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1.  Aerosol Survival of Pasteurella tularensis Disseminated from the Wet and Dry States 
Applied Microbiology  1971;21(3):482-486.
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.
PMCID: PMC377207  PMID: 4994903
2.  PHYSIOLOGICAL ONTOGENY  
As this paper goes to press a complete review of the chemistry of the fertile egg will be appearing (19). The author, Mr. J. Needham, was kind enough to allow me to inspect his manuscript and thus avail myself of the comprehensive bibliography and discussion. It is surprising that no biochemists have estimated the changing water content of the egg during incubation. Many of the analyses reported in Needham's review were expressed in per cent of total weight or per cent of dry solid, and consequently are of questionable value, since these latter functions are themselves changing; the former due to water evaporation and the latter through the addition of shell constituents and the burning of oxidizable organic compounds. Moreover, there has been no statistical treatment of the results, and the reliability of the average, figures obtained has consequently been difficult to estimate. Tangl's work, quoted throughout this paper, except for its lack of statistical treatment is more enlightening. However, his concept of the so called "Energy of Embryogenesis" which he propounds, seems to me misleading and unwarranted. What Tangl measured was the amount and the caloric value of the solid material burned and thus the quantity of energy lost during the embryonic period. The latter is equivalent to the usual measurements of catabolism. In the case of the embryo it is not basal metabolism which is being estimated, since the conditions are not basal. The embryo is absorbing and assimilating nutriment all the while at a relatively rapid rate. The calorific value of the oxidized solid, which is in truth the amount of energy lost during a certain chosen interval, in Tangl's judgment stands for the energy of embryogenesis; i.e., the energy of development (growth + differentiation). We believe that this conception is erroneous. The two processes, anabolism and catabolism, occur together and undoubtedly have some relationship, but surely one is not a measure of the other. In a starving animal, and so probably in a starving embryo, there is a considerable amount of so called basal metabolism. Thus if the "Embryogenetic Energy" were measured under these conditions a figure would be obtained for which there was no growth to correspond, or in other words there would be a value for something which did not exist. It will be seen in our later communications that the changes with age of metabolic rate and growth rate do not coincide. The amount of catabolism under certain circumstances does not accelerate growth or anabolism, but seems rather to be a limiting factor. It is as if when the absorbed energy were constant an increase of catabolism would make inroads upon the amount of energy which otherwise would remain for storage (growth). If, as Pembrey's (20) experiments would tend to show, there is an increase of metabolism in the oldest embryos when the outside temperature is lowered, one would find at the end of incubation in such cases that there was a greater amount of so called "Energy of Development" but smaller embryo. It seems that the potential energy amassed as growth comes from that remaining after the needs of the body have been satisfied. The results of the experiments described in this paper have formed the basis for judgment in the selection of suitable standard conditions for the incubation of hen's eggs. Standardization was necessary so that in future experiments the more important environmental factors might be kept uniform within a certain appropriate range and therefore not be held accountable for deviations observed in the embryos. Henceforth in this series of papers the term "standard incubation conditions" will signify that (1) the temperature was constantly at 38.8 ± 0.4°C., (2) the humidity at 67.5 ± 2.5 per cent, (3) there was a continuous flow of warm air into the incubator to provide the necessary circulation, and (4) the eggs were rolled once a day within the constant temperature room. The incubator, a double-walled copper cabinet, stands in a constant temperature room, the fluctuations of which are ± 1.0°C. The space between the walls of the incubator is filled with water which serves as a buffer to outer variations. It might be repeated that all the eggs are from White Leghorn hens, are incubated 2 days after laying, and that they are kept cold during the interval necessary for transportation. With the figures from our chemical analyses and metabolic rate experiments, it was possible to calculate values for the concentration of total solids, fat, and nitrogen throughout the incubation period. These data were necessary as a general chemical background for further work. The results of the calculations are obviously rough. Because of the great variability of the eggs a satisfactory degree of accuracy could not have been attained without a very large