Survival of microorganisms after drying and storage

Bacteria, yeasts and fungi suspended in a dextran solution were added to ampoules containing strips of filter paper which were dried without vacuum conditions. The ampoules were sealed and stored in the dark at room temperature. Viability counts were made of the original suspension immediately after drying and after storage periods of 3–48 months. Although bacterial cultures of many genera did not show much resistance against dry conditions, bacteria of 13 other genera had survived well or moderately after 4 years of storage. Most of the dried yeast cultures had survived after this period. Of the 16 fungal genera tested, species of 6 genera exhibited growth after 4 years. Results of this study were compared with those of two other preservation methods by which the same microorganisms were used.     

The Preservation of Plant Pathogenic Bacteria

Some 160 cultures were preserved by freeze drying, under mineral oil and in soil. After storage for 5 years all freeze dried cultures were viable; most cultures of xanthomonads were viable under oil and in soil; pseudomonads survived well in soil but only moderately well under oil; soft-rotting Erwinia spp. survived poorly but storage under oil was better than in soil; other Erwinia spp. and most Corynebacterium spp. survived well in soil and under oil. The mean half lives in years ( h ) calculated for freeze dried cultures of groups of closely related bacteria were: Erwinia 'chrysanthemi group', 0·40; Erwinia 'carotovora group', 0·51; Pseudomonas 'syringae group', 0·50; Xanthomonas spp., 0·84 years. Estimated half lives for Corynebacterium spp. ranged from 1·8 to 6·5 years. There was no evidence that bacteria which had been in culture for more than 3 years before being freeze dried had a longer storage life than those freeze dried within 3 years of isolation. Cultures of the Pseudomonas 'syringae group'had a longer storage life when freeze dried by Greaves'method ( h = 0·73) than when freeze dried by Annear's method ( h =0·50). There appeared to be no general correlation between half life in storage and either the proportion of cells surviving the freeze drying process or the viable cell count immediately after freeze drying. Most of the variation in the results could be attributed to variation in viable cell count between different ampoules of the same batch of a culture.     

Survival of Freeze Dried Bacterial Cultures

The survival of 100 strains of bacteria, representing 15 genera, on freeze drying and during storage in the freeze dried state for 10 years has been assessed. Gram-positive organisms tended to survive better than Gram-negative. Viable organisms were recovered from all but one of the cultures.     

Virulence and viability of Yersinia pestis 25 years after lyophilization.

When equal volumes of 6% lactose and a broth culture of Yersinia pestis were mixed before freezing, approximately 50% of the cells survived lyophilization and reconstitution on the following day. Concomitantly, the number of viable cells per 50% lethal dose increased from about 16 to 125 organisms. On subsequent storage of the lyophilized cells under vacuum in glass ampoules at 4 degrees C for 25 years, more than 25% of the cells remained viable. When stored cultures were assayed immediately after reconstitution, virulence for mice was significantly reduced (as many as 4,000 cells/50% lethal dose), but the virulence was fully restored when reconstituted cultures were held for 24 h at room temperature, or when a subculture was prepared in fresh medium.     

Liquid Nitrogen Preservation of Standard Inoculum: Gas-Phase Storage

Liquid nitrogen storage was the most satisfactory of several methods tested for supplying standard Streptomyces viridoflavus inoculum for laboratory and pilot plant experimentation. Shake-flask cultures were subdivided into sterile cotton-plugged ampoules and stored in the gas space of a liquid nitrogen refrigerator. There were no detectable changes in viability or in candidin-producing capacity over a 12-month test period. The procedure also proved satisfactory with all other organisms tested.     

