Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.
Common preliminary treatments of samples of decaying material can involve changes in water content (e.g., via storage in relatively dry air or rinsing) that could conceivably result in loss or gain of fungal membranes and, consequently, ergosterol. A related problem is that collecting of ergosterol content data from widely distributed locales by shipment of samples ideally requires an inexpensive, safe alternative to submerging the samples in methanol for prevention of ergosterol loss. Experimental testing showed that fungal occupants of decaying salt marsh grass leaves did not exhibit loss or gain of ergosterol during air drying (to a water potential of <-8 MPa) or rewetting (to -0.8 MPa). Wet leaves of one grass species (Juncus roemerianus, black needlerush) could be fixed and dried for shipment by microwaving, or by fully drying after alcoholic or pentane fixation, without ergosterol loss, but those of smooth cordgrass (Spartina alterniflora) lost about 40% of their ergosterol content by all three of these drying methods. Ergosterol content of wet leaves of cordgrass could be maintained by alcoholic fixation and subsequent drying down to a thin film of alcohol.
Zimmerman, Leonard (U.S. Army Biological Laboratories, Fort Detrick, Frederick, Md.). Survival of Serratia marcescens after freeze-drying or aerosolization at unfavorable humidity. I. Effects of sugars. J. Bacteriol. 84:1297–1302. 1962.—Suspensions of Serratia marcescens were subjected to freeze-drying or to aerosolization at unfavorable humidity levels. The survival of the cells during one or the other of these treatments was markedly improved in the presence of common sugars, but no one sugar stabilized the cells against both stresses. The protective effects of the sugars were correlated with their penetrability into cells; minimally penetrable sugars stabilized cells against aerosolization, and freely penetrable sugars stabilized cells during freeze-drying. These results were attributed to the modifications of intracellular water content induced by the presence of the sugars in the cell suspensions.
Suspensions of Serratia marcescens (ATCC strain 14041) in water were aerosolized in a rotating drum in the presence of various concentrations of oxygen. The colony-forming ability of aerosolized organisms was rapidly destroyed by contact with 0.25% or more oxygen at 40% relative humidity (RH) and 25 C, but was almost unimpaired for at least 5 hr in nitrogen containing not more than 10 ppm of oxygen. Completely hydrated organisms were insensitive to oxygen at pressures up to 100 psi for 4 hr. No loss in viability occurred in aerosols of washed cells in air at 97% RH. It is proposed that dehydration of the aerosolized cell results in sensitization to lethal effects of oxygen, but is not the primary cause of death. Mn++, Co++, glycerol, and thiourea enhanced the biological stability of aerosols in air. Numerous similarities between the effects of oxygen in this system and in systems using freeze-dried or irradiated organisms or cell-free enzymes support the hypothesis that closely related mechanisms are involved.
Dried Serratia marcescens ATTC 14014 and Escherichia coli ATTC 4157 cells were exposed to various partial pressures of purified water vapor. The colony-forming ability of the S. marcescens was unimpaired when the dried organisms were stored in water-vapor atmosphere such that P/P0 < 0.55 or P/P0 = 1.0 (where P is the pressure of the water vapor in contact with the organisms, and P0 is vapor pressure of pure water at 25 C). During storage under water-vapor atmospheres with P/P0 between 0.6 and 1.0, the colony-forming ability of the dried S. marcescens was destroyed. The inactivation by water vapor followed the expression — ln N/N0 = Kt1/2, where N0 and N are the number of viable organisms before and after exposure, respectively, t is time, and K is a pseudo constant which is dependent upon the partial pressure of the water vapor at 25 C. Similar results were obtained with dried E. coli. The addition of solutes to the suspending media before freeze-drying was found to influence the stability of the organisms during exposure to water vapor.
