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PubMed1996

Specimen preparation of the human cerebellar cortex for scanning electron microscopy using a t-butyl alcohol freeze-drying device.

A freeze-drying device was applied to t-butyl alcohol substituted nerve cells, fibers and synaptic terminals. Ten percent formalin-fixed human cerebellar cortex specimens were postfixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer solution and were rinsed three times in 5% sucrose solution in the same buffer. After the postfixed specimens were dehydrated using a graded series of ethanols and then transferred into a graded series of t-butyl alcohols (freezing point 25.4 degrees C), the t-butyl alcohol substituted specimens were freeze-dried at 15 degrees C and at high vacuum (5 x 10(-2) Torr). The freeze-dried specimens were sputter coated with gold. Scanning electron microscopy revealed synaptic terminals on the surfaces of a Golgi cell and a small flat polygonal cell. Rough somatic surfaces of granule cells were also observed.

Aged↗
PubMed1988

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying.

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Adhesion↗
PubMed1970

The intrarenal distribution of tritiated para-aminohippuric acid determined by a modified technique of section freeze-dry radioautography.

Section freeze-dry radioautography has been used to examine the intrarenal distribution of a water-soluble organic acid (para-aminohippuric acid (PAH-3H)) under constant-infusion, steady-state conditions in mouse and rat kidney in vivo. The technique described here has the following advantages: (a) Sectioning and freeze-drying are accomplished in a closed cryostat at temperatures below -40 degrees C; (b) Handling of the section is facilitated by mounting of the section-to-be on adhesive-coated Saran Wrap prior to cutting; (c) Unembedded freeze-dried sections are attached to photographic film at ambient temperature in the dark room; (d) Fixation follows completion of radioautographic exposure and precedes photographic development; (e) Permanent close contact is maintained between tissue and film. Morphologic preservation compared favorably with that obtained by optimal fixation techniques, which, however, permit diffusion. Cellular accumulation of PAH-3H during secretion was demonstrated in the proximal tubule under steady-state conditions in vivo. The cellular concentration of PAH-3H was uniform throughout the length of the proximal tubule in mouse and rat kidney.

Aminohippuric Acids↗
PubMed2005

Freeze-drying process design by manometric temperature measurement: design of a smart freeze-dryer.

PURPOSE: To develop a procedure based on manometric temperature measurement (MTM) and an expert system for good practices in freeze drying that will allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment. METHODS: Freeze drying was performed with a FTS Dura-Stop/Dura-Top freeze dryer with the manometric temperature measurement software installed. Five percent solutions of glycine, sucrose, or mannitol with 2 ml to 4 ml fill in 5 ml vials were used, with all vials loaded on one shelf. Details of freezing, optimization of chamber pressure, target product temperature, and some aspects of secondary drying are determined by the expert system algorithms. MTM measurements were used to select the optimum shelf temperature, to determine drying end points, and to evaluate residual moisture content in real-time. MTM measurements were made at 1 hour or half-hour intervals during primary drying and secondary drying, with a data collection frequency of 4 points per second. The improved MTM equations were fit to pressure-time data generated by the MTM procedure using Microcal Origin software to obtain product temperature and dry layer resistance. Using heat and mass transfer theory, the MTM results were used to evaluate mass and heat transfer rates and to estimate the shelf temperature required to maintain the target product temperature. RESULTS: MTM product dry layer resistance is accurate until about two-thirds of total primary drying time is over, and the MTM product temperature is normally accurate almost to the end of primary drying provided that effective thermal shielding is used in the freeze-drying process. The primary drying times can be accurately estimated from mass transfer rates calculated very early in the run, and we find the target product temperature can be achieved and maintained with only a few adjustments of shelf temperature. The freeze-dryer overload conditions can be estimated by calculation of heat/mass flow at the target product temperature. It was found that the MTM results serve as an excellent indicator of the end point of primary drying. Further, we find that the rate of water desorption during secondary drying may be accurately measured by a variation of the basic MTM procedure. Thus, both the end point of secondary drying and real-time residual moisture may be obtained during secondary drying. CONCLUSIONS: Manometric temperature measurement and the expert system for good practices in freeze drying does allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment.

Freeze Drying↗
PubMed1979

Induction of mutation in Escherichia coli by freeze-drying.

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.

Arginine↗
PubMed1987

Improved preservation of cartilage extracellular matrix by freeze dried embedding.

A freeze dried embedding procedure is described and used to study the extracellular cartilage matrix of the epiphyseal growth plate. This method provides improved fine structural preservation of the extracellular matrix over conventional fixation and appears comparable to that attained by freeze substitution. The trabecular networks of expanded proteoglycan filaments are illustrated in stereo-images.

Animals↗
PubMed1993

Freeze-drying of cephalothin sodium: granularly agglomerated crystallization during freezing. I.