Survival curves for microbial species stored by freeze-drying

The survival of a variety of species of microorganism following storage for up to 20 years has been analyzed. The organisms were freeze-dried, sealed in ampoules under vacuum (<1 Pa) and stored in the dark at 5 degrees C. The yeast that was tested, Saccharomyces cerevisiae, showed only 8% survival when recovered shortly after freeze-drying, but subsequent loss during storage was the least among all the tested microorganisms. The decrease in the logarithm of survival per year (log survival) was -0.010, which corresponds to a survival rate of 97.7% per year. The Gram-negative bacteria tested, Escherichia coli, Pseudomonas putida, and Enterobacter cloacae, showed 42.6, 33.5, and 50.8% survival shortly after freeze-drying, which was higher than the corresponding survival of S. cerevisiae, but the subsequent loss during storage was greater than S. cerevisiae, the log survival figures being -0.041, -0.058, and -0.073 per year. These values correspond to survival rates of 91.0, 87.5, and 84.5% each year. The Gram-positive bacteria tested, Lactobacillus acidophilus and Enteroccoccus faecium, showed 62.5 and 85.2% survival shortly after freeze-drying, which was even higher than that of the Gram-negative species, and these organisms also showed better survival during storage than Gram-negative bacteria; their log survival rates were -0.018 and -0.016 per year, which corresponded to survival rates of almost 96% per year. Comparison of these results with other published data for different drying conditions suggests that survival during storage is strongly influenced by the degree of vacuum under which the ampoules were sealed. The excellent survival after freeze-drying of each species might be attributable to the high level of desiccation and to sealing under vacuum.     

Production of Soy Sauce Koji Mold Spore Inoculum in Plastic Bags

An innovation is described for producing soy sauce koji mold spore inoculum by using inexpensive autoclavable plastic bags and reuseable plastic enclosures to make culture vessels. After growth, the spore mass could be dried and packaged in the same bag after removing the enclosure. Broken rice was used as the substrate for mold cultivation. Viable spore counts of 10⁹ spores per g were obtained under optimal conditions. After drying at 50°C for 6 h, the moisture content of the spore mass decreased from 35.22 to 6.32% with no significant effect on spore viability. The dry spores could be stored in the refrigerator or at room temperature for at least 3 months.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

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.     

Survival of Azotobacter in Dry Soil

Detection of viable Azotobacter in soils stored in the laboratory for more than 10 years suggests that these bacteria can remain dormant in nature for prolonged periods of time. Studies of dried cultures show that cysts of A. vinelandii 12837 remain viable for at least 10 years, whereas vegetative cells do not survive for even 1 year under the same conditions.     

A New Drying Method of Plant Specimens for Scanning Electron Microscopy the t-Butyl Alcohol Freeze-Drying Method

The t-butyl alcohol freeze-drying method (lnoue and Osatake 1988) is a simple drying method of biological materials for scanning electron microscopy Fixed specimens were immersed in t-butyl alcohol after dehydration throgh a graded series of ethanol. Specimens in the alcohol were then forzen in a refrigerator. They were placed in the bell jar of a vacuum evaporator and simply evacuated with a rotary pump. The samples were completely dried within 40–60 min after the frozen alcohol was sublimated in the vacuum, when the specimen was examined by scanning electron microscopy (SEM), both surface and intracellular structures were demonstrated in three-dimension without any significant drying artifacts. Careful comparison of the results indicated that the SEM imayes obtained by this method were either superior or equal to those obtained by the critical point drying method.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

EFFECTIVE METHOD FOR DRY INOCULATION of BACTERIAL CULTURES

An effective way for inoculation of bacteria into dry foods/ingredients that gives a uniform mixture was developed. In the first part of this experiment, Salmonella typhimurium was successfully inoculated into chalk. Chalk tubes were weighed then soaked in a broth with S. typhimurium and allowed to dry back to their original weight. the dried chalk was made into a powder form. A viable cell count of this chalk, using a selective media for S. typhimurium , showed that the organism survived the drying while entrapped in the chalk. the "charged" chalk was used in an experiment as a dry inoculum where it was mixed with a low-moisture poultry feed. In comparison to a liquid inoculum, the "charged" chalk was a superior way of inoculating into dry particles because it created a more homogenous mixture with the feed without altering any properties of the feed itself. the second part of this experiment entailed a shelf-life study of the "charged" chalk. the same procedures for inoculating chalk were done using ten different cultures including Bacillus cereus, Clostridium perfringens, Escherichia coli, Enterobacter aerogenes, Lactobacillus plantarum, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus , and Streptococcus faecalis. Data showed that the cultures are stable in the chalk for at least six months.     

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold shock during liquid production increases storage shelf-life of Cryptococcus nodaensis OH 182.9 after air-drying

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60-70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C.     

Preservation of microorganisms

Cultures of bacteria, yeasts and fungi were grown on common agar media in normal culture tubes. About 1 week after maximum growth the cotton plugs of the tubes were replaced by sterile rubber seals. The cultures were stored in the dark at room temperature. Tests for viability of cultures were made after periods between 1 and 10 years. The results of this simple method show long survival periods of many bacteria and yeasts.     

