Effect of storage conditions on population variability and activity of Streptomyces recifensis var. lyticus

The results of the study on survival and variation of Streptomyces recifensis var. lyticus 2435 producing lytic enzymes are presented. The culture was maintained for 2.5 years under a layer of vaseline oil, at a temperature of -20 degrees C and in lyophilized state. It was shown that irrespective of the storage method strain 2435 preserved its viability. However, the most intensive growth was observed in the lyophilized cultures. During the storage the content of the productive colonies characteristic of the morphological type culture in the population decreased while the number of the low active variants increased. Lyophilization of the strain spores in the sucrose-gelatine medium provided insignificant morphological variation of the culture and preservation of the initial level of its lytic activity against a number of test-microbes except S. aureus and M. lysodeikticus. Storage of the culture under vaseline oil and at a temperature of -20 degrees C resulted in lowering of its lytic activity against all the test-microbes used. For long-term maintenance of Streptomyces recifensis var. lyticus 2435 the method of lyophilization in the sucrose-gelatine medium is recommended.     

Results of the long laboratory storage of Streptoverticillium hachijoense (Jamaguchi) Baldacci, the producer of trichomycin]

Viability, morphologo-cultural features and antibiotic properties of Sv. hachijoense, strain LIA-0052 stored for 10 years in a dry state and in the state of a resting culture were studied. Spores and mycelium of 2-week strains most stable to some chemical and physical factors were used for drying. It was found that viability of strain LIA-0052 was maintained for a longer period of time after lyophilization, in garden soil and agar culture under a layer of mineral oil. By the end of the observation period the viability of the soil culture decreased and the morphologo-cultural properties were stabilized. When the strain was cultivated on media with sucrose, the level of its antibiotic activity increased.     

Experience with studying suspension media in actinomycete lyophilization]

Viability, cultural features and antibiotic-production properties of the organisms producing tetracycline, chlortetracycline, erythromycin, neomycin, oxytetracycline and polymyxin were studied after their storage for 2 years in ampoules at a temperature of 4--10 degrees in lyophilized state with the use of sodium glutamate, polyvinylpyrrolidone, their combination and horse serum. The highest growth rate was observed in most of the cultures lyophilized in sodium glutamate. The growth of the cultures lyophilized in the solution of polyvinylpyrrolidone alone was mainly scanty or moderate. The antibiotic production level in some strains lyophilized in sodium glutamate or its combination with polyvinylpyrrolidone was after storage for 2 years somewhat higher than that in the control. The cultural features, i.e. the colour of the aerial and substrate mycelium and pigment secretion did not significantly differ in the lyophilized cultures and the cultures maintained on agarized media.     

Experiment to study some suspension media for the lyophilization of actinomycetes]

Viability and cultural properties of 59 actinomycetes and 17 bacteria lyophilized in polyvinylpyrrolidone (PVP), sodium glutamate, their combinations and horse serum were studied after storage for 2 years at a temperature of 4-10 degrees. A 5 per cent solution of sodium glutamate had a high protective effect on viability of the above organisms. The solution containing 3 per cent of sodium glutamate and 3 per cent of PVP was somewhat less effective. The cultures lyophilized in 5 per cent solution of sodium glutamate had the same viability levels as those lyophilized in horse serum, while the latter had better growth rates on their plating out on nutrient media. A 5 per cent solution of PVP had no advantages over sodium glutamate or horse serum with respect to preservation of the organism viability. No significant differences in the cultural properties: colour of the aerial and substrate mycelium and pigment production were noted in the actinomycetes lyophilized in various protective media and the analogous control cultures maintained by means of passages on fresh nutrient media.     

