Design of a Temperature Gradient Incubator

S ummary . A temperature gradient incubator heated and cooled by circulating liquids is described which is suitable for incubating micro-organisms in liquid media and on solid media in roll tubes. The maximum deviation from a linear temperature gradient is 0·35% of the range (0·13°) even when used at temperatures up to 30° above ambient. The temperature of any set of sample wells is uniform to within 0·07° (3·5% of the difference between adjacent rows).     

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.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH ≥ 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12° and 16°C and pH values between 5·2 and 5·5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5·5 and incubated at 16°C, corresponding to a probability of growth from a single spore within 14 d of 1·6 × 10^-5. The probability of growth in 28 d in the remaining conditions was <9 × 10^-6. After transfer of inoculated media from 12° to 30°C growth occurred at pH 5·2–5·5 within 19 d. After transfer of inoculated media from 12° to 20°C growth occurred at pH 5·5 and 5·4 but not at pH 5·3 or 5·2 in 40 d. Growth at pH 5·2–5·5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

Rate of energy depletion and overwintering mortality of juvenile walleye pollock in cold water

The winter energy deficit and mortality of juvenile walleye pollock at extremely cold temperature were examined by field observations and laboratory experiments. In the Doto area, along the northern coast of Japan, juvenile walleye pollock resided on the continental shelf despite extremely cold temperatures (mean 0·4° C) during the latter half of winter (March to April). Measurements of the rate of energy depletion (equivalent to the routine metabolic rate) revealed that juvenile walleye pollock consumed 37% less energy at 0·5° C than at 2·0° C, suggesting an energetic benefit of residence in cold water (<1·0° C) over the shelf during winter. Prior to the starvation experiments, temperatures and ration level in the holding tanks were adjusted to create two different body condition groups of fish. Under the thermal condition of the latter half of winter (0·5° C), fish with a mean condition factor of 0·6 and 0·5 suffered 19·1 and 74·5% mortality, respectively, at the end of the experiments (after 56 days). The residual analysis of total body energy demonstrated that the cause of mortality was mainly associated with the depletion of energy reserves. When a logistic regression model for mortality derived from the experiments was applied to wild fish collected in March, the estimated overwintering mortality in 2004 and 2005 was 25·4 and <2·3%, respectively, assuming no feeding during the winter. Considering that juvenile walleye pollock feed during winter as shown in previous studies, intense overwintering mortality induced by energy depletion is improbable during the latter half of winter in the Doto area.     

Early development and growth of the laboratory reared north-east Atlantic mackerel Scomber scombrus L.

The early development, growth and morphological changes of mackerel Scomber scombrus were investigated at different incubation temperatures (8, 10, 13, 15 and 18° C). Details on the early life history are illustrated with special reference to morphological transformations. Culture techniques to rear larval mackerel stages are described using laboratory cultured foods. Artificially fertilized eggs were hatched after 80·6 h at 18·4° C and 256·8 h at 8·7° C. The standard length ( L _S) of the individuals at first feeding was 4·71 ± 0·18 mm. Four mortality critical periods and cannibalistic behaviour were identified. A maximum average larval size of 37·5 ± 4·41 mm L _S was attained 30 days post-hatch (dph) at 18·4° C. Development and growth were affected significantly by temperature during both endogenous and exogenous feeding periods. Larvae grew more rapidly at high, than at low temperatures. Daily specific growth rate (in mass) ranged from 2·4% at 10·6° C to 16·9% at 18·4° C. Likewise, average growth rate (in length) ranged from 0·05 mm day⁻¹ at 8·4° C to 0·37 mm day⁻¹ at 18·4° C. The allometric relationship of L _S, with several body measurements was not affected by temperature. Comparison with larvae collected in the Bay of Biscay did not show any significant difference in the dry mass and L _S relationship; conversely, the growth rate in length differed significantly between both laboratory and field conditions. The trends observed in the laboratory are described in relation to some aspects of the year-class strength regulation.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Thermally induced phenotypic plasticity of swimming performance in European sea bass Dicentrarchus labrax juveniles