Salmonella typhimurium survived freeze-drying at a platen temperature of 120 F (48.9 C) and also, though to a much lesser degree, at 160 F (82.6 C). The extent of the survival at these temperatures was dependent on the composition of the model system employed. The incidence of damage immediately after freeze-drying was greater for cells dried at the higher platen temperature and was influenced by the composition of the menstruum in which the cells were dried. In model systems having protein-dominant isotherms, survival during subsequent storage depended greatly on relative humidity, with recovery highest at relative humidities below those corresponding to moisture contents at which a monomolecular layer is formed. In menstrua having a higher sugar content, survival was best at low relative humidities corresponding to a very low equilibrium moisture content in the model system used. Damage during storage tended to be a function of the composition of the gels in which the organisms were freeze-dried, and also depended greatly on the presence of air and on the relative humidity. The maximal percentage of damage usually occurred at the low relative humidities as storage time increased.
Salmonella newport and Pseudomonas fluorescens were dried together in papain digest broth and sucrose-glutamate, and stored in several gases at various water activities (aw) between 0.00 and 0.40 at 25 C for various periods up to 81 weeks. Both S. newport and P. fluorescens, dried in papain digest broth and stored in air, died rapidly if the conditions were very dry (0.00 aw) or moist (0.40 aw). Storage in carbon dioxide and argon gave greater survival than storage in air but lower survival than did storage in nitrogen or in vacuo. When the organisms were dried in a sucrose-glutamate mixture the differences between the gases were very small, and variations in residual water were less important. Of the inert gases, argon gave the best survival when the organisms were dried in papain digest broth, especially at 0.00 aw; the survival in neon and krypton was lower and in xenon and helium it was much lower.
The aerosol survival in air and in nitrogen was measured for Pasteurella tularensis live vaccine strain, disseminated from the wet and dry states. The results showed that most of the loss of viability occurred in less than 2 min of aerosol age, i.e., a rapid initial decay followed by a much slower secondary decay. In nitrogen and air, minimum survival occurred at 50 to 55% relative humidity (RH) for wet dissemination and at 75% RH for dry dissemination. This shift indicated that aerosols produced by wet and dry dissemination were not equivalent and suggested that survival might not be related to bacterial water activity or content. The results showed that rehydration is the key process with regard to survival, but that lysis on rehydration is not a primary death mechanism. The effects of oxygen were complex because it could be either protective or toxic, depending upon other conditions. The protective action of oxygen was through an effect on the spent culture suspending fluid. The latter contained a toxic component, the activity of which is suppressed by oxygen; possibly the component is pumped away during freeze-drying. A toxic effect of oxygen was not found in the presence of spent culture media because the toxicity of the latter masks such an effect. With other bacterial suspending fluids, oxygen was shown to be toxic at low RH. Similar effects with regard to oxygen toxicity were also found with a laboratory strain of P. tularensis. Differences in oxygen toxicity for aerosols generated from the wet and dry states also suggest that bacterial water content and activity do not control aerosol survival.
The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes. In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly. In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years. The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1. At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed. As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells. Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels. Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule. A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins. Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell. There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability. Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so. One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity. There are indications of a growing technology for air-dried cells and enzymes. Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century. The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.
An investigation was made of the survival of six strains of Rhizobium meliloti filtered on membrane filters and held in atmospheres of controlled relative humidities (RH) of from 0 to 100% at 30°C in the presence of air. The rate of water loss in the desiccator was determined by the humidity-controlling solution used. Drying was accelerated by a mild evacuation of the desiccator during the drying step. Survival rates of R. meliloti strains were much higher after slow drying to 0% RH than immediately after rapid drying. Fast drying (drying period less than 3.4 h) was shown to adversely affect the tolerance to storage at all RH values tested (no survival after 2 to 5 days of storage). When survival during storage was measurable (after slow drying), the optimum RH values for storage were 43% for strains A145 and Wu498, 22 to 43% for strains RCR2011, Wu499, and Ar16, and 83% for strain RCR2004. The most favorable drying periods were 8, 9.2, 14.2, and 50.1 h for the subsequent storage of strain RCR2011 at RH values of 0, 22, 43, and 83%, respectively. The damaging effects of rapid drying on the tolerance of strain RCR2011 to storage at different RH values could be prevented either by rehydration and subsequent slow redrying or incomplete rapid drying followed by slow drying. It is suggested that R. meliloti strains are susceptible to desiccation stresses. However, the quantitative differences among strains appear to be large enough to permit selection with regard to tolerance to desiccation and storage in dried states.
