An autoclavable intensive care isolator for the adult gnotobiotic dog.

A lightweight stainless steel isolator which allows 3 investigators simultaneous access to an adult gnotobiotic dog for the purpose of rendering intensive care was designed and constructed. This isolator is 54' high X 60' long X 42' wide. It has an 18' diam entry port at 1 end and a pair of gloves mounted beneath a window on each of the other 3 sides. The chamber rests on a 12' high pallet and is moved about the laboratory by a portable hydraulic platform lift. It can be rapidly cleansed with soap and water and sterilized in a large autoclave. Over the past 2 yr. this intensive care isolator has enabled us to perform a variety of manipulations on the adult gnotobiotic dog in a safe, convenient, and efficient manner.     

An autoclavable electronic interface for germfree isolators

An electronic interface was designed and fabricated for use with germfree isolators. It allows a variety of sensors or electrical instruments inside an isolator to be readily connected to monitoring or activating devices on the outside of an isolator. It was built into a 25 cm diameter cylinder whose open ends were loosely covered by an inner and outer connector panel. These two panels were wired together through an airtight dividing wall placed transversely across the middle of the cylinder. The interface was mounted in an entry port of a stainless steel isolator and steam sterilized with the isolator. It has proven to be a useful, contamination-free, durable device.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Infectivity of scrapie prions bound to a stainless steel surface.

BACKGROUND: The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity. MATERIALS AND METHODS: As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease. RESULTS: Stainless steel wire (0.15 x 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 10(5) LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold. CONCLUSIONS: The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments.     

Application of stripping voltammetry and microelectrodes in vitro biocompatibility and in vivo toxicity tests of AISI 316L corrosion products.

Adsorptive stripping voltammetric procedures, using mercury film microelectrodes, were optimised and applied to quantify total iron, chromium and nickel in samples of osteoblast-like cells culture medium and mice organs (liver, kidney and spleen) obtained from, respectively, in vitro and in vivo 316L stainless steel corrosion products biocompatibility and toxicity studies. The methods were based on the pre-concentration of the iron-catechol complex by adsorption at the potential of -1.80 V (vs. Ag/AgCl), of the chromium-diethylenetriaminepentaacetic acid complex at -1.00 V or -1.15 V (vs. Ag/AgCl) and of the nickel-dimethylglyoxime complex at -0.70 V (vs. Ag/AgCl). The detection limits achieved for each metal ion (i) in the culture medium were 1.93x10(-8) mol/L Fe, 2.80x10(-10) mol/L Cr and 7.70x10(-9) mol/L Ni for a collection time of 30 s, 40 s and 10 s, respectively, and (ii) in the mice organ solutions were 1.37x10(-8) mol/L Fe, 1.54x10(-8) mol/L Cr and 1.58x10(-9) mol/L Ni for an adsorption time of 25 s, 25 s and 15 s, respectively. The accuracy of the proposed procedures was verified by comparison of the results obtained by adsorptive stripping voltammetry with those attained by atomic absorption spectrometry for the same set of samples and good agreement was found. The in vitro study showed that stainless steel corrosion products affect the expression of the osteogenic phenotype. The in vivo mice model, used to investigate the systemic effects provoked by the corrosion products per se, indicated that Fe, Cr and Ni are partially accumulated in the organs studied and that Ni induced the more significant morphological alterations.     

The diamond-tip rasp

A new nasal rasp has been developed from tungsten-carbide steel and is available in eight different cutting grits. One-hundred rhinoplasties were completed using a single tungsten-carbide steel rasp without evidence of dulling or loss of cutting effectiveness. Both a conventional stainless steel Fomon rasp and the diamond-tip tungsten-carbide steel rasp were wear tested on an accelerator for comparison. The testing involving the conventional stainless steel rasp was discontinued after the removal of 20 mm of bone. At this time, there was severe loading of the instrument with bony debris and there was an apparently complete lack of cutting ability. The test involving the diamond-tip tungsten-carbide steel rasp was electively discontinued after the removal of 300 mm of bone, at which time there was no evidence of either debris loading or diminished cutting effectiveness. The scanning electron microscope photographs of the two instruments support our conclusions regarding the clinical and laboratory studies.     

