A Simple Method for Long-term Preservation of Stock Cultures of Lactic Acid Bacteria

Strains of Lactobacillus sp., Leuconostoc sp. and Streptococcus sp. were preserved for 6–24 months at ambient temperature, partially dehydrated on granular pumice stone. To date, all the cultures stored by this technique have remained viable and no changes in the cultural or biochemical characteristics were observed.     

Calcium-dependent adenosine triphosphatase activity preservation in isolated sarcoplasmic reticulum.

ATPase activity in sarcoplasmic reticulum vesicles was measured before and after storage for several weeks and under a variety of conditions. Rapid freezing and storage at-80 degrees C provided optimum protection of enzyme activity. Sarcoplasmic reticulum preparations stored at 0 degrees C or frozen slowly and stored at-20 degrees C were not stable. At 0 degrees C sucrose, glycerol, and dithiothreitol had a stabilizing effect while NaCl, dimethylsulfoxide, and antioxidants afforded little or no protection.     

Survival of Cryptosporidium species in environments relevant to foods and beverages

AIMS: To provide data on the survival of Cryptosporidium oocysts in a range of conditions relevant to foods and beverages. METHODS AND RESULTS: Cryptosporidium parvum and C. hominis oocysts were stored in buffered media at different pH values and with various acids. In addition, neutral solutions with high salt (4.5% w/v), glycerol (20% v/v), sucrose (50% w/v) or ethanol (9 and 40% v/v) were used to determine their effects on survival. After storage periods of between 1 h and 14 days, viability was assessed using sporozoite ratio or infection of MRC-5 cell monolayers (not previously reported for culture of this organism). With all treatments, and with both assay techniques, viable oocysts were found at the end of the storage periods. However, treatments with one of the following additions: high salt, glycerol, sucrose or ethanol showed a negative and statistically significant effect on survival. Decline was noted after 1 day or even 1 h of treatment. CONCLUSIONS: MRC-5 cells are suitable for infection by C. parvum and C. hominis. Both tissue culture and sporozoite ratio gave broadly similar survival results and the greatest effects were seen with addition of components which reduced water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful additional information to the food industry when considering the risk posed by this organism.     

Deterioration of Antarctic Krill Muscle during Freeze Storage

The effects of freezing on the heat-induced gelation, Ca²⁺-ATPase activity and myofibril structure of Antarctic krill muscle were investigated. Muscle from freshly caught krill was immediately stored at-20°C in the presence (to prevent freezing) (glycerol krill) and absence (frozen krill) of glycerol. Several protease inhibitors, monosodium glutamate and Ca²⁺ were individually added to glycerol krill to inhibit endogenous proteolysis. The examinations described above were carried out after about 3-month storage at-20°C. In glycerol krill (unfrozen state), viscoelastic parameters of the heat-induced gels and Ca²⁺-ATPase activities of all the krill samples were similar to those of “surimi” (raw fish meat paste) of Alaska pollack which gave gel of good quality, although the micro structure (Z-lines) of myofibrils was different among the glycerol krill samples. In frozen krill, however, the parameters of the gel were different from those of “surimi”, the ATPase activity was completely lost and disruption of the myofibril structure occurred. Refreezing (-20°C) of glycerol krill after removal of glycerol resulted in a marked decrease in the gelation ability. These results suggest that freezing of krill muscle causes deterioration of the gelation ability.     

Effects of Additives on the Survival of Lactic Streptococci in Frozen Storage

Three single-strain cultures, Streptococcus lactis C₂, S. cremoris R₁, and S. diacetilactis DRC₂, were frozen and stored in skim milk, in skim milk containing apple juice, and in skim milk containing one of the following additives: glycerol (10%, v/v), dimethyl sulfoxide (10%, v/v), l-malic acid (0.5 and 2.0%, w/v), acetamide (0.5 and 2.0%, w/v), or succinimide (0.5 and 2.0%, w/v). Cultures were frozen and stored at -23.3 C, frozen and stored at -196 C in liquid nitrogen, or frozen at -196 C and stored at -23.3 C. Cultures frozen and stored at -196 C in liquid nitrogen gave the greatest recovery of viable cells. The number of cells surviving after storage at -23.3 C was greater when the cells had been frozen in liquid N₂ than when they had been frozen at -23.3 C. All strains stored at -23.3 C showed a decrease in numbers of surviving cells; additives, particularly l-malic acid and apple juice, were advantageous in preserving the viability of the S. lactis C₂ and S. cremoris R₁ strains, but had little or no effect on the survival of S. diacetilactis DRC₂. l-Malic acid and apple juice stimulated acid production for all cultures in activity tests following incubation after thawing, whereas glycerol and dimethyl sulfoxide retarded its development.     

