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.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

Ultrastructural and Chemical Changes in Spores of Bacillus cereus after Action of Disinfectants

Spores of Bacillus cereus subjected to 5% NaOH at 50° and 0.25% NaOCl at 20° undergo a 99% loss in viability in 11 and 19.5 min, respectively. Kinetic studies show that the release of Ca²⁺ and dipicolinic acid occur at different rates and that extensive loss of both ribonucleic and deoxyribonucleic acids occur under these conditions of disinfection. Electron micrographic studies reveal a progressive degradation of the spore integuments. Both chemical and ultrastructural observations made in this study lead to the conclusion that the primary lethal effect of either disinfectant is the modification of outer spore coats leading to a disruption of normal permeability barriers.     

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.     

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 .     

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.     

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 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.     

Factors affecting the germination of spores of Bacillus anthracis

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor O-carbamyl-D-serine was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated optimally at 22°C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germination rate using sera from those animals as the germination medium.     

The effect of temperature on the germination of single spores of Clostridium botulinum 62A

Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability). The experiment was repeated at five different temperatures between 20°C and 37°C to look at the effect of temperature on the biovariability of the spore germination. Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature. The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g. permeabilities) or molecules (e.g. lytic enzymes) in the spore population. The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influenced by different environmental factors such as temperature and other factors that influence germination.     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

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.     

Isolation and properties of psychrotrophic and psychrophilic, pectolytic strains of Clostridium

Fourteen strains of pectolytic clostridia have been isolated that were capable of growth at 5–10°C in 7 d; two strains were psychrophiles and failed to grow at 20°C in 14 d and the remainder were psychrotrophs. The bacteria formed pectate lyase enzymes and were capable of degrading potato tissue; they are therefore a potential cause of soft rot of potatoes stored at low temperatures. Doubling times for representative strains were 15–19 h at 10°C and 21–53 h at 5°C. Twelve strains were classified with Group I Clostridium species and two strains with Group II. In the case of one strain the mature spores were not released from the sporangium. Electron microscopy of ultrathin sections of this strain showed the presence of disorganized lamellar structures associated with the spore coat.     

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.     

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.     

Effects of high hydrostatic pressure on Clostridium sporogenes spores

Spores of Clostridium sporogenes were found to be resistant to ultra high pressure, with treatments of 600 MPa for 30 min at 20 °C causing no significant inactivation. Combination treatments including heat and pressure applied simultaneously (e.g. 400 MPa at 60 °C for 30 min) or sequentially (e.g. 80 °C for 10 min followed by 400 MPa for 30 min) proved more effective at inactivating spores. Pressure cycling (e.g. 60 MPa followed by 400 MPa at 60 °C) also reduced spore numbers. Overall, these pressure treatments resulted in less than a 3 log reduction, and it was concluded that the spores could not be inactivated by pressure alone. This could indicate that for the effective inactivation of bacterial spores, high pressure technology may have to be used in combination with other preservation methods.     

The sporicidal activity and inactivation of chlorhexidine gluconate in aqueous and alcoholic solution

The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range. Activity was not observed at 20°C even with concentrations as high as 10% chlorhexidine. Temperatures of 37°–70°C in combination with such high concentrations were required for reductions in spore viability. No viable spores were recoverable after 4 h contact at 55°C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution. Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results. The spores of other Bacillus species examined ( B. cereus, B. megaterium and B. stearothermophilus ) proved to be considerably less resistant than those of B. subtilis. Presence of organic matter had little effect on the activity.     

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.     

Effect of temperature and sex on growth patterns in nymphs of the mayfly Hexagenia hilineata in the laboratory

SUMMARY. Eggs collected from Hexagenia bilineata females were successfully reared in the laboratory at temperatures of 15, 20, 25 and 30°C. Eggs did not hatch at 10°C and although hatching was successful at 35°C, all nymphs at this temperature died while in early instars.
Survival of nymphs between the approximate size interval of 4–14 mm showed a significant decrease with increased temperatures. Nymphs at 15°C, however, generally did not survive transformation to the subaduit stage.
The growth pattern of individual nymphs was well described by a logistic curve at most temperatures. Furthermore, growth pattern was significantly affected by both temperature and sex.
Rate of development from oviposition to first emergence increased with increasing temperatures in a linear fashion between 15 and 30°C. The relationship was equally well described by a hyperbolic equation and a power-law equation. By extrapolation from the hyperbolic equation, the lower threshold temperature for development was estimated to be 10.1°C3.1°C. The degree (°C)-days required for development from oviposition to first emergence was calculated to be 2337 days with 95% confidence limits of 2045–2727 days under laboratory conditions.