Inactivation of influenza A virus on copper versus stainless steel surfaces

Influenza A virus particles (2 x 10(6)) were inoculated onto copper or stainless steel and incubated at 22 degrees C at 50 to 60% relative humidity. Infectivity of survivors was determined by utilizing a defined monolayer with fluorescent microscopy analysis. After incubation for 24 h on stainless steel, 500,000 virus particles were still infectious. After incubation for 6 h on copper, only 500 particles were active.     

Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists.

Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken. The current procedure lacks precision and accuracy and is not quantitative. Variability associated with carriers and the lack of temperature control were evaluated in this paper. The use of porcelain versus stainless steel carriers was also evaluated. When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale. The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU). The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension. Variations in drying time did not alter the number of cells attached to the carrier. When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96%. Data for B. subtilis spores were similar to those for M. bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers. It was also found that the lack of an exacting temperature control could influence the outcome of the test. Changes in temperature as little as 1 degree C could influence the rate of killing of M. bovis BCG.     

Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists

Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken. The current procedure lacks precision and accuracy and is not quantitative. Variability associated with carriers and the lack of temperature control were evaluated in this paper. The use of porcelain versus stainless steel carriers was also evaluated. When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale. The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU). The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension. Variations in drying time did not alter the number of cells attached to the carrier. When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96%. Data for B. subtilis spores were similar to those for M. bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers. It was also found that the lack of an exacting temperature control could influence the outcome of the test. Changes in temperature as little as 1 degree C could influence the rate of killing of M. bovis BCG.     

Contractile function in vitro of slow-twitch skeletal muscle from weanling mice subjected to wasting malnutrition.

Our hypothesis was that malnutrition sufficient to produce weight loss in weanling mice would decrease the ability of slow-twitch skeletal muscle to develop and maintain force. We isolated muscles from 3 groups (n = 5) of weanling C57BL/6J mice of both sexes (i) mice at 19 days of age serving as zero-time or baseline controls (CONT) (ii) mice fed for the next 14 days with a low-protein diet that produces features of incipient kwashiorkor (LPD) and (iii) mice fed for the next 14 days with a complete diet (NORM). Muscles were also obtained from 5 adult mice 7-9 months of age (MAT). We stimulated the soleus at 50 Hz for 500 ms at 0.6 tetanic contractions per min (tet x min(-1)), 6 tet x min(-1), and 30 tet x min(-1) in Krebs-Henseleit bicarbonate buffer at 27 degrees C gassed with 95% O2 and 5% CO2. The initial developed force (mN x mm(-2)) at 0.6 tet x min(-1) did not differ across groups (CONT 211.7 +/- 16.0, LPD 274.2 +/- 41.6, NORM 246.8 +/- 38.0, MAT 210.8 +/- 10.6). The fatigue rate (mN x mm(-2) x min(-1)) at 6 tet x min(-1) was significantly slower in muscles from CONT (0.6 +/- 0.3) and LPD (0.6 +/- 0.4) than in NORM (2.4 +/- 0.6) and MAT (2.3 +/- 0.2). At 30 tet x min(-1), the fatigue rate (mN x mm(-2) x min(-1)) did not differ across groups (CONT 2.4 +/- 0.5, LPD 2.7 +/- 0.5, NORM 2.5 +/- 0.4, MAT 2.0 +/- 0.2). After stimulation at 6 tet x min(-1) and 30 tet x min(-1), only muscles from CONT and LPD recovered to 100%. Because muscles from LPD mice developed equal force, fatigued less, and recovered from fatigue to a greater extent than muscles from NORM mice, we rejected the hypothesis. The function of the tissue remaining in the muscles from LPD mice approximated that of muscles from mice at 19 days of age rather than muscles from either mice of the same age fed a complete diet or adult mice.     

