抗生素微生物检定法The Antibiotics Microorganism Method

第一法 管碟法    

本法是利用抗生素在琼脂培养基内的扩散作用,比较标准品与供试品两者对接种的试验菌产生抑菌圈的大小,以测定供试品效价的一种方法.

1 Cylinder-plate Method

To mensurate the titer of the specimen, this method compares size of the bacteriostatic rings to the vaccinal test organisms produced by the standard and the tested by using the diffusion effect of antibiotics in agar culture medium.

短小芽孢杆菌悬液 取短小芽孢杆菌的营养琼脂斜面培养物,接种于盛有营养琼脂培养基的培养瓶中,在35-37℃下培养7日,用革兰染色法涂片镜检,应有芽孢85%以上. 用灭菌水将芽孢洗下,在65℃加热30分钟,备用.

Preparation of bacillus pumilus suspension: Inoculate the nutrient agar slant with bacillus pumilus to the culture bottle that contains nutrient agar. Keep it in 35-37℃ for 7 days, and then use Gram-stain method to do smear microscopy. The rate of gemma should be above 85%. Next clean the gemma of a fungus with sterile water and heat it at 65℃ for 30mins.

表1  抗生素微生物检定试验设计表The Antibiotics Microorganism Test Table

抗生素类别Type of Antibiotics   试验菌Test Bacteria  培养基 Medium 编号 No.  Ph值Ph Value 灭菌缓冲液Sterilizing Buffer Solution 抗生素浓度范围 (单位/ml ) Rate of Antibiotics 培养条件 Culture Conditions 温度/℃ Temperature 时间/小时 Time

溶液的制备： 精密称（或量）取供试品适量，用备品种项下规定的溶剂溶解后，再按估计效价或标示量照表1的规定稀释至与标准品相当的浓度。

Preparation of the solution: Take a proper amount of the tested material with precision. Dissolve it in the dissolvent as is prescribed and then dilute it to the same consistency with the standard according to the estimated titer and the labeled amount with the instructions of Table 1.

双碟的制备：  取直径约90mm ,高16-17mm 的平底双碟，分别注入加热溶化的培养基（照表1 ）20ml, 使在碟底内均匀摊布，放置水平台上使凝固，作为底层。另取培养基适量加热溶化后，放冷至48-50℃（芽孢可至60℃），加入规定的试验菌悬液适量（能得清晰的抑菌圈为度）。二剂量法标准溶液的高浓度所致的抑菌圈直径在15-18mm),摇匀，在每1 双碟

中分别加入5ml, 使在底层上均匀摊布，作为菌层。放置水平台上冷却后，在每1双碟中以等距离均匀安置不锈钢小管（内径 6.0mm±0.1mm, 高10.0mm±0.1mm, 外径7.8mm±0.1mm）4个（二剂量法）或6个（三剂量法），用陶瓦圆盖覆盖备用。

Preparation of the double-plate: Take a flat-bottomed double-plate, the diameter of which is about 90mm and the height 16-17mm, and then respectively pour in them the heated dissolved culture medium(See Table 1) 20ml. Make it evenly distributed in the bottom of plate, and then put the plates on a horizontal stand. The congealed medium will be used as the bottom layer. Take another proper amount of culture medium and unfreeze it by heat. Cool it off to 48-50℃(for gemma of a fungus to 60℃). Pour in a proper amount of test bacteria suspension as is prescribed (until clear bacteriostatic ring emerges; the diameter of bacteriostatic ring produced by high-consistence standard solution made according to Dosage Method should be 15-18mm),  shake it well and then pour in 5ml to both plates. Spread it evenly upon the bottom layer and this will be used as the bacteria layer. After it is cooled off on the horizontal stand, install 4 or 6 stainless steel tubelets( inner diameter: 6.0mm±0.1mm,  height: 10.0mm±0.1mm,outer diameter:7.8mm±0.1mm) equidistantly in both plates. After that cover them with terracotta round cover and leave the prepared plates for future use.

检定法：

Test method

二剂量法  取按上述方法制备的双碟不得少于4个，在每1双碟中对角的2个不锈钢小管中分别滴装高浓度及低浓度的标准晶溶液，其余2个小管中分别滴装相应的高低两种浓度的供试晶溶液；高低浓度的剂距为2：1或4：1。在规定的条件下培养后，放入多功能抑菌圈测量仪内，即可得出效价。

2 Dosage Method

Take at least 4 double-plates prepared according to the method above mentioned and drip respectively high-consistence and low-consistence standard solution into the two stainless steel tubelets on the cross, and then drip into the other tubelets high-consistence and low-consistence tested solution. The proportion of the rate of high-consistence to low-consistence solution could be 2:1 or 4:1. After cultivation under prescribed conditions the plates will be put into a multi-functional bacteriostatic ring instrument and the titer will be mensurated.