细菌超声分散计数仪在微生物实验前处理中的应用

doi:10.3969/j.issn.2095-3755.2018.03.009

摘要/Abstract

摘要：

目的探究细菌超声分散计数仪在微生物实验前处理中的应用价值。方法以9种不同菌种为实验对象,通过肉眼观察、革兰染色镜检和菌液浊度检测,对比传统手工分散方法和仪器超声分散方法制备菌悬液的分散效果和对细菌生长活性的影响。结果 7种菌需使用低档功率,2种菌需要使用中档功率。不同菌种超声处理时间不同,最长120s,最短10s。经仪器分散获得的菌悬液普遍均匀度更佳,无明显菌的团聚现象。鲍曼不动杆菌、肺炎克雷伯菌和嗜麦芽窄食单胞菌等菌株经手工分散和仪器分散的效果没有明显差异;超声处理30s即可达到较为理想的分散效果,延长处理时间对大部分菌液浊度没有发生明显变化,但鲍曼不动杆菌、阴沟肠杆菌和大肠埃希菌随超声分散时间的延长而菌液浊度明显下降,肺炎克雷伯菌超声处理60s时菌液浊度达到最大值,延长处理时间浊度逐渐下降。在1∶10 ⁵稀释度下,大肠埃希菌经仪器分散后的平板菌落数均值[(59±6)×10 ⁵菌落形成单位(CFU)/ml)]明显低于手工分散者[(76±8)×10 ⁵ CFU/ml)](F=10.321,P=0.033);诺卡菌属经仪器分散后的平板菌落数均值[(9±6)×10 ⁵ CFU/ml)]明显高于手工分散者[(2±0)×10 ⁵ CFU/ml](F=8.693,P=0.015);其他7种菌种用仪器分散和手工分散获得的菌悬液不存在明显的生长活性差异。结论与手工分散方法相比,细菌超声分散计数仪对各参试菌种的分散效果好,耗时较短,不会明显影响菌体的生长活性,能为菌悬液制备提供一套安全、高效、标准化的解决方案,在微生物实验前处理中具有较高的应用价值。

关键词: 超声处理, 微生物学技术, 标本制备, 对比研究

Abstract:

Objective To explore the application value of bacterial ultrasonic dispersion counter in pretreatment of microbiology experiment. Methods The bacterial suspensions of nine different types of bacteria species were prepared by using the traditional manual dispersion method and the new instrument ultrasonic dispersion method, and then the dispersion effects of those bacterial suspensions which prepared by two methods were compared through the macroscopic observation, gram’s stain microscopy and turbidity measure of bacterial suspension. The impacts of these two methods on the activity of bacteria were also compared in this study. Results Seven types of bacteria could be dispersed sufficiently in low level power, while the other two types of bacteria were dispersed in medium level power. Different types of bacteria had different ultrasonic processing time, with the longest 120 s and the shortest 10 s. The degree of uniformity of the bacterial suspensions prepared by the instrument ultrasonic dispersion method was better and there was no obvious bacterial aggregation. The dispersion effects of the bacterial suspensions of Acinetobacter baumannii, Klebsiella pneumoniae and Stenotrophomonas maltophilia, etc. prepared by the two methods had no statistically significant difference. The ideal dispersion effect of the bacterial suspensions could be obtained at 30 s of ultrasonic dispersing; the extension of ultrasonic dispersing time, the turbidity of most of bacterial suspensions didn’t change significantly. However, as for Acinetobacter baumannii, Enterobacter cloacae and Escherichia coli, the turbidity of bacterial suspensions significantly decreased with the extension of ultrasonic dispersing time, while for Klebsiella Pneumoniae, the turbidity of bacterial suspension got top at 60s and then gradually declined with the extension of ultrasonic dispersing time. Under the 1∶10 ⁵ dilution degree, the mean number of plate bac-terial colonies of Escherichia coli dispersed by the instrument ((59±6)×10 ⁵ CFU/ml)) was significantly lower than that of the manual dispersions ((76±8)×10 ⁵ CFU/ml)), F=10.321, P=0.033; the mean number of plate bacterial colonies of Noka bacteria dispersed by the instrument ((9±6)×10 ⁵ CFU/ml)) was significantly higher than that of the manual dispersions ((2±0)×10 ⁵ CFU/ml)), F=8.693, P=0.015; there was no significant difference on growth activity in bacterial suspensions of other 7 types of bacteria species prepared by using the instrument ultrasonic dispersion method and the traditional manual method. Conclusion Compared with traditional manual dispersion method, the dispersion effect of bacterial ultrasonic dispersion counter for the tested bacteria species is more ideal, and instrument ultrasonic dispersion need shorter time and have no influence to bacteria growth activity at the same time. The bacterial ultrasonic dispersion counter is able to provide a set of safe, efficient, and standardized solution for bacterial suspension preparation, which has high application value in pretreatment of microbiology experiment.

Key words: Sonication, Microbiological techniques, Specimen handling, Comparative study

冯通明,肖美方,王威,崔小颖,吴智龙,周杰. 细菌超声分散计数仪在微生物实验前处理中的应用[J]. 结核病与肺部健康杂志, 2018, 7(3): 188-193. doi: 10.3969/j.issn.2095-3755.2018.03.009

Tong-ming FENG,Mei-fang XIAO,Wei WANG,Xiao-ying CUI,Zhi-long WU,Jie ZHOU. Application of bacterial ultrasonic dispersion counter in pretreatment of microbiology experiment[J]. Journal of Tuberculosis and Lung Health, 2018, 7(3): 188-193. doi: 10.3969/j.issn.2095-3755.2018.03.009

图/表 4

表1

图1

图2

表2

各类细菌采用不同分散方法处理后的生长活性比较

细菌类别	生长活性(×10⁵ CFU/ml, $x ˉ$ ±s)		F值	P值
细菌类别	分散仪法	手工法	F值	P值
鲍曼不动杆菌	144±56	130±22	0.323	0.582
阴沟肠杆菌	60±12	48±8	4.356	0.063
大肠埃希菌	59±6	76±8	10.321	0.033
肺炎克雷伯菌	131±19	179±23	15.534	0.003
奈瑟菌属	188±15	236±112	1.087	0.322
诺卡菌属	9±6	2±0	8.693	0.015
人苍白杆菌	277±93	238±80	0.620	0.449
玫瑰单胞菌	286±91	280±177	0.006	0.941
嗜麦芽窄食单胞菌	85±72	97±89	0.070	0.797

表2

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