Influence of the load of nylon capsules on their passage through the digestive tract and specific gravity

The passage and specific gravity of nylon capsules were evaluated in five trials. In individual trials, different lactating cows were fed the same diet consisting of maize silage, alfalfa hay and concentrate. In each trial the different feeds (or no feed) were used to fill the capsules. The capsules were made of nylon cloth (42 microm pore size, 10 mm external diameter). The different weights of the load (L1-L5) were obtained using a combination of 2 and 3 mm stainless steel balls. The highest recovery of the capsules was obtained with the L3 and L4 loads (91.4 and 92.3%, resp.). After 14 hours of incubation in the rumen, the calculated values of functional specific gravity of the capsules ranged from 0.92 to 2.05 g x cm(-3). It was concluded that L3 (one 2 mm and one 3 mm ball) was the suitable weight of the load.     

Enantioseparation of dipeptides and tripeptides by micro-HPLC comparing teicoplanin and teicoplanin aglycone as chiral selectors

Chiral separation of glycyl- and diastereomeric dipeptides and tripeptides was performed by micro-HPLC using macrocyclic antibiotics as chiral selectors. Teicoplanin was compared with teicoplanin aglycone (TAG) regarding selectivity, efficiency and separation time. The stationary phases are based on teicoplanin and TAG chemically bonded to 3.5 mum silica gel. The material was packed into 10 cm x 1 mm stainless steel microcolumns. Different mobile phases were checked using the reversed phase mode. Both teicoplanin and TAG were found to show good chiral separation ability for dipeptides. Glycyl-dipeptides were baseline resolved and most of the diastereomeric dipeptides and tripeptides were separated into their four isomers. In this study, teicoplanin was found to be advantageous compared to TAG regarding separation time, although TAG showed the higher resolution power. Baseline resolution for some glycyl-dipeptides was obtained within 3 min, diastereomeric dipeptides were resolved in 7 min. This method was also shown to be applicable for enantiomer purity control.     

Ultrasonic methodology coupled to ATP bioluminescence for the non-invasive detection of fouling in food processing equipment--validation and application to a dairy factory

The use of an ultrasonic apparatus (40 kHz) for the non-destructive, rapid and reproducible removal of biofilm from standard materials (stainless steel and polypropylene) in a dairy factory was investigated. The application of ultrasound with the tested conditions (10 s and 40 kHz) was found not to be detrimental for standard ATP (concentration ranging between 5 x 10(-9) and 10(-5) mol 1(-1)) and for prokaryotic cells, including both rods and coccoid-shaped bacteria (Escherichia coli and Staphylococcus aureus). It allowed the use of the ATP bioluminescence measurement for quantifying the biofilm removal. The repeatability of industrial milk removal was determined on fouled stainless steel and polypropylene sheets. The variability of the results with the sonication method was constant, +/-24% (coefficient of variation) for both surfaces, and was variable with the swabbing method, +/-42% for the stainless steel sheet and +/-74% for the polypropylene sheet. The ultrasonic apparatus removed twice the amount of industrial milk biofilm compared with the swabbing method in the case of the polypropylene sheets. The apparatus was used to validate the industrial cleaning protocols of a milk factory.     

New chiral stationary phases based on xanthone derivatives for liquid chromatography

Six chiral derivatives of xanthones (CDXs) were covalently bonded to silica, yielding the corresponding xanthonic chiral stationary phases (XCSPs). The new XCSPs were packed into stainless‐steel columns with 150 x 4.6 mm i.d. Moreover, the greening of the chromatographic analysis by reducing the internal diameter (150 x 2.1 mm i.d.) of the liquid chromatography (LC) columns was also investigated. The enantioselective capability of these phases was evaluated by LC using different chemical classes of chiral compounds, including several types of drugs. A library of CDXs was evaluated in order to explore the principle of reciprocity as well as the chiral self‐recognition phenomenon. The separation of enantiomeric mixtures of CDXs was investigated under multimodal elution conditions. The XCSPs provided high specificity for the enantiomeric mixtures of CDXs evaluated mainly under normal‐phase elution conditions. Furthermore, two XCSPs were prepared with both enantiomers of the same xanthonic selector in order to confirm the inversion order elution.     