number of analyses supplemented by complete statistical treatment. The necessity for such a comprehensive study was not evident, and it is our belief that the approximations reached in this paper are sufficiently close to serve our present purposes. The chief facts that have been ascertained in this investigation are (1) Loss of water by the egg during incubation is a function of the atmospheric humidity in its immediate environment. More rapid circulation of air lowers the humidity around the egg and thus increases evaporation. Other facts influencing evaporation are (a) atmospheric temperature, (b) thickness and surface area of the shell, and (c) conditions within the egg, the most important of which, it is suggested, is the amount of heat produced by the embryo. The latter factor, in turn, depends upon its size and age, and a significant change does not become apparent until the last 3 or 4 days of incubation, that is to say, when the embryo is of sufficient mass to exert a measurable force. (2) The surface area of the eggs in sq. cm. may be approximately represented by the formula S = K W⅔, where K = 5.07 ± 0.10, and W = the weight of the whole egg in gm. (3) There is a loss of weight by the shell during incubation. This is most noticeable near the end of the cycle, when the loss seems to parallel in general the weight of the embryo. (4) There is also a loss of solid matter during incubation. Chemical analyses indicate that about 98 per cent of the material oxidized is fat. This conclusion is corroborative of previous work by Hasselbalch, Hasselbalch and Bohr, and Tangl. (5) Carbon dioxide may be measured with relative accuracy. When it is assumed that it is derived from the oxidation of fat, satisfactory corroboration of the chemical analyses is obtained. These experiments have furnished the data from which the values have been calculated for total solids, fats, and protein in the whole egg throughout incubation. The figures may be used later for comparison with the concentration of these substances within the embryo.
PMCID: PMC2140783  PMID: 19872226
3.  Preservation of Serratia marcescens by High-Vacuum Lyophilization 
Applied Microbiology  1966;14(4):561-567.
Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized in an all-glass system capable of evacuation to pressures of less than 5 × 10-6 torr. Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabilized washed organisms compared with 10 to 20% when the cells were lyophilized at pressures of about 2.5 × 10-2 torr. At the latter pressures, 45 to 65% survival was obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspensions before lyophilization. However, the stabilizers failed to increase significantly the levels of survival compared with water suspension when cells were lyophilized at pressures less than 10-5 torr. The high survival rates obtained by the high-vacuum technique may be attributed to the reduction of traces of molecular oxygen which has been reported to be destructive of the dried bacteria. Survival of unstabilized dried S. marcescens after 1-day storage increased markedly with decreasing sealing pressure. Under the highest vacuum attained, survival of the dried bacteria was not impaired by storage for up to 1 month at Dry Ice temperatures; at higher temperatures, viability losses occurred. Exposure of the dried unstabilized bacteria to dry air resulted in rapid viability loss. The inactivation could be stopped almost immediately by evacuation to pressures of less than 10-5 torr, but the evacuation failed to reverse the viability losses that occurred during exposure.
PMCID: PMC546782  PMID: 5332950
4.  Inactivation kinetics of some microorganisms subjected to a variety of stresses. 
Loss of viability in aerosols of Escherichia coli B, E. coli commune, E. coli Jepp (in nitrogen atmospheres), and Semliki forest virus (in air) was determined as a function of relative humidity at 26.5 C. The decay patterns could be accounted for accurately by means of an equation derived from a postulated mechanism involving population distributions and first-order denaturation kinetics. Analyses of published curves describing loss of viability (all of which were semiexponential, ie., J-shaped) for various microorganisms stressed by different techniques showed that the proposed mechanism also provided an explanation for effects of the following factors (in the absence of open air, oxygen, or radiation): (i) influence of relative humidity upon aerosol susvival; (ii) dissemination of aerosols from the wet and dry states; (iii) protecting additives; (iv) relative humidity change before reconstitution; (v) reconstituting fluids; (vi) water content of freeze-dried product; (vii) storage gas; and (viii) storage temperature. The date indicate that low temperatures and high pressures were likely to be conducive to the preservation of viable bacteria and viruses, provided that cold shock and decompression shock were absent.
PMCID: PMC169843  PMID: 779645
5.  Desiccation tolerance of prokaryotes. 
Microbiological Reviews  1994;58(4):755-805.
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.