病原性丝状真菌的菌种保藏方法

目的 探讨常见病原性丝状真菌的菌种保藏方法.方法将73株病原性丝状真菌经过纯化后,接种于马铃薯葡萄糖琼脂斜面分别在4℃和-80℃冷冻管保存,均用10％ (v/v)的丙三醇作为保护剂.结果经过1 a左右的保存,将菌株复活,发现4℃斜面保藏法菌株的存活率为100％,-80℃冷冻管保藏法菌株的存活率为98.6％,有些毛癣菌属...     

Storage and preservation of temperate mushroom cultures, agaricus bisporus and pleurotus Florida

Two temperate mushroom cultures namely Agaricus bisporus (U-3) and Pleurotus florida (PAU-5) were evaluated for their physiological (linear growth and biomass production), biochemical (β-1,4 endoglucanase production) and fruiting behaviour after preservation in 10% (v/v) glycerol and storage at room temperature (25–35°C), −20°C and −196°C for 6 months with the objective of establishing the recovery/changes in these fungi after storage. Studies indicated that the viability and recovery of A. bisporus and P. florida is affected by the storage conditions. Both the fungi could be best stored in liquid nitrogen for longer durations but for regular use, conventional sub-culturing was appropriate.     

Storage of in vitro cultures of Prunus rootstocks

In vitro cultures of three Prunus clones (d. 1869, GF 677 and CAB 11E) were successfully stored at +8°, +4° and-3°C following the proliferation phase.Survival of cultures was dependent upon interactions of storage temperature, light, and age of subculture. Up to 100% of the cultures survived at the end of the trials after 170 (at +4°C) and 200 (at-3°C) days storage. Complete dardness appeared more suitable than 16-h (hour) photoperiod for successful storage at-3°C for up to 10 months. One or two weeks in normal growth room vefore storage at-3°C for up to 10 months. One or two weeks in normal growth room before storage enhanced the survival S^-1. The proliferation of the cultures following storage at-3°C in the first subculture appeared similar to those under standard growth room conditions.Part of the results were presented as a poster at the 10th Congress of Eucapia in Wegeningen, The Netherlands, 19–24 June 1983.This paper in No. 504 of the Istituto Coltivazioni Arboree and No. 232 of the Centro Studi Tecnica Frutticola. The research was partially supported by National Research Council (Roma), G.L. Difesa risorse genetiche delle specie arboree.     

Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol

Glycerol was used as a substrate in the bio-production of poly(hydroxyalkanoates) (PHAs) in an effort to establish an alternative outlet for glycerol and produce value-added products. Pseudomonas oleovorans NRRL B-14682 and Pseudomonas corrugata 388 grew and synthesized poly(3-hydroxybutyrate) (P3HB) and medium-chain-length PHA (mcl-PHA) consisting primarily of 3-hydroxydecanoic acid (C(10:0); 44 +/- 2 mol %) and 3-hydroxydodecenoic acid (C(12:1); 31 +/- 2 mol %), respectively, from glycerol at concentrations up to 5% (v/v). Cellular productivity maximized at 40% for P. oleovorans in 5% (v/v) glycerol and 20% for P. corrugata in 2% (v/v) glycerol after 72 h. Increasing the glycerol media concentration from 1% to 5% (v/v) caused a 61% and 72% reduction in the molar mass (M(n)) of the P3HB and mcl-PHA polymers, respectively. Proton-NMR analysis of the glycerol-derived P3HB revealed that the M(n) decrease was the result of esterification of glycerol onto the polymer in a chain terminating position. However, no evidence of glycerol-based chain termination was present in the mcl-PHA. The growth patterns of P. oleovorans and P. corrugata on glycerol permitted their use as mixed cultures to produce natural blends of P3HB and mcl-PHA. By incorporating a staggered inoculation pattern and varying the duration of the fermentations, P3HB/mcl-PHA ratios were achieved that varied from 34:66 to 96:4.     

A simple,efficient method for purification of degraded PCR primers

Summary A simple, efficient method was used to purify PCR primers which had degraded during storage at-20°C/-9°C. The primers were eiectrophoresed on 3% (w/v) agarose gel, the main band was electroeluted via a trough cut in the gel. The primers were recovered by isobutanol extraction followed by ethanol precipitation. The yield was 20–40% and the A₂₆₀/A₂₈₀ ratio was greater than 1.8. The purification resulted in good amplification.     

Long-term preservation of some Rhodospirillaceae by freeze-drying

Lyophilization of phototropically grown cultures is difficult as it is essential to maintain strict anaerobic conditions or to avoid the exposure of the cultures to light. During a systematic investigation it was observed that several species of Rhodospirillaceae are able to grow heterotrophically in darkness on organic media and are not damaged by air oxygen under such conditions. Based on this character several oxygenic species of Rhodospirillaceae were grown under heterotrophic conditions and were lyophilized using raffinose (5% w/v) along with skim milk (20% w/v) as a protective agent. More than 30 strains from nine species of Rhodospirillaceae were successfully freeze-dried with this method. All tested cultures proved viable and showed 10–100% survival after lyophilization. During 2–3 years of storage at 9°C no further loss in viability was observed. In such lyophilized cultures no loss in photoautotrophy, diazotrophy or other desirable characters like hydrogen production, pigmentation, etc. was detected. The method is not suited to such anoxygenic Rhodospirillaceae which are not able to grown aerobically in darkness.     

