Experiments with an apparatus for lyophile desiccation of micro-organisms

Summary Lyophyle desiccation is an extremely valuable method of preserving micro-organisms. The apparatus described above proves satisfactory. The material should be frozen as rapidly as possible at a temperature of −35°C. or lower, and the apparatus should be constructed in such a way that the pressure remains approximatively constant. During the sealing of the ampoules care should be taken that no dry material passes from the latter to the apparatus. A time limit for the desiccation process is hard to fix. When water has condensed on the outside surface of the ampoules, the apparatus is left untouched for about an hour. This brings the total duration of the desiccation as a rule up to 1 1/2 to 2 hours. A volume of 0.25 ml skimmed milk suffices for the preservation of a strain. It is advisable to start from a fresh culture that has been grown on a solid medium and to suspend the required amount in skimmed milk at a pH of 7.4–7.8. The suspension should directly be frozen in a thin layer. Bofore the frozen ampoules are submitted to the desiccation process, they may be kept for some time at a temperature of −35°C. For this as well as for many valuable indications we are greatly indebted to the Staff of the Kamerlingh Onnes Laboratory.     

Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state

Two endothermic peaks could be observed for five commercial samples of bovine serum albumin (BSA). The smaller peak observed by differential scanning calorimetry (DSC) corresponded to enthalpy relaxation. This peak was followed on storage of BSA, in its glassy state, after it had been heated above its denaturation temperature. Enthalpy and peak temperature increased with duration of storage. On storage for one week at 60 degrees C, a sample at 8.3% moisture showed a peak at 100 degrees C with an energy value of approximately 2 J per g protein. BSA samples were heated within the DSC sufficiently to eliminate the lower enthalpy peak but without altering the denaturation enthotherm. The amount of physical aging shown by these BSA samples was similar to that of the heat-denatured samples. It was concluded that the heating endotherms of dry BSA reflect both the storage and thermal history of the sample. Possible implications of the enthalpy relaxation of BSA on the behavior of this important protein are considered.     

Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or cryopreservation

The effects of in vitro storage on the sperm's ability to undergo the acrosome reaction (AR) have never been studied in avian species despite its major importance for reproduction management.The ability of chicken sperm to undergo the AR was measured after liquid storage at 4 °C and after cryopreservation, and its relationship with other semen quality parameters, including viability, mass motility and objective motility parameters measured by computer semen analyser (CASA) was analysed in two different flocks. The percentage of intact acrosome-reacting spermatozoa (IAR) was dramatically decreased by 48 h liquid storage (loss of 2/3 among the spermatozoa initially able to undergo the AR) whereas motility, viability and morphological integrity were reduced by 10-15%. By contrast, cryopreservation did not affect the induction of AR in flock 1 (29% IAR) whereas it was strongly affected in flock 2 (7% IAR). Motility parameters, viability and morphology were considerably altered by freezing in every case (more that 50% loss). Positive correlations were found between the percentage of intact acrosome-reacting spermatozoa and viability, mass motility and many objective motility parameters.Our results showed that the sperm's ability to undergo the AR was much more affected than other sperm functions after storage at 4 °C, while cryopreservation only had an effect in semen with the lowest initial quality. These results raise questions regarding the specific features of chicken sperm biology that must be taken into account in the treatment of semen.     

High-performance liquid chromatographic method for the determination of benzodiazepines in plasma or serum using the column-switching technique

A column-switching high-performance liquid chromatographic method for the simultaneous determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-μl plasma sample was directly injected into a BioTrap 500 MS (hydrophobic polymer) column. After a washing step with a mixture of phosphate buffer and acetonitrile, the retained benzodiazepines were back-flushed into a reversed-phase (LiChrospher Select B C₈) column with a mobile phase of acetonitrile–phosphate buffer. The method showed excellent linearity from 50 to 1000 ng/ml for clonazepam, flunitrazepam and midazolam and from 50 to 5000 ng/ml for diazepam and oxazepam. The recoveries were around 98% for all the benzodiazepines studied. The relative standard deviation for between- and within-day assay was <20% for low concentrations close to the values of the limit of quantification and <4% for high concentrations. The procedure described is relatively simple and rapid because no off-line manipulation of the sample is required: the total analysis time is approximately 30 min.     