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PMCID: PMC372989  PMID: 7854254
6.  Preservation of Bacteria by Circulating-Gas Freeze Drying 
Applied Microbiology  1963;11(3):244-248.
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.
PMCID: PMC1057981  PMID: 13998202
7.  The Evaporative Function of Cockroach Hygroreceptors 
PLoS ONE  2013;8(1):e53998.
Insect hygroreceptors associate as antagonistic pairs of a moist cell and a dry cell together with a cold cell in small cuticular sensilla on the antennae. The mechanisms by which the atmospheric humidity stimulates the hygroreceptive cells remain elusive. Three models for humidity transduction have been proposed in which hygroreceptors operate either as mechanical hygrometers, evaporation detectors or psychrometers. Mechanical hygrometers are assumed to respond to the relative humidity, evaporation detectors to the saturation deficit and psychrometers to the temperature depression (the difference between wet-bulb and dry-bulb temperatures). The models refer to different ways of expressing humidity. This also means, however, that at different temperatures these different types of hygroreceptors indicate very different humidity conditions. The present study tested the adequacy of the three models on the cockroach’s moist and dry cells by determining whether the specific predictions about the temperature-dependence of the humidity responses are indeed observed. While in previous studies stimulation consisted of rapid step-like humidity changes, here we changed humidity slowly and continuously up and down in a sinusoidal fashion. The low rates of change made it possible to measure instantaneous humidity values based on UV-absorption and to assign these values to the hygroreceptive sensillum. The moist cell fitted neither the mechanical hygrometer nor the evaporation detector model: the temperature dependence of its humidity responses could not be attributed to relative humidity or to saturation deficit, respectively. The psychrometer model, however, was verified by the close relationships of the moist cell’s response with the wet-bulb temperature and the dry cell’s response with the dry-bulb temperature. Thus, the hygroreceptors respond to evaporation and the resulting cooling due to the wetness or dryness of the air. The drier the ambient air (absolutely) and the higher the temperature, the greater the evaporative temperature depression and the power to desiccate.
doi:10.1371/journal.pone.0053998
PMCID: PMC3546976  PMID: 23342058
8.  Influence of Platen Temperatures and Storage Conditions on the Survival of Freeze-dried Salmonella typhimurium1 
Applied Microbiology  1967;15(1):22-30.
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.
PMCID: PMC546837  PMID: 5340168
9.  Rates of Drying and Survival of Rhizobium meliloti Strains During Storage at Different Relative Humidities 
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.
PMCID: PMC238605  PMID: 16346846
10.  A new device for 100 per cent humidification of inspired air 
Critical Care  2000;4(1):54-60.
A new humidifier for use during mechanical ventilation in endotracheally intubated patients is described and tested. The humidifier is based on a heat-moisture exchanger, which absorbs the expired heat and moisture and releases it into the inspired air. External heat and water are then added at the patient side of the heat-moisture exchanger, so that the inspired gas should reach 100% humidity (44 mg/l) at 37°C. In bench tests using constant and decelerating inspiratory flow and minute volumes of 3–25 l the device gave an absolute humidity of 41–44 mg/l, and it reduced the amount of water consumed in eight mechanically ventilated patients compared with a conventional active humidifier. During a 24-h test period there was no water condensation in the ventilator tubing with the new device.
Introduction:
Devices for active humidification of the inspired air in mechanically ventilated patients cause water condensation in the ventilator tubing, which may become contaminated or interfere with the function of the ventilator. The present study describes and tests the performance of a new humidifier, which is designed to eliminate water condensation.
Objectives:
To test the performance of the new humidifier at different ventilator settings in a lung model, and to compare this new humidifier with a conventional active humidifier in ventilator-treated critically ill patients.
Materials and methods:
The humidifier (Humid-Heat; Louis Gibeck AB, Upplands Väsby, Sweden) consists of a supply unit with a microprocessor and a water pump, and a humidification device, which is placed between the Y-piece and the endotracheal tube. The humidification device is based on a hygroscopic heat-moisture exchanger (HME), which absorbs the expired heat and moisture and releases it into the inspired gas. External heat and water are then added to the patient side of the HME, so the inspired gas should reach 100% humidity at 37°C (44 mg H2O/l air). The external water is delivered to the humidification device via a pump onto a wick and then evaporated into the inspired air by an electrical heater. The microprocessor controls the water pump and the heater by an algorithm using the minute ventilation (which is fed into the microprocessor) and the airway temperature measured by a sensor mounted in the flex-tube on the patient side of the humidification device.
