The development of spaceflight experiments with Arabidopsis as a model system in gravitropism studies

Experiments with Arabidopsis have been developed for spaceflight studies in the European Space Agency's Biorack module. The Biorack is a multiuser facility that is flown on the United States Space Shuttle and serves as a small laboratory for studying cell and developmental biology in unicells, plants, and small invertebrates. The purpose of our spaceflight research was to investigate the starch-statolith model for gravity perception by studying wild-type (WT) and three starch-deficient mutants of Arabidopsis. Since spaceflight opportunities for biological experimentation are scarce, the extensive ground-based testing described in this paper is needed to ensure the success of a flight project. Therefore, the specific aims of our ground-based research were: (1) to modify the internal configuration of the flight hardware, which originally was designed for large lentil seeds, to accommodate small Arabidopsis seeds; (2) to maximize seed germination in the hardware; and (3) to develop favorable conditions in flight hardware for the growth and gravitropism of seedlings. The hardware has been modified, and growth conditions for Arabidopsis have been optimized. These experiments were successfully flown on two Space Shuttle missions in 1997.     

Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Future ESA missions in biology

A survey is given of the life sciences research program sponsored by the European Space Agency (ESA). This program rests on a number of facilities originated by ESA: Spacelab, Space sled, Biorack, Anthrorack, Eureca and its Botany — and Protein Crystallization facilities. They are all to be brough into space and returned by one of the NASA Space Shuttles. With these facilities a wide range of space biology research will be covered: cell biology, developmental biology, botany, human physiology, radio-biology, exobiology and biotechnology. Information is given on how to prepare, submit and execute an experiment proposal.     

Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space

We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.     

Application of real-time radiation dosimetry using a new silicon LET sensor

A new type of real-time radiation monitoring device, RRMD-III, consisting of three double-sided silicon strip detectors (DSSDs), has been developed and tested on-board the Space Shuttle mission STS-84. The test succeeded in measuring the linear energy transfer (LET) distribution over the range of 0.2 keV/micrometer to 600 keV/micrometer for 178 h. The Shuttle cruised at an altitude of 300 to 400 km and an inclination angle of 51.6 degrees for 221.3 h, which is equivalent to the International Space Station orbit. The LET distribution obtained for particles was investigated by separating it into galactic cosmic ray (GCR) particles and trapped particles in the South Atlantic Anomaly (SAA) region. The result shows that the contribution in dose-equivalent due to GCR particles is almost equal to that from trapped particles. The total absorbed dose rate during the mission was 0.611 mGy/day; the effective quality factor, 1.64; and the dose equivalent rate, 0.998 mSv/day. The average absorbed dose rates are 0.158 mGy/min for GCR particles and 3.67 mGy/min for trapped particles. The effective quality factors are 2.48 for GCR particles and 1.19 for trapped particles. The absorbed doses obtained by the RRMD-III and a conventional method using TLD (Mg(2)SiO(4)), which was placed around the RRMD-III were compared. It was found that the TLDs showed a lower efficiency, just 58% of absorbed dose registered by the RRMD-III.     

The development of spaceflight experiments withArabidopsis as a model system in gravitropism studies

Experiments withArabidopsis have been developed for spaceflight studies in the European Space Agency's Blorack module. The Biorack is a multiuser facility that is flown on the United States Space Shuttle and serves as a small laboratory for studying cell and developmental biology in unicells, plants, and small invertebrates. The purpose of our spaceflight research was to investigate the starch-statolith model for gravity perception by studying wild-type (WT) and three starch-deficient mutants ofArabidopsis. Since spaceflight opportunities for biological experimentation are scarce, the extensive ground-based testing described in this paper is needed to ensure the success of a flight project. Therefore, the specific aims of our ground-based research were: (1) to modify the internal configuration of the flight hardware, which originally was designed for large lentil seeds, to accommodate smallArabidopsis seeds; (2) to maximize seed germination in the hardware; and (3) to develop favorable conditions in flight hardware for the growth and gravitropism of seedlings. The hardware has been modified, and growth conditions forArabidopsis have been optimized. These experiments were successfully flown on two Space Shuttle missions in 1997.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