The vulnerability of embryonic and larval stages of European sea bass Dicentrarchus labrax to environmental temperature and the longer-term consequences for the early juveniles was demonstrated. This phenotypic plasticity was highlighted by subjecting D. labrax at 15·2 ± 0·3 or 20·0 ± 0·4° C (mean ± s . d .) up to metamorphosis and then at the same temperature (18·5 ± 0·7° C). After 4–6 weeks at the same temperature, the measurement of critical swimming speed at four exercise temperatures (15, 20, 25 and 28° C) showed a significantly higher swimming capacity in the fish initially reared at 15° C than for fish initially reared at 20° C. This performance was correlated with significant differences in the phenotype of red muscle. Thermally induced phenotypic plasticity was clearly demonstrated as an important mechanism controlling swimming performance in early juveniles of D. labrax .     

The critical thermal limits for juvenile Arctic charr Salvelinus alpinus

The chief objective was to determine the critical thermal limits for alevins, fry and parr of Arctic charr, Salvelinus alpinus , (L.) from four races living in Windermere (northwest England). The experimental fish were reared in a hatchery but were the progeny of wild parents. As comparisons between tethal temperatures at four acclimation temperatures (5, 10, 15, 20° C) revealed few significant racial differences, the data were pooled to estimate the lethal values for survival over 7 days (incipient lethal temperature) and over only 10 min (ultimate lethal temperature) for each life stage. Upper lethal values increased with acclimation temperatures for alevins but this effect was negligible for fry and parr, Alevins were generally less tolerant than fry and parr at lower, but not higher, acclimation temperatures; e.g. after acclimation at 5° C, mean upper ultimate values were 23·3, 25·1 and 25·7° C and mean upper incipient values were 18·7, 21·5 and 21·5° C for alevins, fry and parr respectively; after acclimation at 20° C, mean upper ultimate and incipient values were 26·2, 26·1 and 26·6° C and 20·8, 20·8 and 21·6° C for alevins, fry and parr respectively. The area of the temperature tolerance polygon (expressed as ° C²) for juvenile Arctic charr is amongst the lowest recorded for salmonids; being 409, 439 and 461° C² for alevins, fry and parr respectively. These low values are due to lower upper tolerance limits, not high lower tolerance limits; the latter being close to 0° C (<1°C for parr and fry, <0·3° C for alevins) at all acclimation temperatures. Arctic charr are therefore amongst the least resistant of salmonids to high temperatures but probably the most resistant to low temperatures.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Thermal effects on growth and time to starvation during the yolk-sac larval period of Atlantic mackerel Scomber scombrus L.

The effect of incubation temperature (8·6, 11·1, 13·2, 15·1 and 16·8° C) on north-east Atlantic mackerel Scomber scombrus development, growth and age at starvation during the yolk-sac larval period was investigated. Standard length at hatch was found to be inversely proportional to incubation temperatures within the natural thermal ranges of this species; it ranged from 3·76 mm at 11·1° C to 3·30 mm at 17·8° C. Following hatch, however, larval growth rate was positively related to temperature. Individual logistic models, as a function of temperature and age, were fitted to the development processes of gape, eye pigmentation, jaw mobility and yolk exhaustion. Thereafter, development was classified into different ordered stages and an extended continuation model was fitted to the multinomial ordered stage classification. In all cases, there was a difference of >23 h between the first and the last individual developing in certain stage. The probability of survival decreased with age and was inversely related to temperature. Yolk utilization varied from 4·5 to 8·6 days and individuals died between 7·9 and 12·2 days from 17·8 to 11·1° C. The study demonstrated the significant impact that temperature has on development, growth and survival rates, during the early life history.     

