Effect of the ethanol content of beer on the heat resistance of a spoilage Lactobacillus

D -values of a heterofermentative beer spoilage lactobacillus were measured at 55°C, 60°C and 65°C in beers containing <0·05% to 4·4% v/v ethanol. Z -values for the different beers varied between 9·17 and 12·13°C. At each temperature an increase in ethanol reduced the measured D -value. The maximum, 5·01 min was observed in alcohol-free beer (<0·05%) at 55°C and the minimum, 0·31 min, at 60°C and 65°C in beer containing 4·4% ethanol. D -values could be increased by prior growth in the presence of ethanol. They could be reduced by adding ethanol to alcohol-free beer or by increasing its hop extract content. The implications for the pasteurization of low-alcohol beers are discussed.     

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.     

The Survival of Starter Organisms in Concentrated Suspensions

S ummary : Two representative lactic acid bacteria were grown in a rich organic medium, the cells were harvested by centrifugation, and the organisms were suspended in skimmilk at concentrations of 25–55 × 10⁹/ml. The pH was adjusted to different values and the suspensions were stored, with and without the addition of 20% (v/v) glycerol, at 4° and - 20°.
Both organisms survived better at pH 7°0 than at pH 5°0. Glycerol protected the cells at the lower pH value but offered no benefit at neutrality. At 4° about half the cells died within 5 or 6 weeks. At - 20°, however, there was no appreciable loss of viability or acid producing ability up to 9 months at pH 7°0. Suspensions stored for 9 months at - 20° produced excellent cultured buttermilk within the normal incubation period from an inoculum comparable to that used for a fresh culture. Cells harvested early in the maximum stationary phase of the growth cycle were more active than older cells, and they survived better than older cells during storage in the frozen state.     

Temperature-dependent development rates of cod Gadus morhua eggs

Temperature development relationships were determined for batches of Irish Sea cod Gadus morhua eggs incubated in flow-through incubators. Hatching began 16·4 days after fertilization (DAF) at 6° C, 10·3 DAF at 8° C, 9·4 DAF at 10° C and 7·4 DAF at 12° C. Egg mortality increased at the higher temperatures, but survival was >80%. Results were compared with published data at four comparable stage end points: the end of blastula, the end of gastrula, the point of growth of the embryo completely surrounding the yolk and the point when 50% of the eggs were hatched. All the studies showed a curvilinear relationship between age at stage and temperature. There was a 12 day inter-study difference in time to 50% hatch at 2° C and 4 day difference at 10° C. There were no consistent trends that differentiated eastern v. western, or northern v. southern populations. A single model for cod egg incubation time from fertilization to 50% hatch was derived based on data from six cod populations, but it is recommended that individual stock relationships should be used where possible.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

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 .     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Utilization of glucose, fructose and malic acid by malolactic bacteria: effect of ethanol and formation of mannitol and volatile acids

Five lactic acid bacteria capable of carrying out the malolactic fermentation were studied at 15°C and 25°C. Results indicated that at 15°C hardly any glucose, fructose and l -malic acid was utilized over a 9d period. After 9d at 25°C very little glucose and fructose and most (if not all) of the l -malic acid was degraded. The addition of ethanol markedly affected the l -malic acid utilization by the four Leuconostoc oenos strains at 25°C. Large differences in their ability to convert l -malic acid were found amongst the four strains in media containing 5% and 10%(v/v) ethanol.     

The recovery of heat-stressed Escherichia coli in lake water microcosms

C.-H. LIM AND K.P. FLINT. 1995. Escherichia coli was heat stressed at 55, 60 or 65°C in sterile flasks of lake water. After 6 h at these temperatures the viable count on nutrient agar had dropped below the limits of detection (1 colony in 100 ml). The flasks were transferred to a 15°C incubator and left for 7 d. Recovery of the stressed E. coli was shown to occur within 48 h at this temperature. Recovery also occurred in microcosms amended with 5o (v/v) synthetic sewage. The stressed E. coli multiplied in the amended but not in the unamended microcosms.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5°C and 11°C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5°C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11°C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5°C and 11°C. The DEFT count following pre-incubation correctly classified > 80% of pasteurized milks on the basis of KQ.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Effects of environmental factors on survival, growth, and production of American shad larvae

Episodic increases in temperature of 5°C above 20° C, over 48 h or declines in pH of 1·0 unit from pH 7·0 reduced survival of yolk-sac and feeding-stage larvae of American shad Alosa sapidissima . Over 16 days all measures of survival, growth, and production were more favourable at each higher temperature in the 15–25° C range. More favourable responses were also obtained at the higher prey level (500 v . 50 Artemia nauplii l^-1) and at the higher pH (7·5 v . 6·5). Combinations of high temperature and high prey levels, at pH 7·5, led to highest larval production. Little growth or production occurred at 15° C, regardless of pH or prey level. The effect of pH was strong with respect to survival, but weak with respect to growth. In attempts to restore American shad populations by larval stocking, release times and sites can be critical to optimize survival and eventual returns. Releases of larvae potentially will be most effective when made at temperatures >20° C, pH>7·0, and prey levels >50 1^-1. These conditions are most likely to occur in Maryland tributaries of Chesapeake Bay between mid-May and early June.     