Changes of viability and composition of the Escherichia coli flora in faecal samples during long time storage

Long-time storage of faecal samples is necessary for investigations of intestinal microfloras. The aim of the present study was to evaluate how the viability and the composition of the Escherichia coli flora are affected in faecal samples during different storage conditions. Four fresh faecal samples (two from calves and two from infants) were divided into sub-samples and stored in four different ways: with and without addition of glycerol broth at -20 degrees C and at -70 degrees C. The viability and the phenotypic diversity of the E. coli flora in the sub-samples were evaluated after repeated thawings and after storage during 1 year. The samples stored for 1 year without thawing were also kept at room temperature for 5 days and subsequently analysed. According to phenotyping (PhP analysis) of 32 isolates per sample on day 0, all four samples contained two dominating strains of E. coli each, and between one and eight less common strains. Samples that were stored at -70 degrees C in glycerol broth showed equal or even higher bacterial numbers as the original samples, even after repeated thawings, whereas samples stored at -20 degrees C showed a considerably lower survival rate, also with addition of glycerol. Sub-samples containing glycerol broth that were kept at room temperature after storage for 1 year showed a clear increase in the number of viable cells as well as in diversity. The diversities in each sub-sample showed a tendency to decrease after several thawings as well as after storage. Generally, the E. coli populations in samples stored at -20 degrees C were less similar to the population of the original sample than that in samples stored at -70 degrees C. Samples that had been mixed with glycerol broth had an E. coli flora more similar to that in the original sample than those without glycerol broth. Furthermore, the sub-samples that were kept at room temperature after storage for 1 year generally were more similar to the original samples than if they were processed directly. We conclude that for long time storage of faecal samples, storage at -70 degrees C is preferable. If samples have to be thawed repeatedly, addition of glycerol is preferable both for samples stored at -70 degrees C and for samples stored at -20 degrees C. Our data also have indicated that when E. coli isolates from faecal samples are selected for, e.g. analysis of virulence factors, it is necessary to pick several isolates per sample in order to obtain at least one isolate representing the dominating strain(s).     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Purification and Storage of Ribulose 1,5-bisphosphate Carboxylase from Rice Leaves

Ribulose 1,5-bisphosphate (RuBP) carboxylase was purified fromrice leaves. By using a buffer containing 12.5% (v/v) glycerolthroughout purification, the enzyme was protected from coldlability and was obtained at a high yield (5.5 mg/g fresh wt).The purified enzyme exhibited different rates of CO₂/Mg²⁺-activationby temperature pretreatment/storage. The purified enzyme was stable for at least one year in phosphatebuffer containing 12.5% (v/v) glycerol at 4°C or 50% (v/v)glycerol at –20°C. (Received March 1, 1983; Accepted June 27, 1983)     

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Thermoluminescence in pumice stone collected from the Mediterranean coast

Pumice is a low-density, light-coloured volcanic rock (igneous rock) formed when the magma from a volcanic eruption is suddenly cooled. It has a porous structure and can be different colours and have different densities. Pumice stone, unlike other rock, does not sink in water because it has a low density. In the present study, the thermoluminescent (TL) dosimetric characteristics, such as the dose–response, TL signal fading as a function of storage time, heating rate, and reusability of pumice collected on the Mediterranean coast were investigated. From this study, it was concluded that the pumice stone showed TL properties with a TL glow curve including an obviously wide peak at ∼200°C. A wide linear dose–response region up to 144 Gy, low fading of the TL signal when it was kept in a dark room for a long time, and poor reusability properties were observed for dosimetry use.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methane production using a bio-reactor packed with pumice stone on an evaporator condensate of a kraft pulp mill

When an evaporator condensate stream from a kraft pulp mill was treated by anaerobic methane fermentation, the stream could not ferment continuously. Therefore, after the raw evaporator condensate was treated by various procedures, the treated samples were tested in a batch fermentation system. There seemed to be an inhibitor, an oily material, and the evaporator condensate could be fermented easily, after removal of the oily material, in a bio-reactor packed with pumice stone. In continuous operation, the minimum hydraulic retention time was 0.64 days, the maximum volumetric BOD removal rate was 11.47 kg/m³/d, and the maximum gas production rate for the theoretical volume was 97%. The efficiency of a bio-reactor using pumice stone was studied. The pumice stone has a large number of connected pores. The major pore sizes are 20–100 μm. The cell density of the pumice stone used in the bio-reactor was 18–19 g per liter.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.