Microbial Penetration Through Three Types of Double Wrappers for Sterile Packs

Microbial penetration of sterile packs was studied by using double-wrap (two layers each) muslin, single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC wrappers to wrap 20 gauze sponges (2 by 2 in.). These packs were stored on open shelves of a central sterile supply department of a hospital and processed for sterility at weekly intervals. Microorganisms penetrated the double-wrap muslin as early as 28 days, the single-wrap muslin and single-wrap two-way crepe paper combination in 77 days, and the single-wrap muslin and single-layer BAR-BAC combination in 63 days.     

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

Summary A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of Parafilm^TM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.     

The influence of storing beef aerobically or in vacuum packs on the shelf life of mince

Aims: To investigate the influence of aerobic or vacuum pack storage of beef trimmings on the microbiology, colour and odour of subsequently produced mince. Methods and Results: Trimmings stored aerobically for 7 or 10 days and in vacuum packs for 7, 10, 14 or 22 days at 0 or 5°C were minced, stored aerobically at 0 or 5°C for up to 7 days and examined daily to determine Total viable, Pseudomonas, Lactic acid bacteria, Brochothrix thermosphacta, and Enterobacteriaceae counts, colour and odour. Mincing reduced counts, particularly of Pseudomonas, B. thermosphacta and Enterobacteriaceae, probably because of the action free radicals released from muscle and bacterial cells. Storage of vacuum‐packed trimmings for 22 days resulted in improved mince colour and inhibition of the growth of Pseudomonas. Conclusions: The shelf life of mince from trimmings is directly influenced by the trimmings storage conditions, and longer‐term vacuum storage of trimmings produced improvements in mince quality. Significance and Impact of the Study: There appears to be no scientific rationale for limiting the storage of vacuum packaging beef trimmings to 15 days, prior to mince production, as stated in EU 835/2004. This study identifies advantages in storing trimmings in vacuum packs for at least 21 days prior to mincing, in terms of improved mince quality.     

Effects of shelf architecture and parasitoid release height on biological control of Plodia interpunctella (Lepidoptera: Pyralidae) eggs by Trichogramma deion (Hymenoptera: Trichogrammatidae)

The effects of shelving type, packaging, and release height on success of Trichogramma deion Pinto & Oatman (Hymenoptera: Trichogrammatidae) parasitizing Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) eggs was studied under laboratory conditions. In trials on multipletiered gondola-type or open shelving units, with or without packaging, foraging success was evaluated by comparing parasitism and total mortality rates of sentinel egg disks among shelves after a single point-release of T. deion. Results showed that T. deion parasitized more egg disks and killed more total eggs on open shelves than on gondola shelving. The presence of packaging had no effect on parasitoid foraging on open shelves; however, packaging did interfere with parasitism of P. interpunctella eggs on gondola shelving. Egg parasitism and mortality patterns among shelves were not as evenly distributed on gondola-type shelving compared with open shelving. On gondola shelves without packages, changing the release point of T. deion from the middle to the lowest shelf shifted the distribution of parasitism toward the floor. Gondola shelving, especially in the presence of packaging, reduced foraging efficiency of T. deion for P. interpunctella eggs. Thus, to attain adequate control of P. interpunctella, it may be necessary to use two release heights on gondola shelving.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Territory fidelity, space use, and survival rates of wild coyotes following surgical sterilization

Sterilization of wild canids is being used experimentally in many management applications. Few studies have clearly demonstrated vasectomized and tubal-ligated canids will retain pair-bonding and territorial behaviors. We tested whether territory fidelity, space use, and survival rates of surgically sterilized coyote (Canis latrans) packs were different from sham-operated coyote packs. We captured and radio-collared 30 coyotes in December 2006. Sixteen of these animals were sterilized via vasectomy or tubal ligation, and 14 were given sham-surgeries (i.e., remained intact). We monitored these animals using telemetry and visual observations through 2 breeding seasons and 1 pup-rearing season from December 2006 to March 2008. Mean pack size was not significantly different between sterile and intact coyote packs. We found no difference in home range size between sterile and intact coyotes. We found differences in home range and core area overlap between sterile and intact coyote packs in some seasons; however, this difference may have existed prior to sterilization. Home range fidelity was not significantly different between sterile and intact coyotes. All coyotes had higher residency rates during the breeding season, with no differences between sterile and intact coyotes. Survival rates were correlated with biological season, but there were no differences in survival rates between sterile and intact coyotes. We concluded that surgical sterilization of coyotes did not affect territory fidelity, survival rates, or home range maintenance.     

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.     