Long term storage of callus cultures at low temperatures or under mineral oil layer

Plant tissue cultures from various species were stored at low temperatures or under mineral oil overlay for 4 to 6 months without subcultures. After transfer to normal culture conditions, it was checked, with 3 strains, that growth characteristics and secondary metabolite production were preserved. The storage with a mineral oil overlay (easy to run and economical method) could be a possible alternative to cryogenic or low temperature storage for a large number of strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - TLC Thin layer chromatography - HPLC High performance liquid chromatography - CAS Ceric ammonium sulfate     

Storage procedures for yeast preservation: phenotypic and genotypic evaluation

The aim of this study was to evaluate the morphological and biochemical characteristics of yeasts during storage. SixCandida spp. standard strains were stored in agarized medium with mineral oil in distilled water, frozen at ?70°C and freeze dried. Strains were phenotypically characterised before being stored and then periodically for up to 18 months. Randomly Amplified Polymorphic DNA (RAPD) was carried out in time zero, 6, 12 and 18 months of storage. The viability of all samples was preserved except for the strain ofCandida dubliniensis after 12 months of storage with mineral oil. No phenotypic alterations were observed in any of the methods employed. However, variations were observed in some phospholipase or proteinase activities. Changes in the RAPD patterns were not detected. These results seem to indicate that the maintenance methods tested were able to preserve the stability of the yeast phenotypic and genotypic characteristics.     

Survival, growth and pathogenicity of Gaeumannomyces graminis var. graminis with different methods of long-term storage

The fungal plant pathogen Gaeumannomyces graminis var. graminis was preserved with 12 different storage methods. Five strains, each with unique morphological and pathological characteristics, were used for comparison of the methods. The storage treatments included potato-dextrose agar slants, with or without mineral oil, stored at either 4 C, 28 C or ambient temperature; colonized agar plugs placed in glycerol solution at either -75 C or -20 C; colonized agar plugs placed in sterile deionized water at either 4 C or ambient temperature; and mycelial growth on intact or precut pieces of filter paper, desiccated and stored at ambient temperature. Survival was evaluated at 6, 12, 24, 36, 48 and 120 mo. The three best treatments for survival were PDA slants, with or without mineral oil, and colonized agar plugs stored in water, all at ambient temperature. All five fungal strains were recovered from all four replicates at each sampling date for agar plugs stored in water at ambient temperature. The worst treatments were agar slants and agar plugs in water stored at 4 C and agar plugs stored in glycerol at -20 C. Morphological characteristics were not affected by storage treatments. In general, there were minimal or no effects on growth and pathogenicity for all strains for all storage treatments with survival. Colonized agar plugs stored in water at ambient temperature provides an economical storage method (materials and labor) that does not need an electrical power for long-term maintenance.     

Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

Comparative investigation of different methods of storage of lactic acid bacteria

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the generaLactobacillus andLactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacteriumLactococcus lactis subsp.lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum     

Microbial degradation of organophosphonates by soil bacteria

Bacteria that can utilize glyphosate (GP) or methylphosphonic acid (MPA) as a sole phosphorus source have been isolated from soil samples polluted with organophosphonates (OP). No matter which of these compounds was predominant in the native habitat of the strains, all of them utilized methylphosphonate. Some of the strains isolated from GP-polluted soil could utilize both phosphorus sources. Strains growing on glyphosate only were not isolated. The isolates retained high destructive activity after long-term storage of cells in lyophilized state, freezing to ?20°C, and maintenance on various media under mineral oil. When phosphorusstarved cells (with 2% phosphorus) were used as inoculum, the efficiency of OP biodegradation significantly increased (1.5-fold).     

A brief review of lyophilization damage and repair in bacterial preparations

The principal concern of those responsible for the maintenance of culture collections has been to preserve viability, but nonlethal damage should not be ignored. Whether disruption of any element of a cell is nonlethal depends on the ability of the cell to repair the damage. Subsequent repair of DNA damage can lead to mutation.The protective effect of additives, as measured by survival after lyophilized cultures are reconstituted, sometimes depends upon the interval between making the addition and freezing. A rhythmic variation in the extent of viability has been observed, but increases in number of viable cells cannot be attributed to a repair mechanism. Instead, the changes in survival appear to be associated with a physiological condition of the cell at the instant of freezing.Virulence is generally maintained by lyophilized bacteria, but when stored cultures of Y. pestis were assayed immediately after reconstitution, virulence for mice was significantly reduced (as many as 4000 cells per 50% lethal dose). The virulence could be fully restored by holding reconstituted cultures for 24 hr at room temperature, or by subculture in fresh media. Obviously, the injury induced by lyophilization and storage is not to the DNA.     