This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75°C) water followed by lactic acid (2%, 55°C), vacuum packaged, stored at 4 (28 days) or 12°C (16 days), and periodically transferred to aerobic storage (7°C for 5 days). During storage, samples were exposed to sequential heat (55°C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4°C was lower than that of cells on nondecontaminated meat stored at 12°C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12°C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4°C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12°C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.
This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN2) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.
Storage methods, which can be taken into consideration for red blood cells and platelets, include liquid storage, cryopreservation and freeze-drying. Red blood cells can be hypothermically stored at refrigerated temperatures, whereas platelets are chilling sensitive and therefore cannot be stored at temperatures below 20 °C. Here we give an overview of available cryopreservation and freeze-drying procedures for blood cells and discuss the effects of these procedures on cells, particularly on cellular membranes. Cryopreservation and freeze-drying may result in chemical and structural modifications of cellular membranes. Membranes undergo phase and permeability changes during freezing and drying. Cryo- and lyoprotective agents prevent membrane damage by different mechanisms. Cryoprotective agents are preferentially excluded from membrane surfaces. They decrease the activation energy for water transport during freezing and control the rate of cellular dehydration. Lyoprotectants are thought to stabilize membranes during drying by forming direct hydrogen bonding interactions with phospholipid head groups. In addition, lyoprotectants can form a glassy state at room temperature. Recently liposomes have been investigated to stabilize blood cells during freezing and freeze-drying. Liposomes modify the composition of cellular membranes by lipid and cholesterol transfer, which can stabilize or destabilize the low temperature response of cells.
Biopreservation; Storage; Erythrocyte; Thrombocyte; Cryopreservation; Lyophilization; Freeze-drying; Membrane; Red blood cell; Platelet
As plant roots take up water and the soil dries, water depletion is expected to occur in the vicinity of roots, the so called rhizosphere. However, recent experiments showed that the rhizosphere of lupines was wetter than the bulk soil during the drying period. Surprisingly, the rhizosphere remained temporarily dry after irrigation. Such water dynamics in the rhizosphere can be explained by the drying/wetting dynamics of mucilage exuded by roots. The capacity of mucilage to hold large volumes of water at negative water potential may favor root water uptake. However, mucilage hydrophobicity after drying may temporarily limit the local water uptake after irrigation. The effects of such rhizosphere dynamics are not yet understood. In particular, it is not known how the rhizosphere dynamics vary along roots and as a function of soil water content. My hypothesis was that the rewetting rate of the rhizosphere is primarily function of root age. Neutron radiography was used to monitor how the rhizosphere water dynamics vary along the root systems of lupines during drying/wetting cycles of different duration. The radiographs showed a fast and almost immediate rewetting of the rhizosphere of the distal root segments, in contrast to a slow rewetting of the rhizosphere of the proximal segments. The rewetting rate of the rhizosphere was not function of the water content before irrigation, but it was function of time. It is concluded that rhizosphere hydrophobicity is not uniform along roots, but it covers only the older and proximal root segments, while the young root segments are hydraulically well-connected to the soil. I included these rhizosphere dynamics in a microscopic model of root water uptake. In the model, the relation between water content and water potential in the rhizosphere is not unique and it varies over time, and the rewetting rate of the rhizosphere decreases with time. The rhisosphere variability seems an optimal adaptation strategy to increase the water uptake of young root segments, which possibly reached new available water, and partly disconnect the old root segments from the already depleted soil.
drying/wetting cycles; mucilage; neutron radiography; rhizosphere; root water uptake
Background and Aims
Despite differences in physiology between dry and relative moist seeds, seed ageing tests most often use a temperature and seed moisture level that are higher than during dry storage used in commercial practice and gene banks. This study aimed to test whether seed ageing under dry conditions can be accelerated by storing under high-pressure oxygen.