Effects of double bands on Magellanic Penguins

ABSTRACT Banding penguins is controversial because bands can alter the survival, reproduction, and behavior of marked individuals. The effects of bands are not consistent among band types and, although stainless steel is thought to be better than other materials, tests of the long‐term impact of bands on tag‐loss rates and the reproduction and survival of individuals are needed. We tested three types of external tags on Magellanic Penguins (Spheniscus magellanicus) to measure band effects and tag‐loss rates. In 1993, we double‐tagged 300 penguins with aluminum flipper bands, stainless‐steel flipper bands, or small (2 mm × 10 mm) metal tags attached to foot webbing. We searched for double‐tagged birds for 13 of 15 yrs (1994–2008). Aluminum bands deformed, caused feather wear, injured and killed some penguins, and were lost more often than stainless‐steel bands or web tags. During the first 2 yrs of our study, at least nine penguins lost one aluminum band (N= 71 penguins resighted), but no penguins lost a stainless‐steel band (N= 84) or a web tag (N= 88). During the next 13 yrs, five penguins lost one of their two web tags (N= 89), but none lost a stainless‐steel band (N= 84). Females laid eggs of similar size before they carried a band and in the year following tagging (P= 0.09). The type of tags a female carried did not significantly change egg size (P > 0.22). During the first breeding season after tagging, penguins with aluminum bands had lower reproductive success than penguins with stainless‐steel bands or web‐tags (P= 0.04). The annual survival of females with two stainless‐steel bands was lower (0.79) than that of males with two stainless‐steel bands or males and females with two web‐tags (0.87). Aluminum bands injured Magellanic Penguins, were lost at high rates, and should not be used. Double stainless‐steel bands had no apparent effects on adult male Magellanic Penguins, but reduced survival rates of adult females. A single stainless‐steel band would likely have less impact than two bands, and our results suggest that the impact of a single band would be difficult to measure.     

Synthesis and evaluation of a selective molecularly imprinted polymer for the contraceptive drug levonorgestrel

A molecularly imprinted polymer (MIP) has been prepared using levonorgestrel (LEV) as template. The polymer was synthesised in a non-covalent approach using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linking monomer via a free radical polymerization. An equivalent blank polymer was also synthesised in the absence of the template compound. Batch adsorption experiments were used to evaluate the binding affinity of the imprinted polymer. After packing MIP into a stainless steel column (150 mm x 4.6 mm i.d.), retention and elution of the template and related compounds were evaluated by high-performance liquid chromatography (HPLC). This LEV imprinted polymer was further applied for selective solid phase extraction (SPE) of LEV from human serum. It was confirmed that the binding ability of the prepared MIP for LEV was essentially sufficient in the presence of other compounds coexisting in serum sample. Therefore, as a selective and efficient solid phase material, LEV imprinted polymer has a high potential application in analysis of this steroidal hormone in clinical purposes.     

Electrical stimulation of human embryonic stem cells: Cardiac differentiation and the generation of reactive oxygen species

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H₂O₂. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.     

Heat transfer analysis of Staphylococcus aureus on stainless steel with microwave radiation

Staphylococcus aureus (NCTC 6571; Oxford strain) on stainless steel discs was exposed to microwave radiation at 2450 MHz and up to 800 W. Cell viability was reduced as the exposure time increased, with complete bacterial inactivation at 110 s, attaining a temperature of 61.4 degrees C. The low rate of temperature rise, RT, of the bacterial suspension as compared with sterile distilled water or nutrient broth suggests a significant influence of the microwave sterilization efficacy on the thermal properties of the micro-organisms. The heat transfer kinetics of thermal microwave irradiation suggest that the micro-organism has a power density at least 51-fold more than its surrounding liquid suspension. When the inoculum on the stainless steel disc was subjected to microwave radiation, heat conduction from the stainless steel to the inoculum was the cause of bacteriostasis with power absorbed at 23.8 W for stainless steel and 0.16 W for the bacteria-liquid medium. This report shows that the microwave killing pattern of Staph. aureus on stainless steel was mainly due to heat transfer from the stainless steel substrate and very little direct energy was absorbed from the microwaves.     