Effects of temperature on developmental performance, survival and growth of sea lamprey embryos

This study assesses the influence of thermal regime on the development, survival rates and early growth of embryos of sea lamprey Petromyzon marinus incubated at five constant temperatures (7, 11, 15, 19 and 23° C). The time from fertilization to 50% hatching and from hatching to 50% burrowing were inversely related to incubation temperature. All the embryos incubated at 7° C died at very early stages, while those maintained at 11° C did not attain the burrowing stage. Survival from fertilization to hatching was 61, 89, 91 and 89% at 11, 15, 19 and 23° C, decreasing to 58, 70 and 70% from hatching to burrowing at 15, 19 and 23° C, respectively. Larvae reared during the first 3 months of exogenous feeding in a common environment at constant 21° C, revealed maximum survival for an incubation temperature of 15° C (43% of burrowed larvae) decreasing strongly at 19° C (16%) and 23° C (one suvivor among 240 larvae). Body length at the burrowing stage was maximum for embryos incubated at 19° C, but body mass increased in the interval 15–23° C. Mean incubation temperatures experienced by 117 broods during the embryonic development in the source river were estimated in 15·3±2·30° C and 16·7±1·76° C (mean±1 s.d .) for the periods fertilization-to-hatching and hatching-to burrowing, respectively.     

Changes in the acidity and lactic acid content of 'Nono', a Nigerian cultured milk product

Changes in the pH value and lactic acid content of the uninoculated, cultured Nigerian whole milk product, 'Nono', during incubation at room temperature (27°± 2°C) for 120 h were monitored. The pH decreased from 6·78 ± 0·19 to 4·30 ± 0·11 while the lactic acid content increased from 0·32 ± 0·04% to 2·50 ± 0·02% (w/v). This was accompanied by souring and curdling of the milk particles.     

The effect of temperature and fish size on growth and feed efficiency ratio of juvenile spotted wolffish Anarhichas minor

The growth properties of juvenile spotted wolffish Anarhichas minor reared at 4, 6, 8 and 12° C, and a group reared under 'temperature steps', (T‐step) i.e . with temperature reduced successively from 12 to 9 and 6° C were investigated. Growth rate and feed efficiency ration was significantly influenced by temperature and fish size. Overall growth rate was highest at 6° C (0·68% day⁻¹) and lowest at 12° C (0·48% day⁻¹), while the 4 and 8° C, and the T‐step groups had similar overall growth rates, i.e . 0·59, 0·62 and 0·51% day⁻¹ respectively. Optimal temperature for growth ( T _{opt G} ) and feed efficiency ratio (T_{opt FCE}) decreased as fish size increased, indicating an ontogenetic reduction in T _{opt G} and T _{opt FCE}. The results suggest a T _{opt G} of juvenile spotted wolffish in the size range 135–380 g, dropping from 7·9° C for 130–135 g to 6·6° C for 360–380 g juveniles. The T _{opt FCE} dropped from 7·4° C for 120–150 g to 6·5° C for 300–380 g juveniles. A wider parabolic regression curve between growth, feed efficiency ratio and temperature as fish size increased, may indicate increased temperature tolerance with size. Individual growth rates varied greatly at all time periods within the experimental temperatures, but at the same time significant size rank correlations were maintained and this may indicate stable size hierarchies in juvenile spotted wolffish.     

Effects of temperature, body size and feeding on rates of metabolism in young‐of‐the‐year haddock

The mean rate of oxygen consumption (routine respiration rate, R _R, mg O₂ fish⁻¹ h⁻¹), measured for individual or small groups of haddock Melanogrammus aeglefinus (3–12 cm standard length, L _S) maintained for 5 days within flow‐through respiratory chambers at four different temperatures, increased with increasing dry mass ( M _D). The relationship between R _R and M _D was allometric ( R _R = α  M ^b ) with b values of 0·631, 0·606, 0·655 and 0·650 at 5·0, 8·0, 12·0 and 15·0° C, respectively. The effect of temperature ( T ) and M _D on mean R _R was described by     indicating a Q ₁₀ of 2·27 between 5 and 15° C. Juvenile haddock routine metabolic scope, calculated as the ratio of the mean of highest and lowest deciles of R _R measured in each chamber, significantly decreased with temperature such that the routine scope at 15° C was half that at 5° C. The cost of feeding ( R _SDA) was c . 3% of consumed food energy, a value half that found for larger gadoid juveniles and adults.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

