酶标仪分光光度法在微生物耐性研究上的应用

微生物在环境中的耐性是该微生物在相应环境发挥作用的重要基础。为了获得一种用于微生物耐性分析的快速简便方法,传统平板分离法和酶标仪分光光度法被用于评估其在细菌和链霉菌紫外耐受水平检测中的差异,并分析了酶标仪分光光度法在微生物其他耐性水平检测中的适用性。结果显示,两种检测方法均能体现细菌和链霉菌对紫外线的耐受水平,前者经过稀释、涂布、培养、菌落计数,获得的是菌株在紫外线照射后的存活浓度,而后者经过接种96孔培养板、培养、吸光度检测,获得的是菌株经紫外线照射后的生长曲线,并从生长曲线获知菌株的生长速率、增殖能力等信息。此外,酶标仪分光光度法同样适用于细菌对pH和盐的耐受水平分析,对于链霉菌耐受性分析有一定的适用性。酶标仪法除了能获得与平板分离法相似的耐性水平检测外,还能获得菌株在不同耐性水平上的增殖潜力,且在操作上比平板分离法省时、省力,可用于微生物耐性分析、高通量筛选等研究工作。     

Carvone and less volatile analogues as repellent and deterrent antifeedants against the pine weevil, Hylobius abietis

Abstract:  The monoterpenoid carvone ( 1 ) has been shown to have strong antifeedant effects on Hylobius spp. However, because of the high volatility of carvone, long-time protection of conifer seedlings in the field using this compound has not been possible. We demonstrate, in several bioassay steps, that less-volatile, heavier analogues retain their pre-ingestive feeding inhibition activity in the large pine weevil, Hylobius abietis (L.) for a longer time. The first step in the evaluation of the biological activity of 12 carvone analogues was a micro-assay, a choice test lasting 4 h. Compounds active at 100 nmol/cm² were further dose–response tested to give the effective dose needed to inhibit feeding by 50% (ED50). Of the 14 compounds tested, including both carvone enantiomers, seven heavier analogues were active at low doses (had low ED50 values). As expected from their lower vapour pressure compared with carvone, the heavier analogues proved more resistant to evaporation before testing. Thus, whereas the effect of 8-hydroxy- p -menth-en-6-one 4 declined after 2 days, some of the compounds with high molar masses, such as the alkylhydroxymenthenones 6 and 8 , retained a stable activity for 4 days. The retained activity at 4 days was strongly correlated to molecular mass and boiling point. When tested on natural material (host Pinus sylvestris L. twig sections for 48 h), the heavier analogues showed a rather low activity. Probably, the activity of the more volatile compound carvone ( 1 ) is due to a repellent effect (olfactory mode) rather than the deterrent effects (gustatory mode) of the heavier compounds. In agreement with the relatively low activity on twigs in the laboratory, the hydroxymenthenone 4 was not active in the field when tested for 2 months as a 1 : 9 mixture with a polar wax.     

Effects of exposing bovine sperm to bovine serum albumin,or freeze-thawing,on sperm-bound amidase activity

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P < .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5°C or layered onto a solution of 6% BSA in extender at 37°C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5°C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P < .01). However, the amount of exposed sperm-bound amidase also was greater (P < .05) this was a deleterious change. Immediately after thawing, more (P < .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37°C there was no difference. After freezing and thawing of sperm, amidase activity was higher (P < .05) for the isolated sperm than for nonisolated cells. Thus, isolation of sperm using a 6% BSA gradient increased the proportion of progressively motile sperm, but decreased the percentage of sperm with an intact acrosome, based on measurements of amidase activity.