The performance characteristics were tested in a lung model ventilated with a constant flow (inspiratory:expiratory ratio 1:2, rate 12–20 breaths/min and a minute ventilation of 3–25 l/min) or with a decelerating flow (inspiratory:expiratory ratio 1:2, rate 12–15 breaths/min and a minute ventilation of 4.7–16.4 l/min). The device was also tested prospectively and in a randomized order compared with a conventional active humidifier (Fisher & Paykel MR730, Auckland, New Zealand) in eight mechanically ventilated, endotracheally intubated patients in the intensive care unit. The test period with each device was 24 h. The amount of fluid consumed and the amount of water in the water traps were measured. The number of times that the water traps were emptied, changes of machine filters, the suctions and quality of secretions, nebulizations, and the amount of saline instillations and endotracheal tube obstruction were recorded. In order to evaluate increased expiratory resistance due to the device, the airway pressure was measured at the end of a prolonged end-expiratory pause at 1 h of use and at the end of the test, and was compared with the corresponding pressure before the experiment. The body temperature of the patient was measured before and after the test of each device.
Results:
Both with constant flow and decelerating flow, the Humid-Heat gave an absolute humidity of 41–44 mgH2O/l at 37°C, with the lower level at the highest ventilation. In the patients, both Humid-Heat and the conventional active humidifier (MR730) maintained temperatures, indicating that they provided the intended heat and moisture to the inspired air. With both devices, the body temperature was maintained during the test period. There was no difference in the amount of secretions, the quality of the secretions and the frequency of suctions, saline instillations or nebulizations between the test periods with the two devices. There was no endotracheal tube obstruction, and after 1 h of use and at the end of the test no increased airway resistance was found with either device. When the MR730 was used, however, the water traps needed to be emptied six to 14 (mean eight) times (total amount of fluid in the traps was 100–300 ml) and the machine filters were changed two to six (mean four) times due to an excessive amount of condensed water with flow obstruction. No condensation of water was found in the tubing with the Humid-Heat. The water consumption was 23–65 ml/h (mean 30 ml/h) with the MR730 and 4–8 ml/h (mean 6 ml/h) with the Humid-Heat (P < 0.0008). The same relations were found when the water consumption was corrected for differences in minute ventilation.
Discussion:
The new humidifier, the Humid-Heat, gave an absolute humidity of 41–44 mg/l at 37°C in the bench tests. The tests in ventilated patients showed that the device was well tolerated and that condensation in the tubing was eliminated. There was no need to empty water traps. The test period was too short to evaluate whether the new device had any other advantages or disadvantages compared with conventional humidifiers.
PMCID: PMC29037  PMID: 11056746
airway humidification; heated humidifier; intensive care; mechanical ventilation
11.  FREE RADICAL FORMATION AND SURVIVAL OF LYOPHILIZED MICROORGANISMS1 
Journal of Bacteriology  1963;85(5):961-966.
Heckly, Robert J. (University of California, Berkeley), R. L. Dimmick, and J. J. Windle. Free radical formation and survival of lyophilized microorganisms. J. Bacteriol. 85:961–966. 1963.—A correlation between death and spontaneous free radical production, measured by an increase in the relative electron paramagnetic resonance (EPR) signal, was shown to exist for several species of microorganisms stored in the freezedried state, but the relationship between the free radical concentration and number of dead cells was not a simple proportion. Lactose added to Sarcina lutea reduced radical production and increased stability when dry preparations were stored in air. Death and free radical formation were more extensive when lyophilized Streptococcus lactis cultures were stored in air than in vacuum. Free radicals were also produced by dry yeast. Few, if any, free radicals were produced by bacteria or yeast stored in vacuum. It was shown that the observed free radical production was not caused by exposure to light. The EPR signal produced by dry Serratia marcescens decreased rapidly when cells were exposed to a humid atmosphere but the EPR signal slowly increased after cells were redried and exposed to oxygen.