Recent European measurements inside Biorack

The dosimetric package used inside Biorack on board STS76, STS81 and STS84 comprises passive detector stacks built from plastic nuclear track detectors (PNTDs), thermoluminescence detectors (TLDs) and one or two active DOSTEL (DOSimetric TELescope) units using planar silicon detectors. Five passive detector stacks were exposed at different places inside the BIORACK incubators and in different stowage positions. DOSTEL units were exposed inside the 22 degrees C incubator in all flights. Mission integrated dose measurements, particle fluence rates and neutron doses are obtained from the passive detector stacks. These results are complemented by time resolved particle counts and dose rates and linear energy transfer (LET) spectra separately for the contribution of the trapped particles and the galactic cosmic rays (GCR) as a result of the DOSTEL measurements. In addition, it was possible to investigate the anisotropy of the radiation field inside Biorack by the use of a second DOSTEL unit on STS84. Since all exposures are during a solar minimum period, the total radiation exposure is of a similar extent for all flights, although position differences in dose rate up to a factor of two are observed. Particle fluence rates show lower variations. Mission averaged mean quality factors (Q) determined from the LET spectra are 2.0+/-0.1; the deduced dose equivalent rates range from 631 to 716 microSv/day.     

Prostaglandin secretion by endometrium of pregnant and cyclic cattle at day 17 after oestrus in response to in-vitro heat stress

Bilateral perifusion devices were utilized to measure prostaglandin F-2 alpha (PGF) secretion by bovine endometrium in response to in-vitro heat stress. Tissues were collected at Day 17 after oestrus from cyclic (N = 4) and pregnant (N = 5) cows, placed into 3 perifusion devices, perifused (3 ml/10 min, Krebs-Ringer-bicarbonate [KRB]) for 5 h, and fractions were collected every 10 min. Endometrial tissues within each device were subjected to a different temperature and oxytocin (1 i.u./ml KRB) treatment sequence: (1) control-oxytocin: 1 h at 39 degrees C; 2 h at 39 degrees C, 0.5 h at 39 degrees C with oxytocin, 0.5 h at 39 degrees C and 1 h at 39 degrees C; (2) heat-oxytocin: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C with oxytocin, 0.5 h at 42 degrees C and 1 h at 39 degrees C; (3) heat-KRB: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C, 0.5 h at 42 degrees C and 1 h at 39 degrees C. Regardless of reproductive status, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. Oxytocin induced an increase (P less than 0.01) in PGF secretion for endometrium from cyclic cows regardless of temperature. Endometria from pregnant cows did not respond to oxytocin when perifused at 39 degrees C. However, PGF secretion rates from endometrium of pregnant cows increased (P less than 0.01) in response to oxytocin when perifused under heat stress conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic immunoreactivity in the pedal ganglia of the pulmonate snail Megalobulimus abbreviatus after thermal stimulus: A semi-quantitative analysis

Using an immunohistochemical procedure and optical densitometry, the distribution of neurons containing serotonin (5-HT) was investigated in the pedal ganglia of Megalobulimus abbreviatus after thermal "non-functional stimulus" (22 degrees C) and stressful thermal conditions (50 degrees C).The animals were sacrificed at different times (3 h, 6 h and 24 h) following these stimuli. In control animals, the results showed the location of these serotonergic immunoreactive elements (5HT-ir) in this ganglion to be similar to those shown in other studies, where the anterior region of ventral sections showed the largest number of 5HT-ir neurons. In the anterior neurons, significant differences (p < 0.01) were observed between the groups of animals stimulated at 50 degrees C and 22 degrees C and sacrificed after 6 h. In the medial neurons, significant differences (p < 0.05) were observed between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 6 and 24 h. Neuropilar area 1 showed differences (p < 0.01) in 5HT-ir between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 3 and 24 h. Neuropilar area 2 showed a significant difference (p < 0.05) between the groups of animals stimulated at 22 degrees C and sacrificed after 3 and 24 h. These results suggest the involvement of 5-HT in the nociceptive circuit of M. abbreviatus, mainly that of the medial neurons and neuropilar area 1, which showed increases in 5HT-ir after thermal aversive stimuli. These results could be helpful in drawing cellular homologies with other gastropods.     

Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35 degrees C and/or freeze-thawing (48 h at -20 degrees C; 4 h at 4 degrees C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log10-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35 degrees C treatments. In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4 degrees C, 2 h at 25 degrees C, or 1 h at 35 degrees C or 6 h at 35 degrees C alone. A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35 degrees C plus freeze-thawing, SA plus 12 h at 25 degrees C plus freeze-thawing, SA plus 6 h at 35 degrees C, and SA plus 4 h at 35 degrees C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35 degrees C. Cider with no added organic acids treated with either 6 h at 35 degrees C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

Progesterone metabolism during storage of blood samples from Gyr cattle: effects of anticoagulant, time and temperature of incubation

Ten Gyr cows with a functional corpus luteum were used to evaluate the effects of time and temperature of incubation of blood samples on progesterone (P4) concentrations detected in plasma or serum. From each cow, a blood sample was collected into a flask containing no anticoagulant, another into an heparinized flask and a third into a flask containing sodium fluoride. The blood from each flask was divided into 46 aliquots. One of them was centrifuged within 5 min of collection. The remaining 45 aliquots were divided into three groups and kept at three different temperatures: 4 degrees C, 17 degrees C, or 37 degrees C. For each anticoagulant, aliquots from every cow and incubation temperature were centrifuged every 30 min for 6 h, and then at 8, 12 and 24 h. Plasma or serum were separated immediately after centrifugation and were kept frozen at -20 degrees C until assayed for progesterone. The mean initial concentration of P4 in serum (8.3 ng/ml) significantly diminished (P<0.05) to 6.7 ng/ml after 5 h of incubation at 4 degrees C, 3 h at 17 degrees C, or 2 h at 37 degrees C. In plasma from heparinized blood the initial concentration (7.8 ng/ml) declined significantly after 6 h of incubation at 4 degrees C, 2 h at 17 degrees C, or 1 h at 37 degrees C. Sodium fluoride used as anticoagulant prevented the degradation of P4 since the initial concentration of P4 (6.7 ng/ml) never declined during incubation at either 4 degrees C or 37 degrees C; the only significant reduction occurred after 24 h of incubation at 17 degrees C.     

Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila.

The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48 degrees-65 degrees C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7 degrees C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30 degrees C) were twice as resistant as those in the early stage (5 h at 30 degrees C), and the maximum D-value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z-values significantly. The heat resistance of cells incubated for 48 h at 30 degrees C increased (twice) after holding at 7 degrees C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30 degrees C) did not influence their D-values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 to 4.0 reduced D-values by a factor of 5. Although the strain studied was heat-sensitive (D55 degrees C = 0.17 min; z = 5.11 degrees C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Factors affecting the pheromone composition of voided boar saliva

The pheromone binding protein 'pheromaxein' which binds the pheromonal 16-androstene steroids in the saliva of the male pig (boar), was degraded and lost its binding activity in saliva incubated in air for 72 h at 21 degrees C and 37 degrees C. However, pheromaxein and its binding activity were retained in saliva incubated for 168 h at 4 degrees C. When the 3H-labelled pheromones 5 alpha-androst-16-en-3 alpha-ol (3 alpha-androstenol), 5 alpha-androst-16-en-3-one (5 alpha-androstenone) and 5 alpha-androst-16-en-3 beta-ol (3 beta-androstenol) were incubated with boar saliva for 168 h at 21 degrees C, 3 alpha-androstenol was primarily converted to 5 alpha-androstenone and 5 alpha-androstenone to 3 beta-androstenol; 3 beta-androstenol was unchanged. Evidence was obtained for microorganisms being responsible for these steroid transformations.     

Rapid cold hardening in the western flower thrips Frankliniella occidentalis

A rapid cold hardening process is reported in first instar larvae of Frankliniella occidentalis. When larvae are transferred directly from 20 degrees C to -11.5 degrees C for 2h there is 78% mortality, whereas exposure to 0 degrees C for 4h prior to transfer to -11.5 degrees C reduces mortality to 10%. The response can also be induced by exposure to 5 degrees C for 4h or by gradual cooling at rates between 0.1 and 0.5 degrees C min(-1.) The acquired cold tolerance is transient and is rapidly lost (after 1h at 20 degrees C). Rapid cold hardening extends survival times at -11.5 degrees C and depresses lethal temperatures in short (2h) exposures. Rearing at 15 degrees C (12L:12D), (a cold acclimation regime for F. occidentalis), does not protect against the cold shock induced by direct transfer to -11.5 degrees C (which rapid cold hardening does) but does extend survival time at -5 degrees C (i.e. increased chill tolerance) whilst rapid cold hardening does not. The rapid and longer term cold hardening responses in F. occidentalis therefore appear to have different underlying mechanisms.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20 degrees C for 24 h and then stored at either 20 degrees C or 5 degrees C for 96 h. Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20 degrees C and 5 degrees C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10(7)-10(9)/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10(3)-10(6) and 10(2)-10(5) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Higher maximum numbers, 10(9)-10(10), of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10(5)-10(8) and 10(4)-10(7) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.