faeces was similar to that in bulk milk and the total spore content varied seasonally, the spore content of faeces being on the average a hundred times greater during indoor feeding than during the period when the cattle were fed outside. A faecal infection of the milk in the ratio of 1:10⁴ would infect the milk with spores at about the same concentration as they are found in bulk raw milk, and it is suggested that bovine faeces could be a primary source of spore formers in milk supplies.     

Laboratory studies on food intake, growth and food conversion of young herring, Clupea harengus (L.)

Food intake, growth and food conversion of young, O-group herring were studied at two temperatures and feeding regimes over a period of 19 weeks. The food intake of fish fed to satiation twice daily showed considerable variation. Food intake per fish at 14.5° C was about three times that at 6.5° C, and was generally much higher than in most other species of fish studied. The mean increase in wet weight over the 19-week period was 0.581 g/week at 14.5° C and 0.236 g/week at 6.5° C in fish fed to satiation and 0.094 g/week at 6.2° C and a ration of 1.3% of the body weight. Growth depensation was found to occur even in fish fed to satiation. The changes in specific growth rate, that is the percentage increase in weight/day, showed similar trends at different temperatures and food regimes. The mean conversion efficiency of fish on a ration of 1.3% at 6.2° C was higher than that of fish fed until satiation, at 14.5 and 6.5° C. The conversion efficiency of fish fed to satiation at 14.5° C showed a distinct decrease with increasing weight while at 6.5° C such a clear trend was not observed. In general, the conversion efficiency of young herring were found to be much lower than that of most other species studied. The weight exponent of the quantitative relationship between food intake and body weight at 14.5° C was 0.744. The total metabolic expenditure at 14.5° C, calculated using Winberg's (1956) 'utilization coefficient, gave a weight exponent of 0.773.     

The effect of sodium chloride and temperature on the rate and extent of growth of Clostridium botulinum type A in pasteurized pork slurry

A selective medium was used to enumerate Clostridium botulinum growing in the presence of natural spoilage organisms in a model cured pork slurry. The growth responses of a mixed spore inoculum of six strains of Cl. botulinum type A were studied at 15°, 20° and 27°C with 1˙5, 2˙5, 3˙5 or 4˙5% (w/v) salt added (a_w range 0961–0990). Gompertz and logistic curves, which have a sigmoid shape, were fitted to the data and lag times, growth rates, generation times and time to maximum growth rates were derived. Variation in germination rates of the spores occasionally gave a falsely extended lag time resulting in an exceptionally high estimate for growth rate. Products containing 4˙5% (w/v) NaCl would be capable of supporting growth of proteolytic strains of Cl. botulinum , even at 15°C, although the lag period would be extended. In products where absence of Cl. botulinum cannot be assured additional preservative measures are essential. The information obtained provides a framework to investigate the effects of a wider range of additives or variables on the growth responses of Cl. botulinum .     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

Cell-type Conversion at Low Temperature in Dictyostelium discoideum

The effects of low temperature (5°C) on cell-type conversion in whole slugs of Dictyostelium discoideum and their anterior prestalk- and posterior prespore-isolates were examined immunohistochemically and electronmicroscopically. When slugs were incubated for nine days at 5°C, the proportion of cells containing spore-antigens increased from about 75% to 85%. More important, the proportion of prespore and spore cells increased from about 3% to 40% in anterior prestalk isolates incubated at 5°C for 12 days, but no cell-type conversion from prespore to prestalk cells occurred in posterior prespore isolates. Therefore, the mechanism regulating the proportions of cells that operates at 21 °C does not operate at low temperature. The cells with full competence for stalk differentiation could change into stalk cells even at 5°C, because a short stalk was always formed when early culminants were transferred to low temperature. The effects of low temperature on several sequential steps of cell differentiation are discussed on the basis of these findings. The ultrastructural characteristics during the process of cell-type conversion are also described.     

THE GERMINATION AND ENZYMIC ACTIVITIES OF BACILLUS SPORES AT LOW TEMPERATURES

SUMMARY: Well washed spore preparations of Bacillus cereus and B. subtilis were suspended in various nutrient broths, soil extracts, autoclaved soil of various moisture contents, and in two inorganic solutions, phosphate buffer, pH 7·2 and Ringer's solution. These were incubated at 8°, 5°, 1° and 0° for periods up to 270 days. Periodic total and spore counts on plates indicated a progressive decrease in each, associated with germination taking place in all conditions except in the two inorganic media. Enzymic tests indicated secretion and activity of nitratase and gelatinase and, with spores of B. pasteurii , urease, as a result of germination. B. subtilis germinated to a greater extent than B. cereus in each of the nutrient media. Germination of both organisms at 5° was also observed in L - and D -alanine: in the latter it was probably the result of racemization to the L -form.