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PMCID: PMC278268  PMID: 14044024
12.  Minimizing Ergosterol Loss during Preanalytical Handling and Shipping of Samples of Plant Litter 
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.
PMCID: PMC1388504  PMID: 16535086
13.  Aerosol Survival of Escherichia coli B Disseminated from the Dry State 
Applied Microbiology  1970;19(4):604-607.
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.
PMCID: PMC376747  PMID: 4911439
14.  SURVIVAL OF SERRATIA MARCESCENS AFTER FREEZE-DRYING OR AEROSOLIZATION AT UNFAVORABLE HUMIDITY I.  
Journal of Bacteriology  1962;84(6):1297-1302.
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.
PMCID: PMC278062  PMID: 14003706
15.  Exploring the influence of sterilisation and storage on some physicochemical properties of coconut (Cocos nucifera L.) water 
BMC Research Notes  2011;4:451.
Background
Fresh coconut (Cocos nucifera L) water is a clear, sterile, colourless, slightly acidic and naturally flavoured drink, mostly consumed in tropical areas. It is a rich source of nutrients and has been used for medical purposes. This study was designed to investigate changes in selected characteristics of coconut water after autoclaving, gamma irradiation and storage. Also, the study was designed for assessing the possibility of measuring the growth of bacterial in fresh, stored or sterilised coconut water using turbidity measurements (at wavelengths between 600 nm and 800 nm) or by dry biomass determinations.
Results
Portions of coconut water aseptically extracted from the matured fruit, (average pH of 6.33 ± 0.17) were either stored at 4°C, autoclaved at 121°C for 20 min., or irradiated with gamma rays at 5 kGy. Subsequent changes in selected characteristics were determined. Autoclaving, gamma irradiation and long term storage of coconut water at 4°C resulted both in the development of a pale to intense yellow colour and changes in turbidity. After storage, the dry matter content of fresh, autoclaved and irradiated coconut water by 52.0%, 23.5% and 5.0% respectively. There were also significant differences in the UV spectra before and after sterilisation and during the storage of the coconut water. Although changes in total carbohydrates were observed, they were not significant (p > 0.05).
Conclusions
The enormous differences in the characteristics before and after storage suggests that the use of turbidity and dry biomass measurements for measuring the growth of bacteria in fresh, autoclaved and gamma irradiated coconut water before storage is practicable without any possibility of interference by the innate turbidity, colour and dry matter of the coconut water. However, this is not practicable after storing the coconut waters at 4°C, since there were increases in the turbidity and dry matter of the coconut water to levels that will mask the turbidity of a growing bacteria culture.
doi:10.1186/1756-0500-4-451
PMCID: PMC3213049  PMID: 22032822
16.  Effects of Moisture Content on the Storage Stability of Dried Lipoplex Formulations 
Journal of pharmaceutical sciences  2009;98(9):3278-3289.
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.
doi:10.1002/jps.21846
PMCID: PMC2785107  PMID: 19569198
Stabilization; Gene Delivery; Nonviral vector; freeze-drying; Formulation; Storage; lyophilization
17.  The evaluation of a humidifying device for vitreoretinal surgery 
The British Journal of Ophthalmology  2004;88(12):1582-1584.
Aim: To study the feasibility of humidifying air during vitreoretinal surgery and measure the water content of air before and after intraocular transit.
Methods: The absolute water content of air was measured in a series of six eyes undergoing fluid-air exchange during macular hole surgery. Infrared absorption spectroscopy was used to determine the water content of the air infusing and exiting each eye. After baseline measurements for each eye were recorded, a second fluid-air exchange was performed and the effect of humidifying the air infusion was documented.The humidifying device used in this study was a prototype adapted from a commercially available respiratory humidifier and enables humidified air to be delivered at a controlled temperature.
Results: The water content of air increased following intraocular transit, implying dehydration occurs from the intraocular surfaces. For a standard airline infusion the mean increase in water content of air egressing from an eye was 13.4 mg/l. Humidifying the air reduced the rate of water loss by nearly 90%.
