捐精志愿者精液冷冻保存前后细菌培养的初步研究

Bacterial culture of donor semen before and after cryopreservation:A preliminary study

目的:通过比较捐精志愿者精液冷冻保存前(冻前)和冷冻保存后(冻后)的细菌培养情况,分析导致精液冷冻过程中细菌污染的因素。方法:按照WHO《人类精液检查与处理实验室手册》(第5版)用精子冻存液将精液样本按1∶1(v/v)的比例进行稀释。实验前对取精杯、精子冻存液和哥伦比亚血琼脂培养基进行细菌培养,均无菌生长。对3112例精液样本分别进行冻前和冻后的细菌培养,并按细菌菌落数进行比较,分为正常组(冻前>冻后,且冻前<10^(4) cfu/ml)和异常组(包括2种情况:(1)冻前≥冻后,且冻前≥10^(4) cfu/ml;(2)冻前<冻后),并对可疑菌落进行菌种鉴定。结果:3112例细菌培养结果中,正常组(冻前>冻后,且冻前<10^(4) cfu/ml)2458例,占78.98%,异常组654例,占21.02%(其中(1)冻前≥冻后,且冻前≥10^(4) cfu/ml 263例,占8.45%;(2)冻前<冻后391例,占12.56%)。可疑菌落分离鉴定出217株细菌,检出率为6.97%,以表皮葡萄球菌、溶血性葡萄球菌、极小棒状杆菌、金黄色葡萄球菌、粪肠球菌为主,分别占23.96%、16.59%、11.52%、7.37%、5.99%。结论:精液检出的细菌主要以表皮葡萄球菌、溶血性葡萄球菌、极小棒状杆菌、金黄色葡萄球菌、粪肠球菌为主。导致精液冷冻过程中细菌污染的因素较多,选择无菌、清洁、干燥的实验耗材,恰当的精液样本保存方式,符合标准的实验环境,同时工作人员严格按照SOP标准操作,保持积极工作态度可以减少精液冷冻保存过程中细菌的污染。

Objective:To investigate the factors causing bacterial contamination of donor semen during cryopreservation.Methods:Based on the WHO Laboratory Manual for the Examination and Processing of Human Semen(5 th ed),we diluted donor semen samples with sperm cryopreservation medium in a 1∶1(v/v)ratio.Before the experiment,we cultured bacteria in the sperm cup,sperm cryopreservation medium and Columbia blood a-gar medium to ensure sterile growth.We also conducted bacterial culture of 3,112 semen samples before and after freezing and,according to the number of bacterial colonies(NBC),divided them into a normal group(NBC before freezing<10^(4) cfu/ml and>NBC after freezing)and an abnormal group(NBC before freezing≥10^(4) cfu/ml and≥NBC after freezing,or NBC before freezing<NBC after freezing),followed by bacterial species identification of the suspected colonies.Results:Of the 3112 donor semen samples,2458(78.98%)were included in the normal,and the other 654(21.02%)in the abnormal group(263[8.45%]with NBC before freezing≥10^(4) cfu/ml and≥NBC after freezing,and 391[12.56%]with NBC before freezing<NBC after freezing).In the suspected colonies,217(6.97%)strains of bacteria were isolated and identified,mainly including Staphylococcus epidermidis(23.96%),Staphylococcus haemolyticus(16.59%),Corynebacterium minutissimum(11.52%),Staphylococcus aureus(7.37%),and Enterococcus faecalis(5.99%).Conclusion:The bacteria detected in the donor semen mainly included Staphylococcus epidermidis,Staphylococcus haemolyticus,Corynebacterium minutissimum,Staphylococcus aureus and Enterococcus faecalis.Many factors may cause bacterial contamination of donor semen during cryopreservation,which can be reduced by employing sterile,clean and dry materials,appropriate semen preservation methods,normalized experimental environment,and standard operating procedures.