Soil drying as a model for the action of stress factors on the natural bacterial population

The drying of soil samples reduced the abundance (especially of predominant species) and the diversity of bacteria isolated from these samples, making easier the isolation of rare bacterial species. Some bacterial species that were minor before soil drying became dominant in dried soil samples. In general, soil drying allowed the diversity of soil bacteria to be determined more adequately. The bacteria that were isolated from dried soil samples turned out to be resistant to gamma radiation (with LD90 = 2.8-4.6 kGy) and desiccation. It is concluded that soil drying may serve as a model for the action of stress factors on natural bacterial populations. The hypothesis that periodic desiccation was the primary cause of formation of bacterial radioresistance in nature is discussed.     

Possible storage of Coccinella undecimpunctata (Col., Coccinellidae) under low temperature and its effect on some biological characteristics

Developmental stages of Coccinella undecimpunctata L. were stored at 6.0°C for various storage periods in a refrigerator. Egg hatching was 65.0% after 7 days of storage. However, no egg hatching were observed after 15, 30, 45 and 60 days of storage. The survival of the third and fourth instar larvae was higher than the first and second instar. The survival of larvae declined sharply after 15 days. No larvae survived after 30 or 60 days of storage. Emergence percentage of adults from stored pupae varied from 85.0 to 25.0% after storage for 7 up to 30 days. The survival percentage of adults differed and appeared to depend on prior feeding before storage. From the present results, it appears that the adult stage may be better able to survive extended periods of storage than the other developmental stages. In addition, it was found that prior feeding of adult stage affected the longevity, fecundity and consumption rate.     

Comparative evaluation of three in vacuo dessication procedures on bacterial cultures

The paper presents results obtained for the bacterial cultures preservation (E. coli ATCC 25 922 and S. aureus Wood) by three in vacuo desiccation procedures: freeze-drying (lyophilization or cryo-desiccation at eutectic zone), cryo-desiccation above eutectic zone and direct drying. It has been in view: the survival of liquid cultures as reported to the desiccation procedure per se, the loss of viability of desiccated cultures stored in refrigerator for at least one year and the residual moisture of desiccated cultures. For 3 batches there has been applied the accelerated thermal degradation test. Employing the same protective medium for both cultures, E. coli cultures prove to be more easily affected by lyophilization and cryo-desiccation above eutectic zone as compared to S. aureus cultures, fact that may be due to the differences between the wall structures of G--bacteria and G+ bacteria, respectively. During storage, E. coli and S. aureus cultures proved a quite similar loss of viability. The residual moisture content was quite similar for both E. coli and S. aureus cultures exposed to the same in vacuo desiccation procedure. The lyophilization and the cryo-desiccation above eutectic zone, as compared to direct drying, yielded superior results. The accelerated thermal degradation test provides only informative results, partially confirmed by viable counts determined at stated intervals of storage time in the refrigerator.     

THE RESISTANCE OF TWO STRAINS OF BACTERIUM COLI TYPE I TO DRYING UNDER ATMOSPHERIC CONDITIONS

SUMMARY: Suspensions of two strains of Bacterium coli type I were dried as thin films under atmospheric conditions and the numbers of organisms determined before and after drying. Three methods were used to grow the culture; in two the culture was grown in broth and in the other on agar slopes.
Strain 28.D.10 was less sensitive than strain NCTC 5934. After culturing in broth NCTC 5934 showed irregular daily fluctuations in sensitivity to drying; 28.D.10 became more sensitive for the first 2–3 weeks and thereafter less sensitive. Suspensions prepared from 18 hr plate cultures were more sensitive than from 24 hr plate cultures; a change from peptone water to Lemco broth for daily culturing slightly decreased the sensitivity to drying. With both strains suspensions prepared from broth cultures were more sensitive than those from agar slopes.
Continuous daily culturing of 28.D.10 on a solid medium as compared with broth decreased the sensitivity of suspensions and over a long period the culture appeared to be more stable. When strain NCTC 5934 was grown on a solid medium the suspension was as sensitive to drying as that obtained when broth was used but the daily fluctuation of results appeared to be less.
A decrease in the number of cells in the 24 hr broth culture of 28.D.10 coincided in time with an increase in the sensitivity to drying of the suspension prepared from the same culture.