Open fronted safety cabinets in ventilated laboratories

Open fronted Class I and II microbiological safety cabinets (MSCs) are required by the British Standard 5726 to provide similar levels of operator protection (viz. 10⁵). In laboratories that are naturally ventilated large numbers of both types of cabinets have been shown to exceed this requirement consistently over a number of years. The designs of some mechanically ventilated laboratories, however, produce excessive turbulence and draughts that can prejudice containment at the front aperture. On-site commissioning tests to determine operator protection factor are now well established and are recognized as being essential to the setting up of all open fronted cabinets in both ventilated and unventilated laboratories. This paper shows that where environmental conditions induce unsatisfactory cabinet containment, adjustments to air supply and exhaust systems can be made which will enable both Class I and II cabinets to produce operator protection factors in excess of 10⁵. When compatibility is achieved between the local environment and the cabinets it is demonstrated that disturbances at the front aperture, caused by operator working procedures or by disturbances due to personnel movement within the room, have similar effects on both Class I and II cabinets. Once performance levels have been satisfactorily achieved, regular containment testing has shown that consistent performance can be maintained. These aspects of open fronted safety cabinet performance are discussed in relation to ventilated laboratories suitable for work with the human immunodeficiency virus (HIV). Of paramount importance in the future is the necessity to design laboratory air systems that will be compatible with satisfactory safety cabinet performance—a relatively new requirement in ventilation system specifications.     

Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani

1. During development of Paragonimus westermani, larvae develop during migration within the host, and adult worms feed on pulmonary tissues, causing significant pathology in the mammalian host. In this report acidic extracts of various developmental stages (metacercariae and worms at one, two and three months of development) were examined for cysteine proteinase activity. 2. A soluble thiol-dependent proteinase activity with a native molecular weight of approximately 20,000 was isolated and partially purified. 3. The enzymes purified from the various developmental stages of the parasite had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. 4. Enzymatic activity was stable at pH 5.0 for at least two days when stored at 4 degrees C. 5. It is suggested that these enzymes may be involved in the nutrition of these parasites and/or during penetration and lysis of the tissues.     

Enhancing mating performance after juvenile hormone treatment in Anastrepha fraterculus: a differential response in males and females acts as a physiological sexing system

Methoprene (a mimic of juvenile hormone) treatment can reduce the time required for sexual maturation in Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) males under laboratory conditions, supporting its use as a treatment for sterile males within the context of the sterile insect technique (SIT). We evaluated sexual behaviour, mating competitiveness of methoprene-treated males, and female readiness to mate after methoprene-treatment in field cages. The study involved two strains of A. fraterculus from Argentina and Peru, which show several polymorphisms in relation to their sexual behaviour. We also analyzed whether methoprene treatment affected male and/or female behaviour in the same way in these two strains. Methoprene-treated males were equally competitive with untreated mature males, and became sexually competitive 6 days after emergence (3–4 days earlier than untreated males). In contrast, methoprene did not induce sexual maturation in females or, at least, it did not induce a higher rate of mating in 7-day-old females. These results were observed both for the Argentina and the Peru strains. Altogether, our results indicate that methoprene treatment produces sexually competitive males in field cages. In the absence of a genetic sexing system, and when sterile males and females of A. fraterculus are released simultaneously, the fact that females do not respond as do males to the methoprene treatment acts as a physiological sexing effect. Therefore, in the presence of mainly sexually immature sterile females, released sexually mature sterile males would have to disperse in search of wild fertile females, thereby greatly reducing matings among the released sterile insects and thus enhancing sterile insect technique efficiency.     

In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.     

Bacterial flora in bottled uncarbonated mineral drinking water

A quantitative study of bacterial populations in mineral water was carried out. Samples were stored at 6 and 20 degrees C, and the colony counts were determined on tryptone agar plates incubated at 22 and 37 degrees C. Samples were collected from the spring source in sterile glass flasks and from the bottling factory in conventional plastic and glass containers. In both cases, the initial population (10(1)-10(2) cfu/mL water) increased to 10(5)-10(6) cfu/mL after 3 days storage as determined from plate counts incubated at 22 degrees C. The levels reached by this population were similar to those of samples of mineral water obtained at the market stage. Results from plate counts incubated at 37 degrees C showed that populations in samples collected at the bottling factory reached 10(2)-10(3) cfu/mL. No growth was observed in water collected from spring source. Bacterial multiplication was not stopped even when water was stored at 6 degrees C. Caulobacter was the genus found most frequently in both types of samples, followed by Sphaerotilus-Leptothrix. Acinetobacter calcoaceticus and Pseudomonas fluorescens were frequently found in only two springs, and Pseudomonas putida, Arthrobacter, Aeromonas hydrophilia, and Corynebacterium were isolated less frequently. Janthinobacterium was recovered only once from a single spring. A giant bacterium closely resembling Hyphomicrobium and a budding one similar to Pasteuria were recovered from all samples of a single spring and from some of the commercial samples.     

