Preservation of Mycobacteria: 100% Viability of Suspensions Stored at -70 C

Our earlier studies on long-term preservation of mycobacteria have been expanded to include other species in this genus. Mycobacterium kansasii and M. marinum, like mammalian tubercle bacilli and BCG, survive much better when stored at -70 C. By statistical analysis, M. gordonae, M. scrofulaceum, M. xenopi, M. avium, M. intracellulare, M. terrae, M. fortuitum, and M. smegmatis survived equally well at -20 C or -70 C; however, viable counts of all strains stored at -20 C were always lower than those of paired suspensions stored at -70 C, suggesting that the lower temperature is preferable for prolonged storage periods. Advantages of preservation at -70 C in Tween-albumin liquid medium are: (i) 100% viability of bacterial populations for long periods, (ii) highly reproducible inocula for animal experiments, (iii) minimal clonal selection of undesirable mutants, (iv) maintenance of genetic characteristics, and (v) adaptability to a "seed lot" system. On the basis of available information, a discussion of lyophilization versus freezer storage is presented.     

The preservation of micro-organisms in biological specimens stored at – 70°C

The preservation of micro-organisms that may be found on the skin was studied by storage in liquid media at -70°C. In the first part of the study the performance of 12 varieties of suspending media was evaluated with pure cultures of 17 species of micro-organisms maintained in the laboratory. After storage for 1 year the best medium (Oxoid Nutrient Broth with 15% glycerol) showed a mean survival for all organisms studied of 83.8%, with no significant differences between organisms. Even the worst medium (distilled water) permitted greater than 40% survival at 1 year. No changes in the characteristics of these micro-organisms were detected after 6 months storage in glycerol broth. In the second part of the study nose swabs were suspended in one representative medium (Bacto Nutrient Broth containing 7% glycerol). The mean percentage survival of staphylococci in these suspensions after 1 year's storage at -70°C was 75.4%. These results indicate that coagulase-negative staphylococci in samples of skin flora may be stored under these conditions for long periods, greatly reducing the work-load in epidemiological studies of infection.     

The preservation of micro-organisms in biological specimens stored at-70 degrees C

The preservation of micro-organisms that may be found on the skin was studied by storage in liquid media at--70 degrees C. In the first part of the study the performance of 12 varieties of suspending media was evaluated with pure cultures of 17 species of micro-organisms maintained in the laboratory. After storage for 1 year the best medium (Oxoid Nutrient Broth with 15% glycerol) showed a mean survival for all organisms studied of 83.8%, with no significant differences between organisms. Even the worst medium (distilled water) permitted greater than 40% survival at 1 year. No changes in the characteristics of these micro-organisms were detected after 6 months storage in glycerol broth. In the second part of the study nose swabs were suspended in one representative medium (Bacto Nutrient Broth containing 7% glycerol). The mean percentage survival of staphylococci in these suspensions after 1 year's storage at - 70 degrees C was 75.4%. These results indicate that coagulase-negative coagulase-negative staphylococci in samples of skin flora may be stored under these conditions for long periods, greatly reducing the work-load in epidemiological studies of infection.     

Effect of freezing of water samples on viable counts of environmental mycobacteria

The effect of prolonged storage on mycobacteria and other heterotrophic bacteria in brook water samples was examined by determination of viable counts from fresh samples and again after water concentrates had been stored in nutrient broth at —75°C for 15 months. The counts of mycobacteria were on average three times higher after storage (range of ratio 0·9–10·4). In contrast, the viable counts of other heterotrophic bacteria were reduced by 69%. The increase in mycobacterial counts was probably due to break-up of microcolonies or release of attached bacteria from particles. The possibility of cultivating mycobacteria from frozen samples is of practical help in large-scale field surveys.     

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.     

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 phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.     

Effects of short-term cold storage on recovery of proteases from extracellular products of Aeromonas hydrophila.

Three protease-containing fractions were recovered by gel filtration from concentrated crude extracellular products produced by Aeromonas hydrophila grown in a defined medium. The recovery of a heat-stable protease was differentially prevented when the crude preparation was stored for 48 h at -20 degrees C but was unaffected by storage of the crude preparation at either 4 or -70 degrees C. Once fractionated, the heat-stable protease appeared to be unaffected by subsequent storage at 4, -20, or -70 degrees C.     

Effects of short-term cold storage on recovery of proteases from extracellular products of Aeromonas hydrophila

Three protease-containing fractions were recovered by gel filtration from concentrated crude extracellular products produced by Aeromonas hydrophila grown in a defined medium. The recovery of a heat-stable protease was differentially prevented when the crude preparation was stored for 48 h at -20 degrees C but was unaffected by storage of the crude preparation at either 4 or -70 degrees C. Once fractionated, the heat-stable protease appeared to be unaffected by subsequent storage at 4, -20, or -70 degrees C.     

A note on some factors affecting the survival of Rhizobium cultures during freeze drying and subsequent storage

Viable counts of Rhizobium before and immediately after freeze drying in sucrose-peptone medium (SPM) showed that neither culture age nor cell concentration affected survival. SPM gave greater protection during drying than either dextran-sucrose-glutamate medium or distilled water. The half-lives of freeze-dried cultures stored at 4°C were estimated using an accelerated storage test and were dependent on both the strain and the suspending medium used. It is recommended that Rhizobium cultures, prepared by the procedures used in the Rothamsted Collection of Rhizobium , should be redried at intervals of 30 years.     

