对无菌试验培养基灵敏度试验法准确性的初步研究

Preliminary Study on the Accuracy about the Sensitivity Test of Culture Media for Aerobes,Anaerobes and Fungi

目的:研究《中国生物制品规程》(2000年版)(简称《规程》)中需氧菌和厌氧菌无菌试验培养基灵敏度试验法及真菌无菌试验培养基灵敏度试验法的准确性。方法:按照《规程》中需氧菌和厌氧菌无菌试验培养基灵敏度试验法及真菌无菌试验培养基灵敏度试验法,同时采用平皿[乙型溶血性链球菌采用血琼脂平皿,短芽孢杆菌采用营养琼脂平皿,生孢子梭状芽孢杆菌(简称生孢梭菌)采用硫乙醇酸盐软固体琼脂平皿(厌氧培养),白色念珠菌采用真菌培养基琼脂平皿]计数法,在相同稀释度及培养温度条件下平行进行1mL菌液的菌落形成单位(Colony Forming Units,CFU)计数。将菌液稀释至与标准比浊管相同之浓度,然后作10倍系列稀释。将稀释度为10-6～10-8的短芽孢杆菌、10-6-10-8的生孢梭菌,10-7～10-9的乙型溶血性链球菌菌液各1 mL,分别接种到9 mL硫乙醇酸盐培养基中;将稀释度为10-5～10-7的白色念珠菌菌液各1 mL,分别接种到9mL真菌培养基。每个稀释度至少接种3管,用未接种培养基作对照。将接种短芽孢杆菌的培养基,置35℃培养5 d,接种生孢梭菌或乙型溶血性链球菌的培养基,置35℃培养3 d,接种白色念珠菌的培养基,置25℃培养5 d,同时重复3次试验,记录结果。用每个菌种,按照需氧菌和厌氧菌无菌试验培养基灵敏度试验法及真菌无菌试验培养基灵敏度试验法重复进行3次灵敏度试验。结果:按照《规程》中需氧菌和厌氧菌无菌试验培养基灵敏度试验法及真菌无菌试验培养基灵敏度试验法,同一种培养基,用同一质控菌种重复3次灵敏度试验,结果往往不同,尤其是用乙型溶血性链球菌进行质控时,更为明显。平皿CFU计数与比浊菌数常有显著差异。结论:《规程》中需氧菌和厌氧菌无菌试验培养基灵敏度试验法及真菌无菌试验培养基灵敏度试验法准确性欠佳。

Objective: To investigate the accuracy about the sensitivity test of culture media for aerobes, anaerobes and fungi from Requirements for Biologies of PRC(The People's Republic of China,2000). Methods: According to the sensitivity test of culture media for aerobes, anaerobes and fungi from Requirements for Biologies of PRC, count colony forming units (CFU · mL-1) parallel under the same dilution and culture temperature ( Streptococcus β -hemolyticus inoculated on blood agar plate, Bacillus brevis inoculated on nutrient agar plate, Clostridium sporogenes inoculated on fluid thioglycollate soft - solid agar plate under anaerobic condition, and Candida albicans inoculated on fungi agar plate). First dilute Bacillus brevis, Clostridium sporogenes,Streptococcus β - hemolyticus and Candida albicans to a concentration against the standard opacity,and then carry out 10 -fold serial dilutions. Then add 1 mL of Streptococcus β -hemolyticus at each of dilution of 10-9 - 10-7,Bacillus brevis at each of dilution of 10-8 - 10-6 and Clostridium sporogenes at each of dilution of 10-8- 10-6 into 9 mL fluid thioglycollate medium and 1 mL of Candida albicans at each of dilution of 10 -7 - 10-5 into 9 mL fungi medium to be tested,respectively. Each dilution inoculates at least three tubes. Uninoculated medium is used as the control. Incubate the media inoculated with Bacillus brevis for 5 days at 35 ℃ and the media with Clostridium sporogenes or Streptococcusβ -hemolyticus for 3 days at 35 ℃. The media inoculated with Candida albicans is incubated for 5 days at 25℃. Then repeat three times and note the results. According to the sensitivity test of culture media for aerobes,anaerobes and fungi,the sensitivity test of culture media for the same bacteria or fungi is repeated for three times. Results: According to the sensitivity test of culture media for aerobes, anaerobes and fungi from Requirements for Biologies of PRC, there are not all the same results among three sensitivity tests when the same bacteria or fungi is inoculated at the same culture medium , especially the significantly different results of Streptococcus β - hemolyticus. There are often some striking differences between the counts of CFU·mL-1 and theoretical value of nephelometery. Conclusions; The accuracy about the sensitivity test of culture media for aerobes, anaerobes and fungi from Requirements for Biologies of PRC is unsatisfactory.