Dry barley (Hordeum vulgare), cabbage (Brassica oleracea), lettuce (Lactuca sativa) and soybean (Glycine max) seeds were stored between 2 and 7 weeks in steel tanks under 18 MPa partial pressure of oxygen. Storage under high-pressure nitrogen gas or under ambient air pressure served as controls. The method was compared with storage at 45 °C after equilibration at 85 % relative humidity and long-term storage at the laboratory bench. Germination behaviour, seedling morphology and tocopherol levels were assessed.
The ageing of the dry seeds was indeed accelerated by storing under high-pressure oxygen. The morphological ageing symptoms of the stored seeds resembled those observed after ageing under long-term dry storage conditions. Barley appeared more tolerant of this storage treatment compared with lettuce and soybean. Less-mature harvested cabbage seeds were more sensitive, as was the case for primed compared with non-primed lettuce seeds. Under high-pressure oxygen storage the tocopherol levels of dry seeds decreased, in a linear way with the decline in seed germination, but remained unchanged in seeds deteriorated during storage at 45 °C after equilibration at 85 % RH.
Seed storage under high-pressure oxygen offers a novel and relatively fast method to study the physiology and biochemistry of seed ageing at different seed moisture levels and temperatures, including those that are representative of the dry storage conditions as used in gene banks and commercial practice.
Oxidative stress; seed ageing; seed longevity; seed quality; seed storage; seed test; tocopherol
Peach (Prunus persica (L.) Batsch) fruit was subjected to hot water and moist hot air treatment at varying temperatures. The activities of polyphenoloxidase (PPO) and polygalacturonase (PG) were monitored during storage for 0, 3 and 6 days. PPO activity decreased in all treatments during storage. This decrease was more in hot water treated fruits than in hot air. PPO activity decreased with the increase in treatment duration. However, the PG activity increased in heat treated fruits as well as control. This increase was more in mild heat treatments as compared to severe heat treatment. Both polyphenol and pectin contents decreased during storage in both heat treatments.
Peach; Heat treatment; Pectin; Polyphenol; Polyphenoloxidase; Polygalacturonase
The purpose of this study is to investigate the effects of moisture content on the storage stability of freeze-dried lipoplex formulations. DC-Cholesterol: DOPE (dioleoyl phosphatidylethanolamine) /plasmid DNA lipoplexes were prepared at a 3-to-2 DC-Cholesterol+ to DNA− molar ratio and lyophilized prior to storing at room temperature, 40 °C, and 60 °C for three months. Different residual moistures (1.93%, 1.10%, 1.06% and 0.36%) were obtained by altering the secondary drying temperatures. In addition to moisture content, lipoplex formulations were evaluated after freeze-drying and/ or storage for particle size, transfection efficiency, accumulation of TBARS (thiobarbituric reactive substances), glass transition temperature, DNA supercoil content, and surface area. Lipoplex formulations stored at room temperature for 3 months maintain TBARS concentrations and supercoil contents. At higher storage temperatures, formulations possessing the highest moisture content (1.93%) maintained significantly lower TBARS concentrations and higher supercoil content than those with the lowest (0.36%) moisture content. Curiously, the intermediate moisture contents exhibited marked differences in stability despite virtually identical moisture contents. Subsequent measurements of surface area indicated that the lower stability corresponded to higher surface area in the dried cake, suggesting that there may be an interplay between water content and surface area that contributes to storage stability.