The Effect of Long-Term Storage on Mycobacterium bovis

It was established that when stored for many years (10–13 years) in low-temperature conditions (3°C), without sub-culture on a nutrient medium, Mycobacterium bovis grew as visible colonies along the line of inoculation. However, due to long-term storage in conditions of low temperature (3°C) morphology of mycobacteria differed significantly from initial cultures formed by rod-shaped bacteria. Some of them became pigment-forming and smooth on the surface. Unlike the initial strain of mycobacteria, a perennial bacteria stored under hard conditions did not cause the death of guinea pigs or their sensitization to a purified protein derivative for mammals. Morphological forms of the perennial mycobacteria had the following changes: pigment forming, L-forms of the vesicular type, non-acid-fast thread-like (filamentous) bacillary forms, and elementary bodies when compared to the initial strain. There were also some genetic changes in the target DNA due to the long-term storage of M. bovis. It may indicate a mutation in the pathogen’s DNA. These mycobacteria had altered biochemical activity during storage. The number of passages on the solid nutrient medium did not affect their fermentative activity. However, the low cultivation temperature increases mycobacterial catalase activity and the ability to hydrolyze Tween-80.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Host-Parasite Relationships in Experimental Airborne Tuberculosis II. Reproducible Infection by Means of an Inoculum Preserved at −70 C

The use of an inoculum preserved at low temperature for the infection of guinea pigs by the respiratory route was evaluated. In a preliminary study with Mycobacterium bovis (BCG), some of the conditions required for maximal recovery of viable cells stored at low temperature were examined. Survival of BCG was decreased by rapid freezing to -70 C and by storage at -20 C, but there was no decrease when BCG was frozen slowly and stored at -70 C or -196 C. In a subsequent study, the effect of storage at -70 C on viability and infectivity of M. tuberculosis (H37Rv) was considered. There was no loss of viability of H37Rv cells suspended in Dubos broth and stored 1 year at -70 C. This suspension showed no loss of infectivity as assessed by the number of primary pulmonary lesions initiated in guinea pigs. Constant viability and infectivity of a suspension stored at low temperature assures the reproducibility of the amount of infection and facilitates comparisons between experiments. This advantage, as well as others, of storage at low temperature are discussed.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: I. Preparation of 14C-Labeled Purified Protein Derivative Antigens and Their Adsorption to Glass

Biologically active (14)C-labeled purified protein derivative ((14)C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of (14)C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO(2) developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of (14)C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The (14)C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%.     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

The hibernating 13-lined ground squirrel as a model organism for potential cold storage of platelets

Hibernating mammals have developed many physiological adaptations to extreme environments. During hibernation, 13-lined ground squirrels (Ictidomys tridecemlineatus) must suppress hemostasis to survive prolonged body temperatures of 4-8°C and 3-5 heartbeats per minute without forming lethal clots. Upon arousal in the spring, these ground squirrels must be able to quickly restore normal clotting activity to avoid bleeding. Here we show that ground squirrel platelets stored in vivo at 4-8°C were released back into the blood within 2 h of arousal in the spring with a body temperature of 37°C but were not rapidly cleared from circulation. These released platelets were capable of forming stable clots and remained in circulation for at least 2 days before newly synthesized platelets were detected. Transfusion of autologous platelets stored at 4°C or 37°C showed the same clearance rates in ground squirrels, whereas rat platelets stored in the cold had a 140-fold increase in clearance rate. Our results demonstrate that ground squirrel platelets appear to be resistant to the platelet cold storage lesions observed in other mammals, allowing prolonged storage in cold stasis and preventing rapid clearance upon spring arousal. Elucidating these adaptations could lead to the development of methods to store human platelets in the cold, extending their shelf life.     

Degradation of specific polyunsaturated fatty acids in red blood cells stored at -20 degrees C proceeds faster in patients with schizophrenia when compared with healthy controls

Deficits in red blood cell (RBC) polyunsaturated fatty acids (PUFAs) have been extensively reported in schizophrenia although reports are inconsistent. A possible explanation for this inconsistency is varying storage conditions of blood samples prior to analysis, especially freezer storage temperature. We conducted a prospective investigation of fatty acid degradation rates in RBCs from healthy control subjects when samples from each individual were stored at both -20 degrees C or -70 degrees C. Differences were detected between storage conditions. A second prospective study was conducted to investigate the effect of differential storage conditions on RBC membrane fatty acids from schizophrenic patients. We found that storage at -20 degrees C was associated with reduced levels of PUFAs. Comparison of decay rates suggest that schizophrenics decay approximately twice as rapidly as controls. Furthermore, this phenomenon appears to be specific for the longer chain PUFAs suggesting that an enzymatic process may be responsible, e.g. elevated phospholipase A(2) activity, as opposed to simple chemical oxidation.     

Thermal stability of freeze-dried mammalian interferons. Analysis of freeze-drying conditions and accelerated storage tests for murine interferon.

Conditions of Interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions.The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 m sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacuo with a starting temperature of ?30 °C to a final residual moisture of about 3%. The final product, reference reagent G002-904-511, was stable throughout the course of the LNS test. From an extensive MIS test, this reference interferon was predicted to lose 1000 units of activity in 110 years at 4 °C and 1000 units in 100 days at 37 °C. After 6 years of storage at 37 °C when the predicted residual activity would be about 20% of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test.