Overexpression of eNOS prevents the development of renovascular hypertension in mice

Gene therapy has become an important tool for understanding several cardiovascular diseases. In the present study we investigated the effects of endothelial nitric oxide synthase (eNOS) overexpression on renovascular hypertension. Experiments were carried out in C57BL/6 mice randomly assigned to either a two-kidney one-clip (2K1C) hypertension group or a sham-operated group. At the same time surgery was carried out, both 2K1C and sham mice received an intravenous injection of recombinant adenovirus expressing the functional gene eNOS or the reporter gene beta-galactosidase (beta-gal). Fourteen days later, arterial pressure, baroreflex sensitivity, and cardiac sympathetic and parasympathetic tone were evaluated in conscious mice. Measurement of mean arterial pressure showed arterial hypertension in 2K1C-betagal mice compared with sham-betagal mice (121 +/- 3 vs. 96 +/- 2 mm Hg, p < 0.01), which was prevented by eNOS overexpression (2K1C-eNOS 100 +/- 4 vs. sham-eNOS 99 +/- 3 mm Hg). Linear regression analysis of the reflex tachycardia response to sodium nitroprusside-induced hypotension showed that baroreflex sensitivity was significantly attenuated in 2K1C-betagal mice (5.8 +/- 0.5 vs. sham-betagal 8.0 +/- 0.8 beats.min-1 x mm Hg-1, p < 0.05), but this decrease was not prevented by eNOS overexpression (2K1C-eNOS 7.2 +/- 0.5 vs. sham-eNOS 8.8 +/- 0.7 beats x min-1 x mm Hg-1, p < 0.05). The cardiac sympathetic tone was augmented and the vagal tone was reduced in 2K1C-betagal (152 +/- 17 and 45 +/- 12 beats.min-1, respectively) compared with sham-betagal mice (112 +/- 6 and 89 +/- 7 beats.min-1, respectively), and similar results were observed in 2K1C-eNOS mice compared with sham-eNOS. The data indicate that eNOS overexpression was able to prevent the development of 2K1C renovascular hypertension in mice, without affecting other characteristic cardiovascular dysfunctions.     

Procurement and Maintenance of Germ-Free Swine for Microbiological Investigations

Germ-free swine were routinely procured by both hysterectomy and hysterotomy (Caesarian section). By means of light-weight portable equipment, piglets could be obtained and transported to the laboratory (without contamination) over distances in excess of 100 miles. The isolators employed in rearing were constructed of stainless steel and flexible plastic film. At weekly intervals, fecal swabs and waste from the floor of the isolator were cultured on blood-agar and in thioglycolate broth, as well as being examined microscopically for the presence of bacteria, yeast, and fungi. The presence of pleuropneumonia-like organisms (PPLO) and viruses in such material was not demonstrable, either by the use of enriched PPLO media or primary porcine-kidney cell cultures. Tissues, body fluids, and cecal contents of piglets sacrificed specifically for microbiological examination were also negative for PPLO, viruses, bacteria, yeast, and fungi. Prenatal infestations by ascarids were not observed. Nutritional problems related to rearing of germ-free piglets, such as hypoglycemia, were not encountered, and the use of an autoclaved commercial sow's milk replacer proved quite satisfactory. The temperature to which piglets were subjected during the first few days of life, however, was very important. The isolator design and application of gnotobiotic techniques to the procurement and rearing of a large germ-free animal such as the pig proved feasible and less difficult than anticipated.