Effects of long-term storage on plasmid stability in Bacillus anthracis

The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.     

Creation and use of a liquid substance based on association of petroleum-oxidizing bacteria

An association of four bacterial strains with high oil-oxidizing and bioemulsifying activities, psychrophilicity, resistance to chemical pollutants, and lack of pathogenicity was selected from a collection of natural oil-oxidizing microorganisms. A new liquid preparation containing stabilizers and preservatives that maintain the cell viability and oil-oxidizing activity during long-term storage was developed. A field experiment in oil-polluted sod-podzol and clay sand soils demonstrated that this preparation accelerated the biodegradation of oil and its individual fractions, especially in the presence of mineral and organic fertilizers. Treatment of oil-polluted soil with this preparation and additives decreased the oil-induced suppression of certain groups of soil microflora.     

Viability and sporulating capability of Coelomycetes preserved under a range of different storage regimes

The viability and sporulating capability of 45 Coelomycetes strains were evaluated. Strain subcultures were maintained under mineral oil, in soil and on agar slant for different periods of time lasting as long as 50 years, 39 years and 2 years, respectively. Of the 34 strains preserved under mineral oil, 20 maintained their viability but lost the sporulating capability with exception of one strain of Pestalotiopsis guepinii. Of the 16 strains also preserved in soil only one was viable and it was not able to sporulate. All 12 endophytic strains, 11 preserved on agar slant and one under mineral oil remained viable; however, the strain preserved under mineral oil lost its sporulating capability, while the strains on agar slant were only able to sporulate after culturing on sterilized alfalfa twigs. The results demonstrate that routine monitoring, and the use of different preservation methods, specially with the addition of sterilized plant tissue on the culture media for promoting conidiomata formation, is necessary for the success of the Coelomycetes long-term preservation.     

Development and Application of a Liquid Preparation with Oil-Oxidizing Bacteria

An association of four bacterial strains with high oil-oxidizing and bioemulsifying activities, psychrophilicity, resistance to chemical pollutants, and lack of pathogenicity was selected from a collection of natural oil-oxidizing microorganisms. A new liquid preparation containing stabilizers and preservatives that maintain cell viability and oil-oxidizing activity during long-term storage was developed. A field experiment in oil-polluted sod-podzol and clay sand soils demonstrated that this preparation accelerated the biodegradation of oil and its individual fractions, especially in the presence of mineral and organic fertilizers. Treatment of oil-polluted soil with this preparation and additives decreased the oil-induced suppression of certain groups of soil microflora.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

Comparison of methods of storing lactic acid bacteria

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the genera Lactobacillus and Lactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacterium L. lactis subsp. lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum.     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.     

Survival of Brucella abortus Strain RB51 Lyophilized and as Liquid Vaccine under Different Storage Conditions

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (−25, 4 and 25°C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5×10⁸, 1×10⁹, or 5×10⁹ cfu/ml) and two storage temperatures (4 or 25°C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0·05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0·05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25°C had a more rapid decline in viability (P< 0·05) when compared to vaccine stored at −25 or 4°C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at −25°C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25°C (P< 0·05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0·05) viability during storage at 4 or 25°C. When compared to liquid SRB51 vaccine stored at 25°C, storage at 4°C was associated with a slower decline in viability (P< 0·05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.     

The activity of the carbon metabolism enzymes inchromatium minutissimum after long-term storage

The activity of the enzymes of the tricarboxylic acid cycle and glyoxylate shunt, as well as of some enzymes involved in carbohydrate metabolism, were determined in the purple sulfur bacteriumChromatium minutissimum either maintained by subculturing in liquid medium or stored in the lyophilized state for 36 years. In cultures stored in the lyophilized state, the activities of the key enzymes of the tricarboxylic acid cycle, glyoxylate shunt, and Embden-Meyerhof-Parnas pathway were higher, whereas the activities of glucoses-phosphate dehydrogenase, pyruvate kinase, and ribulose bisphosphate carboxylase were somewhat lower than in cultures maintained by regular transfers.