Upper thermal limits for feeding and growth of 0+ Arctic charr

When Arctic charr Salvelinus alpinus from two diVerent stocks were fed live Neomysis integer , the upper thermal limits for feeding and growth were established in the range 21·5–21·8° C. These critical temperatures might have been underestimates, because fish tend to show increased sensitivity to handling at high experimental temperatures. In the second experiment, the proportion of feeding undisturbed charr from four stocks decreased initially as temperature was raised in steps from 18 to 22° C. At the lower temperatures, 18 and 20) C, almost all fish resumed feeding, but the recovery time was longer and more fish ceased to feed at 20) C than at 18° C. When the temperature was increased to 21° C, 50% of the fish ceased feeding permanently, and all fish ceased feeding within 2 days at 22° C. It is concluded that 0+ charr cease to feed and grow at c .21·5) C and that the critical temperatures for feeding and growth coincide.     

Compensatory growth of juvenile brown flounder Paralichthys olivaceus (Temminck & Schlegel) following thermal manipulation

The compensatory growth of juvenile brown flounder Paralichthys olivaceus (body mass c. 12 g) following different thermal exposure was investigated. Fish were exposed to one of the five temperatures: 8·5 ( T _8·5), 13·0 ( T _13·0), 17·5 ( T _17·5), 22·0 ( T _22·0) and 26·5° C ( T _26·5) for 10 days and fish grew best at 22·0° C. Then the water temperature in all treatments was equably adjusted to 22·0° C over 3 days. At the end of the following 30 days after temperature adjustment, there were no significant differences between body masses of fish in the different treatments (wet body mass at the end of the experiment ranged from 22·13 to 24·56 g). Results indicated that the juvenile P. olivaceus achieved complete compensatory growth. Analysis of the dynamics of the feeding rates and feed conversion efficiencies indicated that compensatory growth of the fish experienced low temperature ( T _8·5, T _13·5 and T _17·5) or high temperature ( T _26·5) exposure was mainly dependent on increasing feed intake (hyperphagia) and possibly by improvement in feed conversion efficiency. The moisture content was not affected by different temperature exposure significantly. The lipid and energy content of juvenile P. olivaceus in T _8·5, however, were significantly lower than other treatment. Results of the current study indicate that a short period of low or high temperature exposure may not affect annual growth, but may affect lipid and energy deposition.     

The influence of the rate of temperature change on the activation of dormant seeds of Rumex obtusifolius L.

1. One temperature shift from 20 to 30°C in darkness induces 30–40% germination in Rumex obtusifolius seeds. The same germination percentages are found with heat treatment varying between 1 and 6h duration, indicating that the total heat sum of the temperature shift is not important.
2. Germination is greatly enhanced by three consecutive heat shifts of 1h at 30°C separated by 1h periods at 20°C.
3. The seeds are activated to a small extent after a slow warming (+2°Ch^–1) from 20 to 30°C, followed by incubation for 1h at 30°C. Germination is much higher after rapid heating (+10°Ch^–1) to 30°C, followed by 1h incubation at this temperature. Repeated fast heating treatments on four consecutive days enhances germination. Moderately rapid heatings (+3·3°Ch^–1) give intermediate results.
4. The rate of cooling does not influence the germination percentage. Cooling alone cannot induce germination.
5. Heating alone from 15 to 25°C without cooling also activates germination. In this temperature range the seeds are more activated by rapid warming than by slow warming.
6. The ecological relevance of the response to different warming rate is discussed. The insensitivity of seeds to a slow warming might keep deeply buried seeds in a dormant stage.