Bacterial sources of putrescine and cadaverine in chill stored vacuum-packaged beef

Of the meat strains of streptobacteria, leuconostocs, Enterobacteriaceae and Brochothrix thermosphacta tested, only Hafnia alvei and Serratia liquefaciens showed diamine-producing potential during growth in pure culture on beef stored in vacuum packs at 1 degree C. Both organisms produced cadaverine at concentrations similar to those reported previously in naturally contaminated beef stored under the same conditions. Putrescine concentrations produced by the two organisms, however, were an order of magnitude lower. During the growth on beef of either H. alvei or S. liquefaciens in mixed culture with arginine-utilizing strains of streptobacteria, putrescine as well as cadaverine concentrations were similar to those detected in naturally contaminated samples.     

Effect of seedling treatment on growth and yield of sugar beet in the field

Sugar-beet seeds were germinated (1) in a growth cabinet at 20°C lit continuously by fluorescent tubes (L), (2) in a cabinet at 20°C lit by fluorescent tubes for 16 h/day (S), (3) in a cage with glass roof and open sides with natural illumination (N), or (4) in the open ground (D). The seedlings from the cabinets and cage were transplanted to the field when they had two true leaves. Samples were taken on six occasions during growth, and leaf areas and dry weights determined. There were no differences between treatments in total number of leaves produced or leaf area duration. Leaf area per plant increased fastest on L plants at first, but from mid-June until end of July drilled plants had the largest leaf surface. From August onwards S plants had the largest area. Although treatment had little effect on growth of the tops, roots grew fastest throughout the season on the plants raised in growth cabinets and the final mean root dry weight of L and S plants was 39% greater than of N and D plants. Throughout the season L and S plants had a larger root:top ratio than plants raised in the cage or drilled directly in the field. The larger roots of plants raised in the cabinets evidently provided a larger sink for carbohydrate and increased the mean photosynthetic efficiency of the leaves over the whole season by 11 % and increased yield of roots by 6 tons/acre.     

Midline fusion in the formation of the secondary palate anticipated by upregulation of keratin K5/6 and localized expression of vimentin mRNA in medial edge epithelium.

Secondary palatal fusion is dependent on targeted removal of the epithelium between the palatal shelves. Aseptically delivered rat embryos 15 through 18 days post coitum (dpc) were probed with DIG-labeled antisense and sense ssDNA probes for spliced exon sequences flanking intron E of cytokeratins K5/6 and spliced exon sequences flanking intron F of vimentin. Cytokeratin K5/6 expression was upregulated in the medial edge epithelium (MEE) prior to rotation of the palatal shelves and in the vomerine epithelium in the region of fusion with the palate. K5/6 expression continued in the medial epithelial seam (MES) and in epithelial islands during breakdown of the MES. Vimentin expression was not detected in the MEE prior to rotation but was specifically upregulated in the MEE following rotation and prior to midline contact and continued in the MES and in epithelial cells identifiable during the breakdown of the MES. Initiation of vimentin upregulation in the MEE prior to contact of the palatal shelves was tested by serum-free organ culture of palates from embryos at 15.5 dpc with the shelves separated by a biocompatible membrane. Vimentin upregulation occurred in the epithelium specifically in the region of anticipated contact. These results are interpreted as indicating that i) cytokeratin K5/6 expression may play a critical role in the integration of the epithelial layers of the MES to ensure subsequent merging of the mesenchyme and ii) epithelial cells in the MEE are specifically 'primed' to upregulate expression of mesenchymal genes prior to integration into and breakdown of the MES.     

Rescue of an in vitro palate nonfusion model using interposed embryonic mesenchyme

The authors previously established an in vitro palate nonfusion model on the basis of a spatial separation between prefusion embryonic day 13.5 mouse palates (term gestation, 19.5 days). They found that an interpalatal separation distance of 0.48 mm or greater would consistently result in nonfusion after 4 days in organ culture. In the present study, they interposed embryonic palatal mesenchymal tissue between embryonic day 13.5 mouse palatal shelves with interpalatal separation distances greater than 0.48 mm in an attempt to "rescue" this in vitro palate nonfusion phenotype. Because no medial epithelial bilayer (i.e., medial epithelial seam) could potentially form, palatal fusion in vitro was defined as intershelf mesenchymal continuity with resolution of the medial edge epithelia bilaterally. Forty-two (n = 42) palatal shelf pairs from embryonic day 13.5 CD-1 mouse embryos were isolated and placed on cell culture inserts at precisely graded distances (0, 0.67, and 0.95 mm). Positive controls consisted of shelves placed in contact (n = 6). Negative controls consisted of shelves placed at interpalatal separation distances of 0.67 mm (n = 6) and 0.95 mm (n = 7) with no interposed mesenchyme. Experimental groups consisted of embryonic day 13.5 palatal shelves separated by 0.67 mm (n = 11) and 0.95 mm (n = 12) with interposed lateral palatal mesenchyme isolated at the time of palatal shelf harvest. Specimens were cultured for 4 days (n = 19) or 10 days (n = 23), harvested, and evaluated histologically. All positive controls at 4 and 10 days in culture showed complete histologic palatal fusion. All negative controls at 4 days and 10 days in culture remained unfused. Five of six palatal shelves separated at 0.67 mm interpalatal separation distance with interposed mesenchyme were fused at 4 days, and all five were fused at 10 days. At an interpalatal separation distance of 0.95 mm with interposed mesenchyme (n = 12), no palates (zero of four) were fused at 4 days, but seven of eight were fused at 10 days. These data suggest that nonfused palatal shelves can be "rescued" with an interposed graft of endogenous embryonic mesenchyme to induce fusion in vitro.     

