The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.     

The function of the suspending medium during the freeze-drying preservation of Escherichia coli

The role of added solutes in the freeze-drying preservation of bacteria is examined. Escherichia coli were washed, suspended in solutions of selected hydroxy-substituted compounds of various molecular weights, and frozen at rates of the order of one degree C per minute. The frozen materials were freeze-dried and rehydrated in several different ways. Freeze-drying survival was correlated with the development and persistence of an amorphous solute matrix and the desorption of residual water. Protection from potentially harmful effects of freeze-drying was attributed to the dispersion, by the aforementioned amorphous matrix, of metabolites released from the cells during the preparation, and freezing of the bacterial suspensions.     

The effect of cooling rate and of dimethyl sulfoxide concentration on low temperature preservation of neonatal rat heart cells

The effect of five cooling rates, 1, 5, 10, 30, and 50 °C/min, and of four DMSO concentrations, 2.5, 5, 7.5, and 10%, on the survival of neonatal rat heart cells after freezing and thawing were studied. Growth area, contracting area and contraction frequency were used as viability parameters. Growth area and contracting area were measured in a number of fields in statistically adjusted locations of the culture dish on the second and on the fifth day of culturing.Without freezing, DMSO concentrations higher than 5% caused a considerable decrease of the growth area and of the contracting area. After freezing and thawing, biphasic survival curves were found with a narrow optimum at 2.5, 5, and 10% DMSO and a broad optimum at 7.5% DMSO. The survival based on the growth area and the survival based on the contracting area were about the same on the second day of culturing but differed on the fifth day. On the second day of culturing the highest survival was 73%, at a cooling rate of 5 °C/min and with 5% DMSO. On the fifth day of culturing the highest survival based on the growth area was 100%, at a cooling rate of 10 °C/min with 7.5% DMSO; the contracting area was the same as on the second day. The cooling rate of 5 °C/min was optimal at all DMSO concentrations tested. There was no correlation between the contracting area and the spontaneous contraction frequency after freezing and thawing when both were expressed as percentages of the control. The contraction frequency after freezing and thawing was independent of the cooling rate and was maximally 50% of the control value.     

The effect of pressure and cooling rate on spermatoza, kidney tissue, and the whole kidney

Application of high hydrostatic pressure as a cryoprotective was evaluated for the bovine sperm cell, kidney tissue, and the whole kidney. Spermatozoa samples were pressurized between 172 and 1724 bar for various lengths of time. The effect of pressure and cooling at 5 °C/min, 35 °C/min, and 55 °C/min to ?60 °C was investigated. Application of pressure to sperm cells resulted in extensive damage, as shown by a decrease in motility, an increase in morphological a bnormalities, and the release of enzymes. Freezing of sperm cells, kidney tissue, and the whole kidney under high pressure was significantly more damaging than freezing at the same rates at atmospheric pressure. Pressure did not act as a cryoprotective agent but instead led to gross cellular damage, either when applied at 4 °C or during freeze-thaw procedures.     

The effect of clutch cooling rate on starling, Sturnus vulgaris, incubation strategy

In avian species where only one parent incubates, that parent must divide its time between the mutually exclusive activities of incubation and foraging in such a way as to maintain both body condition and clutch temperature within certain limits. In a uniparental incubator, the starling, we experimentally reduced the rate at which unattended clutches of eggs cooled down and monitored the resulting changes in the parent's incubation strategy. Opposite to the predictions of standard models of time allocation during incubation, parents spent a much greater percentage of each 24-h period incubating when the rate of clutch cooling was reduced. Incubation bouts lasted significantly longer on experimental nests than on control nests, both during the daytime and overnight. Mean foraging bout duration did not differ between the two groups of nests. These results are consistent with the hypotheses that parental foraging success cues the end of a foraging bout, and that parental energy level cues the end of an incubation bout. However, most previous studies suggest that parents spend less time incubating when the rate of clutch cooling is slow. If parental energy level cues departure, these results can be explained only if the amount of time available for incubation is constrained in these cases by the time a parent must spend foraging in order to maintain body condition. Such parents should take more time away from incubation when the unattended clutch cools slowly, as this is when the cost of being absent is minimized. Copyright 1999 The Association for the Study of Animal Behaviour.     

The effect of cooling rate on the survival of cryopreserved bull,ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines

Spermatozoa from three species, bovine, ovine, and porcine, were frozen using standard techniques in two controlled-rate cooling machines, a commercial instrument and a custom-built device. Ice crystallisation was induced mechanically by touching the straws with a pre-cooled rod. The sperm samples were stored 24h, and then thawed rapidly and evaluated for motility, viability, and acrosomal integrity in the membrane-intact population. The custom-built controlled-rate cooling machine proved significantly better at all cooling rates for all species. This was particularly evident for the ram and the boar spermatozoa. In general, -30 or -50 degrees C/min were better than -1 degrees C/min, with a slight advantage being evident for -30 degrees C/min. However, this became very apparent for boar spermatozoa. It is clear that the higher cooling rates are necessary for successful freezing of spermatozoa from these species, and that careful control of the cooling rate is essential for maximal recovery of viable and functional cells. This is best achieved when the cooling profile is controlled from within a dummy sample.     