Conclusions: Significant water losses can occur from eyes undergoing fluid-air exchange. Humidifying the infused air can substantially reduce the dehydrating effect during an air exchange. This outcome may have a beneficial effect in reducing cataract formation and visual field defects associated with macular hole surgery.
doi:10.1136/bjo.2004.045591
PMCID: PMC1772441  PMID: 15548817
dehydration injury; fluid-air exchange; humidity; macular hole
18.  Responses of sap flow, leaf gas exchange and growth of hybrid aspen to elevated atmospheric humidity under field conditions 
AoB Plants  2014;6:plu021.
We demonstrate that higher air humidity mitigates the effect of low soil water availability on broadleaved trees during dry years by reducing stomatal limitation to photosynthesis, allowing higher net photosynthetic rates and supporting higher growth rates. At the same time, rising atmospheric humidity increases sensitivity of canopy conductance to water deficit and reduces the responsiveness of intrinsic water-use efficiency. The results imply that a future rise in atmospheric humidity at high latitudes may be disadvantageous in evenly rainy/humid years and expose trees to higher dehydration risk during weather extremes, although mitigating the impact of soil water deficit under moderate drought.
An increase in average air temperature and frequency of rain events is predicted for higher latitudes by the end of the 21st century, accompanied by a probable rise in air humidity. We currently lack knowledge on how forest trees acclimate to rising air humidity in temperate climates. We analysed the leaf gas exchange, sap flow and growth characteristics of hybrid aspen (Populus tremula × P. tremuloides) trees growing at ambient and artificially elevated air humidity in an experimental forest plantation situated in the hemiboreal vegetation zone. Humidification manipulation did not affect the photosynthetic capacity of plants, but did affect stomatal responses: trees growing at elevated air humidity had higher stomatal conductance at saturating photosynthetically active radiation (gs sat) and lower intrinsic water-use efficiency (IWUE). Reduced stomatal limitation of photosynthesis in trees grown at elevated air humidity allowed slightly higher net photosynthesis and relative current-year height increments than in trees at ambient air humidity. Tree responses suggest a mitigating effect of higher air humidity on trees under mild water stress. At the same time, trees at higher air humidity demonstrated a reduced sensitivity of IWUE to factors inducing stomatal closure and a steeper decline in canopy conductance in response to water deficit, implying higher dehydration risk. Despite the mitigating impact of increased air humidity under moderate drought, a future rise in atmospheric humidity at high latitudes may be disadvantageous for trees during weather extremes and represents a potential threat in hemiboreal forest ecosystems.
doi:10.1093/aobpla/plu021
PMCID: PMC4052457  PMID: 24887000
Atmospheric humidity; canopy conductance; climate change; net photosynthesis; photosynthetic capacity; relative stomatal limitation; stomatal conductance; water-use efficiency.
19.  γ-irradiation of PEGd,lPLA and PEG-PLGA Multiblock Copolymers: II. Effect of Oxygen and EPR Investigation 
AAPS PharmSciTech  2008;9(4):1110-1118.
The purpose of this research was to evaluate how the presence of oxygen can affect irradiation-induced degradation reactions of PEGd,lPLA and PEG-PLGA multiblock copolymers submitted to gamma irradiation and to investigate the radiolytic behavior of the polymers. PEGd,lPLA, PEG-PLGA, PLA, and PLGA were irradiated by using a 60Co irradiation source in air and under vacuum at 25 kGy total dose. Mw and Mn were evaluated by gel permeation chromatography. The stability study was carried out on three samples sets: (a) polymer samples irradiated and stored in air, (b) polymer samples irradiated and stored under vacuum, and (c) polymer samples irradiated under vacuum and stored in air. The thermal and radiolytic behavior was investigated by differential scanning calorimetry and electron paramagnetic resonance (EPR), respectively. Samples irradiated in air showed remarkable Mw and Mn reduction and Tg value reduction due to radiation-induced chain scission reactions. Higher stability was observed for samples irradiated and stored under vacuum. EPR spectra showed that the presence of PEG units in multiblock copolymer chains leads to: (a) decrease of the radiolytic yield of radicals and (b) decrease of the radical trapping efficiency and faster radical decay rates. It can be concluded that the presence of oxygen during the irradiation process and the storage phase significantly increases the entity of irradiation-induced damage.
doi:10.1208/s12249-008-9150-9
PMCID: PMC2628270  PMID: 18987978
ionizing radiation; multiblock copolymer; polymer stability; radicals; radiolysis mechanism
20.  Effect of Water Vapor on Lyophilized Serratia marcescens and Escherichia coli 
Applied Microbiology  1967;15(6):1299-1302.