Annelid sperm and fertilization biology

This paper provides data on fine particulate organic matter (FPOM) and macroinvertebrates associated to natural and artificial leaf packs in a small woodland stream (Schlaube, Brandenburg). Macroinvertebrate colonisation and the dynamics of FPOM were studied in oven-dried alder leaf packs, air-dried alder leaf packs and packs with artificial leafshaped substrate exposed in the stream during a 68-day period. The importance of FPOM as a potential food source for macroinvertebrates especially in artificial leaf packs was evaluated. Changes in the quantity as well as in the chemical composition of the accumulating FPOM (>63 and <63 μm) was determined using soluble carbohydrates, proteins and chlorophyll a as parameters of the nutritional quality. Mass loss and the chemical changes of alder leaves during the decompositional process were also described. The loss of soluble carbohydrates due to leaching was more rapid in oven-dried alder leaf packs than in air-dried ones. After 3 days of leaf pack exposure weight loss of oven-dried and air-dried leaf packs was nearly comparable, as the similar decay coefficients, k = 0.0228 (oven-dried leaf packs) and k = 0.0214 (air-dried leaf packs), respectively, show. The amount of FPOM per unit leaf area constantly increased in artificial packs, although it remained below that of alder leaf packs at all sampling dates. The nutritional quality of FPOM <63 μm was constantly greater than that of FPOM >63 μm and decreased in both size-fractions with length of exposure. Referring to leaf area the abundance of macroinvertebrates continually increased in all packs till the end of exposure, whereas the numbers in artificial packs remained below that in alder leaf packs. The taxonomic composition of all treatments was very similar with Gammarus pulex being the most abundant taxon in all packs until day 42, while afterwards the caddis fly genus Hydropsyche gained in importance. The amphipod Gammarus pulex in general did not show a preference for air-dried alder leaf packs compared to oven-dried alder leaf and artificial packs. Corresponding dynamics of macroinvertebrate colonisation and FPOM content in artificial packs support the hypothesis that FPOM functions not only as an important food source for macroinvertebrates including gammarideans but also as a control mechanism of macroinvertebrate abundance in stream habitats. Even if the accumulation of FPOM and drifting macroinvertebrates might be influenced by the same abiotic factor (e.g. by reduction in stream velocity inside the packs) it is quite unlikely that only physical properties caused the invertebrates to stay.     

QUALITY OF POWDERED BLACK PEPPER (PIPER NIGRUM L.) DURING STORAGE. I. SENSORY AND PHYSICOCHEMICAL ANALYSES

Black pepper powder (60 mesh) was stored in consumer unit packs of 100g capacity in low density polyethylene (LDPE) films of 100, 300, and 500 gauge at 27°C and 65% RH. Analyses for sensory quality (odor and flavor), volatile oil, oleoresin, piperine content, and TLC were carried out at 15, 30, 45 and 80 days of storage. Significant loss of “top notes”, volatile oil, and hydrocarbons were seen after 15 days of storage itself while the “basic notes” and oxygenated compounds were retained up to 45 days. There was no loss of piperine up to the end of the study. The black pepper powder was not fit for table use after 15 days, though it could be used for other culinary purposes up to 80 days of storage.     

A histamine-releasing factor from activated human mononuclear cells

Human mononuclear cells activated by streptokinase-streptodornase have been shown to elaborate a factor capable of releasing histamine from human basophils. We have developed reproducible methods for its production in large quantities by using cells obtained from leukapheresis packs, by detection utilizing donor basophils known to release well with anti IgE, and by quantitation of histamine by the radioenzyme method. Human histamine-releasing factor (HRF) gave a single peak upon gel filtration with an estimated m.w. of 32,000; SDS gel electrophoresis revealed a single major band as seen at m.w. 30,000. HRF can be resolved into at least two forms separable by ion-exchange chromatography on QAE Sephadex, and two peaks of activity were obtained by chromatofocusing or isoelectric focusing in gels at pH 6.9 and between 7.4 and 8.3. This factor represents an important potential link between cellular immunity and immediate hypersensitivity.