Optimal conditions for the crystallization and subsequent freeze-drying of an aqueous cephalothin sodium (CET-Na) solution of supersaturated concentrations to obtain granular crystalline CET-Na have been discussed, as have the qualities of the product thus obtained. In general, CET-Na in supersaturated aqueous solution is barely recrystallized, even when in its frozen stage. Our previous report revealed that when the solution is kept at low temperatures for a long duration, the molecules begin to change structurally to as condensed a state as that of liquid crystals, and such change facilitates a spontaneous nucleation and seed-independent crystal growth in the frozen solution. These findings prompted the authors to investigate optimal CET-Na concentrations and thermal histories during the crystallization and subsequent freeze-drying process without seeding. It has subsequently been found out that optimization of the latter conditions gives granular agglomerated crystalline CET-Na contaminated with neither the amorphous nor the quasi-crystalline form. The optimized conditions are: 25-28% CET-Na concentrations; storage before the freezing process at 0 degrees C for 2 h, and subsequent storage at 20-25 degrees C for 1 h; cooling in the freezing process at a rate not faster than 0.5 degrees C/min; warming of the solution for facilitating crystallization prior to vacuum application for drying at -4 degrees C. Under these conditions, the freeze-dried product of CET-Na in granular form has been successfully obtained in a shorter freeze-drying cycle, exhibiting a faster reconstitution time than those of CET-Na prepared according to seeded crystallization followed by a conventional freeze-drying.

Cephalothin↗
PubMed1984

Cytoplasmic and cell surface structure of purified liver gap junctions revealed by freeze-drying.

After freeze-drying of purified liver gap junction plaques and vesicles the structural features of the inner and outer aspects of purified gap junctions were investigated. No structural details were seen on the cytoplasmic side of the connexions whereas on the cell surface side the connexions were organized in a paracrystalline pattern and exhibited a central depression or pore. We conclude that the central pore through each connexion varies in diameter along its length and that the closing site is located near the cytoplasmic face.

Animals↗
PubMed1983

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying.

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog - activated by means of a rapid increase in the Ca-concentration - showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6 X 10(-6) M and the mid-point of the pCa-tension curve occurred at 6.3 X 10(-7) M. Addition of phosphate (less than or equal to 10(-2) M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached. Addition of caffeine (10(-2) M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.

Animals↗
PubMed2005

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.

Animal Husbandry↗
PubMed1998

Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freeze-drying and rehydration.

During freeze-drying and rehydration, the activity of the H+-ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H+-ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H+-ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20 degrees C or below, activity was fully retained, while at 30, 40, or 50 degrees C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H+-ATPase was stored at different temperatures for up to 60 days. Storage at 4 degrees C resulted in retention of most of the enzymatic activity, while storage at 20 or 30 degrees C resulted in loss of activity. The protection of the H+-ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling.

Cell Membrane↗
PubMed1994

Protein purification process engineering. Freeze drying: A practical overview.

Freeze drying provides a valuable tool to the formulation scientist by permitting dehydration of heat-sensitive drugs and biologicals at low temperature. The final product is quickly and easily reconstituted, and the process is compatible with aseptic operations. Freezing is a critical step, since the microstructure established by the freezing process usually represents the microstructure of the dried product. The product must be frozen to a low enough temperature to be completely solidified. If the solute crystallizes during freezing, this temperature is the eutectic temperature. If the solute remains substantially amorphous with freezing, the relevant temperature is the collapse temperature. Understanding the physical form of the solute--crystalline or amorphous--after freezing can be important from the standpoint of drying characteristics, appearance of the final product, and even product stability during storage. Supercooling is a significant factor in freezing of formulations intended for freeze drying--prior to both primary and secondary (eutectic) crystallization. The driving force for freeze drying is the difference in vapor pressure of ice between the sublimation zone and the condenser. Because the vapor pressure of ice increases sharply with increased product temperature, it is important from the standpoint of process efficiency to maintain product temperature as high as possible during primary drying without damaging the product. The upper limit of product temperature during primary drying again depends on the physical form of the solute. Exceeding either the eutectic temperature (crystalline solute) or the collapse temperature (amorphous solute) results in loss of the desirable properties of a freeze dried product. Freeze drying is a coupled heat and mass transfer process, where either heat transfer or mass transfer may be rate limiting with respect to the overall drying rate. Heat transfer is often the rate-limiting transfer operation because of the high heat of sublimation of ice and the inefficiency of heat transfer. Conduction is the primary mechanism of heat transfer, as opposed to convection or thermal radiation. The rate-limiting resistance to heat transfer is usually the interfacial, or contact, resistance caused by poor contact between materials--the heated shelf, metal trays, and the bottom surface of glass vials. Since the thermal conductivity of a gas is directly proportional to pressure in the free molecular flow regime, the chamber pressure during primary drying is an important determinant of the overall heat transfer rate. As a result, the drying rate for a heat transfer-limited process increases sharply with chamber pressure up to a pressure where free molecular flow conditions no longer apply.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotechnology↗
PubMed2006

Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties.

Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/ml. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.

Freeze Drying↗
PubMed2001

Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa.

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris.HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.

Animals↗