Stabilization; Gene Delivery; Nonviral vector; freeze-drying; Formulation; Storage; lyophilization
Survival was determined for Escherichia coli B disseminated as an aerosol from the dry state. Survival in nitrogen, like that for wet dissemination, was better at low than at high relative humidity (RH). At high RH, survival was characterized by critical zones of instability in survival as a function of RH, instability occurring at 100, 95, 78, 70, and 60% RH. In air, survival was inferior to that in nitrogen at low RH, whereas the converse was found at high RH. The effect was attributed to oxygen. In general, results support the conclusion that to the first approximation survival is related to bacterial water content, the latter increasing with RH. However, a more detailed analysis of results indicates that survival might not be exactly related to bacterial water content. It is shown that death occurred because of rehydration and that the pretreatment of E. coli B affected its aerosol stability characteristics; i.e., wet and dry disseminated aerosols are not equivalent.
• Background and Aims Hylocereus undatus, a hemiepiphytic cactus cultivated in 20 countries for its fruit, has fleshy stems whose water storage is crucial for surviving drought. Inter-tissue water transfer during drought was therefore analysed based on cell volumes and water potential components.
• Methods In addition to determining cell dimensions, osmotic pressures and water potentials, a novel but simple procedure leading to an external water potential of zero was devised by which cells in thin sections were perfused with distilled water. The resulting volume changes indicated that the parenchyma–chlorenchyma water movement was related to more flexible cell walls in the water-storage parenchyma with its lower internal turgor pressure (P) than in the chlorenchyma.
• Key Results Under wet conditions, P was 0·45 MPa in the chlorenchyma but only 0·10 MPa in the water-storage parenchyma. During 6 weeks of drought, the stems lost one-third of their water content, becoming flaccid. About 95 % of the water lost came from cells in the water-storage parenchyma, which decreased by 44 % in length and volume, whereas cells in the adjacent chlorenchyma decreased by only 6 %; the osmotic pressure concomitantly increased by only 10 % in the chlorenchyma but by 75 % in the water-storage parenchyma.
• Conclusions The concentrating effect that occurred as cellular volume decreased indicated no change in cellular solute amounts during 6 weeks of drought. The ability to shift water from the parenchyma to the chlorenchyma allowed the latter tissue to maintain a positive net CO2 uptake rate during such a drought.
Cell dimensions; cell wall; chlorenchyma; CO2 uptake; drought; hydrostatic pressure; Hylocereus undatus; osmotic pressure; parenchyma; water potential; water relations
By comparing the composition of McCarey-Kaufman (MK) medium before and after corneal storage we attempted to identify specific physiological changes in the medium as predictors of tissue damage. We also tried to determine if hydrocortisone (a lysosomal membrane stabiliser) added to the medium could reduce tissue damage during storage. Corneas (human and rabbit) were stored in the MK medium with and without hydrocortisone for 4 days at 4 degrees C. The water and nitrogen contents of the stored cornea were compared with those of the fresh cornea. The medium was analysed before and after corneal storage to determine the concentrations of glucose, protein, and amino acids as well as pH and osmolarity. Scanning electron microscopy (SEM) was used to estimate the degree of the corneal endothelial cell damage. The nitrogen contents and dry weights of the steroid treated and untreated stored corneas were similar to those of the fresh unstored cornea. The steroid treated cornea contained a lesser amount of water than the untreated cornea. The cornea stored in medium without steroid took up a greater amount of glucose from the medium than the cornea stored in medium with steroid. As compared with their concentrations in the fresh unused medium the concentrations of leucine, lysine, and glycine were lower and that of glutamic acid was higher in both the media used for corneal storage. However, the steroid treated storage medium as compared with the untreated storage medium had a greater reduction in the lowering of leucine, lysine, and glycine, and a lesser reduction in the increase of glutamic acid. Steroid treated medium also had a lesser amount of protein released from the stored cornea. Changes in the pH and osmolarity of the media before and after corneal storage were not remarkable. SEM showed that the endothelial cells of the cornea stored in the medium containing steroid were less damaged than those of the cornea stored in the medium without steroid.