High-performance liquid chromatographic assay for cefepime in serum

A simple, rapid, specific and sensitive high-performance liquid chromatographic method was developed for the determination of cefepime 1-[[6R, 7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo-[4.2.0] oct-2-en-3-yl]methyl]-1-methylpyrrolidinium hydroxide, inner salt, 7²-(Z)-(O-methyloxime) in human serum. Separation was achieved on a reversed-phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4). Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV absorbance at 254 nm. The lower limit of quantitation of cefepime in plasma was 0.5 μg/ml. The average absolute recovery was 106.2±2.1%. The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient greater than 0.999. The within-day C.V.s for human samples were 4.9 and 2.3% for 1 and 50 μg/ml, respectively. The between-day C.V.s for human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50 μg/ml, respectively. Cefepime was found to be unstable in serum at room temperature. For delayed assay, samples must be stored at −80°C.     

High-performance liquid chromatographic assay for meropenem in serum

High-performance liquid chromatographic procedures have been developed for the measurement of meropenem in serum. The separation was performed on an Ultrasphere XL-ODS analytical column (75×4.6 mm I.D.). The mobile phase consisted of 10.53 mmol/l ammonium acetate-acetonitrile (95:5, v/v) (pH 4). The UV detection was at 298 nm. The quantitation limit both in serum and water was 0.25 μg/ml. The method was validated in serum and aqueous solution over the concentration range 0.25–50 μg/ml. The extraction recovery from serum spiked with meropenem was 99.7±3.4%. The intra- and inter-assay coefficients of variation were below 6%. Stored at −80°C for three months at various concentrations in serum and in aqueous solution, meropenem did not reveal any appreciable degradation. After 24 h, it was also stable at 4°C in serum, aqueous solution and supernatant of extraction but not at room temperature. The stability of the drug was also confirmed in serum after repeated freezing-thawing cycles at −80°C on four consecutive days.     

Polypropylene straw ampoules for the storage of microorganisms in liquid nitrogen

Liquid nitrogen storage systems are widely used for long term preservation of a range of microorganisms. Strains are stored in ampoules immersed in nitrogen or held in its evolving vapour. Immersion storage maximises the storage capacity of a vivostat but imposes limits on the type of ampoule that can be used. It is of vital importance that an ampoule can be sealed to prevent leakage of the nitrogen during storage. Several ampoule types are commercially avaailable but all have a number of disadvantages in use. They are costly, wasteful of space and difficult to seal reliably. An ampoule which can be simply made in the laboratory from polypropylene drinking straws has been described which is cheap, space saving and easy to seal. These ampoules have been in use for ten years without any problems and their use in the storage of over 2000 strains, predominantly fungi, is described. They have no detrimental effect on strain retrieval as shown by recovery rates after protracted storage. Straw ampoules provide a cheap and safe alternative to those ampoules that are commercially available and can be recommended for use in liquid nitrogen storage systems.     

Simple and sensitive high-performance liquid chromatographic method for the investigation of dynamic changes in the redox state of rat serum albumin

Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MA(r)) and rat non-mercaptalbumin (NA(r)), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MA(r) fraction in control and exercise groups were 76.2+/-1.8 and 69.0+/-3.5%, respectively. The MA(r) in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress.     

Determination of alosetron in human plasma or serum by high-performance liquid chromatography with robotic sample preparation

A method of analysis for the determination of alosetron in human plasma or serum has been developed. The method was fully automated using a laboratory robot in order to improve analytical precision, efficiency and safety. The assay involved solid-phase extraction with reversed-phase HPLC separation and fluorescence detection. A validation exercise over the concentration range of 0.1 to 20 ng/ml demonstrated the selectivity, linearity, sensitivity, accuracy, precision, extraction efficiency, ruggedness and stability of the method. The method has been applied in support of numerous human pharmacokinetic/biopharmaceutic studies over the last five years.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the chiral reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C₈ column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Preservation of Coombs' serum in liquid nitrogen

Experiments with Coombs-sera as raw and usable sera having different levels of titre were carried out in order to examine their stability in fluid nitrogen. Therewith, significant differences concerning the storing stability did not appear. Generally seen two third of the sera retained their initial titres at the end of one year in store.