幽门弯曲菌的生物学特性的研究

我们对各型胃病患者共300例进行了幽门弯曲菌(Campylobacter pylori 以下简称CP)检查。以10%小牛血清布鲁氏菌肉汤为保存液,6%羊血布鲁氏菌琼脂平皿作培养基,在微氧条件下分离 CP,阳性率为65%。用改良尿素酶快速诊断的(4小时敏感性)阳性率为34.6%,及95.4%(24小时),特异性为100%。胃粘膜研磨液直接涂片,用0.3%碱性复红染色后镜检,二者阳性符合率为87.2%。CP 的生化试验表明,氧化酶、过氧化氢酶、尿素酶呈阳性反应;葡萄糖发酵、硝酸盐还原及3.5%NaCl 均为阴性。药敏试验的结果显示 CP 对庆大霉素、四环素、红霉素、氯霉素、羧苄青霉素,痢特灵、卡那霉素、先锋霉素等敏感;而对磺胺、萘啶酮酸、多粘菌素 B 等耐药。CP 经口感染小鼠 C57、及 BALb/c,与金黄色地鼠,均无致病性表现。     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

The effect of cooling rate and warming rate on the packing effect in human erythrocytes frozen and thawed in the presence of 2 M glycerol

The effect of hematocrit (2 versus 75%) has been studied on human red blood cells frozen and thawed in 2 M glycerol at a range of cooling rates (0.8-850 degrees C/min) and warming rates (0.1-200 degrees C/min). The data obtained at a hematocrit of 2% agree well with the data of R. H. Miller and P. Mazur (Cryobiology 13, 404-414, 1976). The results at a hematocrit of 75% show a decrease in recovery with increased cell packing, primarily dependent on warming rate at cooling rates less than 100 degrees C/min and on cooling rate at higher cooling rates. Rapid warming reduced the packing effect, whereas cooling faster than 100 degrees C/min accentuated it. It has been argued that these effects are unlikely to be due to modulation of the generally accepted mechanisms of freezing injury, that is, solution effects and intracellular freezing. It has been suggested that they may be explained by effects of cooling and warming rates on the dimensions of the liquid channels in which the cells are accommodated during freezing and thawing.     

Proceedings: Protectants in the freeze-drying and the preservation of vaccinia virus

Purified and unpurified vaccinia virus suspension were prepared from calf dermal pulp or chorioallantoic membrane of developing hen eggs, infected with vaccinia virus strain Ikeda. For the preparation of more stable dried product, single and combined protectants composed of a mixture of sodium glutamate with soluble starch, polyvinylpyrrolidone (PVP) or sodium carboxymethyl cellulose (SCMC), were compared in the course of the freeze-drying and preservation process.Single protectants sodium glutamate or peptone were effective in the freeze-drying and the preservation of both purified and unpurificd vaccinia virus products. There was an optimal concentration of sodium glutamate to be added to the suspension for the preservation of the dried products, especially the purified products.Combined protectants were effective in the purified products when the concentration of sodium glutamate exceeded the optimum necessary for the preservation.     

The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link

AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum. METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying. Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation. Citrinin was detected in all cultures for all preservation techniques on YES. The patulin profiles obtained differed with strain and culture media used. CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains. There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.     

Proceedings: The preservation of dermatophytes in parasitic form by freezing and freeze-drying

Preservation of M. canis infecting hair at room temperature, in the frozen state and as freeze-dried material was examined. The isolation of the fungus was equally successful from those hairs preserved at room temperature for 6 months as compared with fresh hair samples, but beyond 6 months the isolation became less successful. No successful isolations were made from hairs preserved at room temperature beyond $\text{2}\text{1}\text{2}$ years.Good recovery of M. canis infecting hair was also obtained after freezing and freeze-drying without any detectable change in morphological and functional characteristics.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

The effect of age at mating on the rate of egg laying in the cricket, Teleogryllus commodus

As female crickets, Teleogryllus commodus (Walker) age there is a progressive change in their ability to produce eggs as indicated by the number deposited. Virgin females lay very few eggs (11/week) when only one week old, but the number increases to an average value of 160/week by the age 3, 5 or 7 weeks post imago. There is essentially no change in the number of eggs oviposited when very young (1 week old) or senescent (7 weeks old) females are mated, but there is a dramatic increase in the long term production of eggs when females are mated at middle age (3 and 5 weeks old). The results are correlated with previously published data from this species on the JH III titres of virgin females and females mated at different ages.

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Zusammenfassung Das Altern von T. commodus-Weibchen bringt eine fortschreitende Veränderung in ihrer Eiproduktion mit sich, die sich in der Anzahl von abgelegten Eiern ausdrückt. Während 1 Woche-alte, unbegattete Weibchen wenige Eier (11/Woche) ablegen, steigt ihre Zahl im Alter von 3, 5 oder 7 Wochen auf eine Durchschnittswert von 160/Woche an. Eine Begattung von jungfräulichen Weibchen im Alter von 1 Woche oder 7 Wochen ändert die Anzahl der abgelegten Eier nicht, wogegen sich bei 3–5 Wochen alten Weibchen zu einem dramatischen Anstieg in der langfristigen Eiproduktion führt. Die Ergebnisse sind mit früher publizierten Daten an T. commodus korreliert, die unterschiedliche JH III Titer in unbegatteten und begatteten Weibchen aufzeigten.