This study demonstrated the complexity of the factors leading to changes in the infectivity titers of freeze-dried canine distemper and poultry infectious bronchitis viral vaccines. The change in moisture content during the storage period was an additional parameter which may influence the infectivity titer. The results emphasized the difficulty of predetermining variations in infectivity titers from the initial residual moisture. The analysis of the variations in infectivity titers during the storage of two vaccines led to the formulation of a hypothesis of the presence of two components of different thermostability. Moreover, the temporary increase in the infectivity titer of infectious bronchitis vaccine stored progressively dissociating during storage concurrent with a progressive inactivation of infectious particles.
Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (aw) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl2 solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The aw of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without aw adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low aws. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low aws. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C).
We studied the survival of Bordetella pertussis in four suspending solutions (Casamino Acids broth, deionized water, phosphate-buffered saline, and serum inositol), subjected to three storage temperatures (4, -20, and -70 degrees C) and two freezing methods (direct freezing and fast-freezing in an ethanol-dry-ice bath). Recovery rates were higher for longer periods for suspensions stored at -70 degrees C than those stored at -20 or 4 degrees C. Serum inositol showed the highest recovery rates for all experimental conditions, followed by Casamino Acids, deionized water, and phosphate-buffered saline. Cell viability was significantly reduced in phosphate-buffered saline suspensions fast-frozen before storage. These results identify optimal conditions for storing B. pertussis cells and are applicable to the collection, transport, and storage of aspirated nasopharyngeal samples for use in the laboratory diagnosis of pertussis.
Air-dried soils which were equilibrated under relative humidities greater than 93% or moistened with liquid water showed marked increases in their capacities to oxidize CO to CO2. Liquid water addition in excess of saturation resulted in lower CO oxidation rates, reflecting the limited diffusion of CO through the aqueous phase. After 35 days' storage under 100% relative humidity, the capacity for CO oxidation decreased to 21% of the value observed with a freshly collected sample. Incubation of this stored soil under an atmosphere containing 200 ppm of CO (250 mg/m3) for 21 days resulted in a sevenfold increase in CO oxidation. A correlation was noted between the CO oxidative activity and the history of previous exposure of soils to high ambient levels of CO. The organisms responsible for CO oxidation apparently comprise a small fraction of the microbial population in the soils. With a roadside soil the oxidation of CO provided the driving force for the assimilation of CO2. The stoichiometry of the oxidative and assimilatory reactions in soil was in the range of values reported from laboratory studies with CO chemoautotrophs (carboxydobacteria). It is proposed that the population and activity of CO-oxidizing microorganisms increase in response to increasing levels of CO in the environment.
The technique for freezing, drying, and preserving in vacuo which is in common use for plasma can be successfully applied to hemoglobin solutions when the hemoglobin is first deoxygenated to the extent of 99.7 per cent or more. In confirmation of Morrison and Hisey, the preliminary deoxygenation of the solution is found necessary to avoid formation of methemoglobin during drying. If a solution of oxyhemoglobin is frozen and dried, 20 to 30 per cent is changed to methemoglobin. Deoxygnated hemoglobin dried and preserved in vacuo retained all its oxygenbinding activity for 180 days, when stored at temperatures from 4° to 30°C. Storage at 38°C. for 92 days, or at 56° for 7 days, caused no loss in activity. The dried hemoglobin had a foam structure which caused it to dissolve immediately upon contact with water. Deoxygnated hemoglobin in the dry state was partly converted to methemoglobin by even momentary contact with oxygen. When, however, the deoxygnated hemoglobin was dissolved before it was exposed to air, the hemoglobin in solution was relatively stable, and could be stored for months at 4° in contact with air without significant loss of activity.