Phenotypic sex differentiation of blue tilapia under constant and fluctuating thermal regimes and its adaptive and evolutionary implications

Oreochromis aureus exposed during the first 28 days of exogenous feeding to constant 35° C, or fluctuating temperatures (day at 35° C, night at 27° C, and vice versa) showed significantly ( P <0·05) faster growth, least size heterogeneity and better survival rates than siblings under constant 27° C. Constant high temperatures had a strong masculinizing effect (M: F sex ratios of 7·33–19·00: 1·00 v . 0·75–0·82: 1·00 in controls reared at 27° C). Fluctuating temperatures had less masculinizing potential but still produced sex ratios significantly skewed to the detriment of females (M: F sex ratios of 2·33–11·50: 1·00). This suggests that ambient temperature may have represented a sufficient environmental pressure for the selection of thermolabile sex-determinism in this species, and presumably in other Oreochromis spp. The evolutionary advantage of thermosensitivity in Oreochromis spp. is discussed, considering a framework where individual advantages oppose, to some degree, to the population or species interest.     

Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus . at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.     

The relative importance of temperature and diet to larval development and adult size of the winter stonefly, Soyedina carolinensis (Piecoptera: Nemouridae)

SUMMARY. 1. Soyedina carolinensis Claassen, a leaf shredding stonefly, was reared in a series of three laboratory experiments from early instar to adult on different species of deciduous leaves and at various constant and fluctuating temperature regimes.
2. Experiment 1, which involved rearing larvae on fourteen different leaf diets at ambient stream temperatures, showed that diet significantly affected larval growth and adult size but did not affect overall developmental time.
3. Experiment 2, which involved rearing larvae on five different leaf diets at each of three fluctuating temperature regimes (viz ambient White Clay Creek (WCC), ambient WCC+3°C, and ambient WCC+6°C), showed that: (i) adding 6°C to the normal temperature regime of WCC was lethal to 99% of the larvae regardless of diet; and (ii) warming WCC by 3°C did not affect developmental time but did significantly reduce adult size relative to adults reared at WCC temperatures on certain diets.
4. Experiment 3, which involved rearing larvae on five different leaf diets at each of five constant temperatures (viz 5, 10, 15, 20, 25°C), showed that: (i) temperature significantly affected the mortality, growth, and development time of larvae whereas diet only affected larval growth and mortality; (ii) temperatures at or near 10°C yielded maximum larval growth and survival for most diets; (iii) at 5°C, larval mortality was high and growth was low resulting in a few small adults for most diets; (iv) larval mortality was at or near 100% at 15°C regardless of diet; and (v) no larvae survived at 20 and 25°C.     

The microflora of soak water during natural fermentation of coarsely ground chickpea (Cicer arietinum) seeds

K. KATSABOXAKIS AND K. MALLIDIS. 1996. The microflora of soak water was studied during natural fermentation of coarsely ground chickpea seeds at 32°, 37° and 42°C. The soak water was slightly salted with NaCl 0.5% (w/v) and its proportion to sample was 10 : 1 (v/w). A gas-producing Clostridium species isolated from Reinforced Clostridium Agar (RCA) plates incubated anaerobically, was found to dominate this fermentation system particularly at the higher temperatures. Gram-negative bacteria and yeasts were not found. Bacillus species were isolated from Plate Count Agar (PCA) plates and Lactobacillus, Corynebacterium, Micrococcus and Pediococcus spp. from RCA plates were incubated aerobically. Butyric was the main acid produced at 37° and 42°C and CO₂ with hydrogen were identified as main fermentation gases.     

Cross-point determination of Penicillium conidia—Characterization of closely related fungi

The cross-points of conidia of Penicillium piceum were determined by partition in aqueous two-phase systems. No differences in cross-points or partition behaviour were registered for conidia subjected to u.v. radiation, 95°C dry heat or 120°C wet heat, compared with untreated conidia. The cultivation of fungi on various types of substrates affected the surface characteristics and thereby the cross-points of the conidia. When the method was applied to three closely related and morphologically similar isolates in the P. viridicatum group, significant differences were observed between the range of cross-points for conidia of P. crustosum, P. camembertii group II and P. camembertii group III grown on various media.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.