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.
Images
PMCID: PMC547187  PMID: 16349738
21.  Parenchyma–Chlorenchyma Water Movement during Drought for the Hemiepiphytic Cactus Hylocereus undatus 
Annals of Botany  2006;97(3):469-474.
• 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.
doi:10.1093/aob/mcj054
PMCID: PMC2803650  PMID: 16390846
Cell dimensions; cell wall; chlorenchyma; CO2 uptake; drought; hydrostatic pressure; Hylocereus undatus; osmotic pressure; parenchyma; water potential; water relations
22.  Humidity Detection and Hygropreference Behavior in Larvae of the Tobacco hornworm, Manduca sexta  
Water is a critical resource for any terrestrial animal, especially for a soft-bodied insect such as larvae of the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae). Strategies for coping with a dry environment might include seeking out regions of high relative humidity that reduce desiccative stress, or to find and imbibe liquid water. Desiccated larvae placed in a linear arena with a humidity gradient preferred the humid end, whereas un-desiccated larvae did not. This behavior was not affected by temperature. Ablation or occlusion of the antennae showed that they are required to mediate this behavior. A series of experiments showed that control larvae oriented towards and imbibed liquid water whereas those whose antennae had been occluded with wax did not. Electrophysiological recordings from the lateral basiconic sensillum of the second antennal segment revealed the presence of at least one hygroreceptive unit that greatly increased its firing rate in response to moist air, decreased firing rates in response to dry air, and showed mild post-stimulatory inhibition.
doi:10.1673/031.007.3901
PMCID: PMC2999434  PMID: 20302460
hygroreceptor; hygroreception; water detection; relative humidity
23.  The social ecology of water in a Mumbai slum: failures in water quality, quantity, and reliability 
BMC Public Health  2013;13:173.
Background
Urban slums in developing countries that are not recognized by the government often lack legal access to municipal water supplies. This results in the creation of insecure “informal” water distribution systems (i.e., community-run or private systems outside of the government’s purview) that may increase water-borne disease risk. We evaluate an informal water distribution system in a slum in Mumbai, India using commonly accepted health and social equity indicators. We also identify predictors of bacterial contamination of drinking water using logistic regression analysis.
Methods
Data were collected through two studies: the 2008 Baseline Needs Assessment survey of 959 households and the 2011 Seasonal Water Assessment, in which 229 samples were collected for water quality testing over three seasons. Water samples were collected in each season from the following points along the distribution system: motors that directly tap the municipal supply (i.e., “point-of-source” water), hoses going to slum lanes, and storage and drinking water containers from 21 households.
Results
Depending on season, households spend an average of 52 to 206 times more than the standard municipal charge of Indian rupees 2.25 (US dollars 0.04) per 1000 liters for water, and, in some seasons, 95% use less than the WHO-recommended minimum of 50 liters per capita per day. During the monsoon season, 50% of point-of-source water samples were contaminated. Despite a lack of point-of-source water contamination in other seasons, stored drinking water was contaminated in all seasons, with rates as high as 43% for E. coli and 76% for coliform bacteria. In the multivariate logistic regression analysis, monsoon and summer seasons were associated with significantly increased odds of drinking water contamination.
Conclusions
Our findings reveal severe deficiencies in water-related health and social equity indicators. All bacterial contamination of drinking water occurred due to post-source contamination during storage in the household, except during the monsoon season, when there was some point-of-source water contamination. This suggests that safe storage and household water treatment interventions may improve water quality in slums. Problems of exorbitant expense, inadequate quantity, and poor point-of-source quality can only be remedied by providing unrecognized slums with equitable access to municipal water supplies.
doi:10.1186/1471-2458-13-173
PMCID: PMC3599692  PMID: 23442300
Diarrhea; Water quality; Water quantity; Contamination; Coliform bacteria; E. coli; Household water treatment; Safe storage; Urban slums; India
24.  Sensitivity of Honeybee Hygroreceptors to Slow Humidity Changes and Temporal Humidity Variation Detected in High Resolution by Mobile Measurements 
PLoS ONE  2014;9(6):e99032.
