摘要
目的:鉴定细菌是否分泌甲壳素酶,并分解利用甲壳素的经典方法是甲壳素缺刻试验,但其需时较长,结果不明显,为解决以上缺点,研制甲壳素微结晶分散体系,拟创造一种新的实验方法甲壳素溶解环试验,并探讨其影响因素。方法:实验于2005-04/2006-12在浙江省医学科学院医学生物工程研究所完成。①通过高温高压、超声等综合手段,制备乳白色的甲壳素微结晶分散体系,并进行透射电镜观察。②采用制备的甲壳素微结晶分散体系,制造甲壳素培养基,取副溶血弧菌和致病性大肠杆菌44815菌悬液106cfu/mL各1μL,分别注入甲壳素培养基中,37℃培养72h,如接种点周围出现明显的透明环,即为阳性,反之为阴性。③制备2%,1%,0.5%3种不同浓度的NaCl及相应8.0,7.0和6.0不同pH值组合的3种甲壳素培养基,分别接种副溶血弧菌菌悬液106cfu/mL各1μL,37℃培养72h后测量溶解环直径。④菌量和培养温度的影响:将3种浓度菌悬液(108,106和104cfu/mL)各1μL注入甲壳素培养基中,37℃培养72h;固定菌量(2.8×106cfu/mL)接种后,分别在40℃、37℃和25℃培养72h,测量溶解环直径。结果:①成功制备了甲壳素微结晶分散体系,甲壳素浓度为4%,微粒粒径<0.5μm。②培养72h后,接种副溶血弧菌甲壳素培基上呈现阳性反应。③在NaCl浓度为2.0%,pH为8.0环境条件下,溶解环直径最大。④在菌量108￣104cuf/mL范围内,菌量与溶解环直径大小呈正比,但最适浓度为106cuf/mL;在37℃环境中溶解环直径最大。结论:①甲壳素溶解环试验是鉴定细菌能否分泌甲壳素酶、并分解利用甲壳素的一种直观、快速、准确的方法。②甲壳素培养基NaCl、pH值、培养温度及菌量等对甲壳素溶解环试验均有直接的影响。
AIM： The chitin erosion test is dominant to identify the release of chitinase from bacteria and decompose the chitin, but it costs longer time and obtains unobvious outcomes. Thus the dispersion system of chitin micro-crystal （DSCMC） is prepared for test of Chitin Soluble Circle （TCSC） to substitute the routine method. Moreover its influencing factors are also explored. METHODS： The experiment was conducted in the Institute of Medical Bioengineering, Zhejiang Academy of Medical Science between April 2005 and December 2006.①lvory DSCMC was prepared by high temperature, high pressure, and ultrasound. The diameter of chitin particle was determined by transmission electron microscope.②l μL （108 cfu/mL） of bacterial suspension （E.coli44815 or Vibrio parahaemolyticus） was inoculated in the ivory agar of chitin, which was then incubated at 37 ℃ for 72 hours. TCSC positivity： The obvious transparent circles appeared surrounding the inoculating area in chitin agar, otherwise it was negative.③1μL （108 cfu/mL） bacterial suspension of Vibrio parahaemolyticus was respectively added to three agar of chitin containing 2%, 1%, 0.5% concentrations of NaCI with the pH values as different as 8.0, 7.0 and 6.0. After incubating at 37 ℃ for 72 hours, the diameter of chitin cycle was measured.④μL （108, 108, 104 cfu/mL） of bacterial suspension was added to the agar of chitin, which was then incubated at 37 ℃ for 72 hours; Moreover the chitin was also respectively incubated at 40 ℃, 37 ℃and 25 ℃at the fixed bacterial quantity （2.8×l 06 cfu/mL）. Afterwards the diameter of chitin cycle was measured. RESULTS：① DSCMC was successfully established at the 4% concentration of chitin and less than 0.5μm diameter of chitin microparticles. ②.At 72 hours of incubation, the positive response appeared in chitin agar with Vibrio parahaemolyticus. ③The soluble cycle reached the highest at the 2.0% concentration of NaCI and 8.0 pH value.④ Bacterial quantity was positively correlated wit
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第40期8131-8133,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
浙江省科技计划重大项目资助(2005F1101)
浙江省科技计划项目资助(2006F12037)~~