The moist cell and the dry cell on the antenna of the male honeybee were exposed to humidities slowly rising and falling at rates between –1.5%/s and +1.5%/s and at varying amplitudes in the 10 to 90% humidity range. The two cells respond to these slow humidity oscillations with oscillations in impulse frequency which depend not only on instantaneous humidity but also on the rate with which humidity changes. The impulse frequency of each cell was plotted as a function of these two parameters and regression planes were fitted to the data points of single oscillation periods. The regression slopes, which estimate sensitivity, rose with the amplitude of humidity oscillations. During large-amplitude oscillations, moist and dry cell sensitivity for instantaneous humidity and its rate of change was high. During small-amplitude oscillations, their sensitivity for both parameters was low, less exactly reflecting humidity fluctuations. Nothing is known about the spatial and temporal humidity variations a honeybee may encounter when flying through natural environments. Microclimatic parameters (absolute humidity, temperature, wind speed) were measured from an automobile traveling through different landscapes of Lower Austria. Landscape type affected extremes and mean values of humidity. Differences between peaks and troughs of humidity fluctuations were generally smaller in open grassy fields or deciduous forests than in edge habitats or forest openings. Overall, fluctuation amplitudes were small. In this part of the stimulus range, hygroreceptor sensitivity is not optimal for encoding instantaneous humidity and the rate of humidity change. It seems that honeybee's hygroreceptors are specialized for detecting large-amplitude fluctuations that are relevant for a specific behavior, namely, maintaining a sufficiently stable state of water balance. The results suggest that optimal sensitivity of both hygroreceptors is shaped not only by humidity oscillation amplitudes but also according to their impact on behavior.
doi:10.1371/journal.pone.0099032
PMCID: PMC4047084  PMID: 24901985
25.  Climatic influences on intra-annual stem radial increment of Pinus sylvestris (L.) exposed to drought 
Trees (Berlin, Germany : West)  2010;24(5):887-898.
Within a dry inner Alpine valley in the Eastern Central Alps (750 m a.s.l., Tyrol, Austria) the influence of climate variables (precipitation, air humidity, temperature) and soil water content on intra-annual dynamics of tree-ring development was determined in Scots pine (Pinus sylvestris L.) at two sites differing in soil water availability (xeric and dry-mesic site). Radial stem development was continuously followed during 2007 and 2008 by band dendrometers and repeated micro-sampling of the developing tree rings of mature trees. Daily and seasonal fluctuations of the stem radius, which reached almost half of total annual increment, primarily reflected changes in tree water status and masked radial stem growth especially during drought periods in spring. However, temporal dynamics of intra-annual radial growth determined by both methods were found to be quite similar, when onset of radial growth in dendrometer traces was defined by the occurrence of first enlarging xylem cells. Radial increments during the growing period, which lasted from early April through early August showed statistically significant relationships with precipitation (Kendall τ = 0.234, p < 0.01, and τ = 0.184, p < 0.05, at the xeric and dry-mesic site, respectively) and relative air humidity (Pearson r = 0.290, p < 0.05, and r = 0.306, p < 0.05 at the xeric and dry-mesic site, respectively). Soil water content and air temperature had no influence on radial stem increment. Culmination of radial stem growth was detected at both study plots around mid-May, prior to occurrence of more favourable climatic conditions, i.e. an increase in precipitation during summer. We suggest that the early decrease in radial growth rate is due to a high belowground demand for carbohydrates to ensure adequate resource acquisition on the drought prone substrate.
doi:10.1007/s00468-010-0458-1
PMCID: PMC3191526  PMID: 22003269
Dendrometer; Drought; Dry inner Alpine valley; Pinus sylvestris; Radial growth; Xylem cell analysis

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