生物负荷回收验证-生物负荷回收方法Ⅱ

Recovery Methods
回收方法
1. Solutions and Raw Materials-Total Count
溶液和原料—总计数

Qualitative or quantitative recovery methods for bioburden can be performed via membrane filtration. Membrane filtration is applicable in testing non-bacteriostatic or non-fungistatic liquids and soluble powders, as well as those items which possess inherent bacteriostatic/fungistatic properties. In performing the test, care must be exercised to minimize the potential for extraneous contamination and to assure complete rinsing/neutralization of the drug product on the membrane when inhibitory substances are present.
定性或定量回收方法通过膜过滤进行。膜过滤适用于检测非抑菌或非抑制真菌溶液和可溶性粉末，以及那些本身带有抑菌或抑制真菌的特性的物质。在检测过程中存在抑制物时，必须要注意减少潜在的外界污染，并确保在膜上完成药物产品的冲洗/中和。

Therefore, validation testing to confirm the adequacy of the technique by 
recovery of known, low-level, inoculated sample "test" solutions as compared to inoculated "control" solutions，should be performed.
因此，验证测试通过对已知的、低水平的回收，接种样品"测试"方案与接种"控制"方案对比，证实技术的充分性，应该进行验证测试。

a. Quantitative Recovery-Filtration
定量回收—过滤
The filtration unit should consist of a reservoir and receptacle in which a membrane, with a nominal porosity of 0.45±0.02 μm and a diameter of 47
millimeters, is used. The entire unit may be assembled and sterilized with the filter in place, or a sterile filter will be aseptically put in place at the time of the test. Aseptically transfer the appropriate volume of a liquid sample (to achieve 10-100 colony forming units) into a membrane filter funnel.
使用的过滤设备应该包括蓄水池和带有薄膜的容器，薄膜孔隙度为0.45±0.02 μm，直径为47mm。测试的时候，组装，消毒整个装置并在适当位置装上过滤器，或者在合适的位置安装无菌过滤器。无菌转移适量的液体样本（达到10-100菌落形成单位）到膜过滤器过滤漏斗。

In the case of raw materials which are soluble solids, make an appropriate dilution of the sample by placing an appropriate amount of the raw material into the solubilizing agent prior to filtration. (Note, one must predetermine if the filter integrity will be affected by the solubilizing agent.) Pass the specimen through the filter with the aid of vacuum or pressure. If the product possesses any static or cidal properties, the filter should be rinsed thoroughly with a solution which will neutralize or remove the product solution. 
就可溶性固体原材料而言，在过滤前对样品进行适当的稀释，将适量的原材料放入增溶剂中（注意，必须事先确定是否增溶剂会影响过滤器整合性）。样本在真空或压力下通过过滤器。如果产品拥有任何静态或灭菌酚的特性，那么应该用溶液彻底清洗过滤器，这样产品溶液会被中和或除去。

Aseptically remove the membrane and place it on Soybean-Casein Digest agar medium, or appropriate growth medium. Incubate at the appropriate temperature for an adequate time to observe microbial growth. (Typically, incubation at 30-35℃ for hours is used. Reference Section 1I.C for additional information.)
无菌移膜并将它放在大豆消化酪素琼脂培养基上，或者恰当的培养基中。在适当的温度下培养足够的时间以观察微生物生长。（在30-35℃下培养48-72小时，其他信息参考章节1I.C）

b. Quantitative Recovery-Pour Plate
定量回收—倾倒平板法

Dissolve a sample, if it is a solid, or take a sample, if a liquid, and place it into sufficient volume of Fluid Soybean-Casein Digest Medium or other appropriate diluting fluids. Prepare serial tenfold dilutions, if necessary, so that when plating a sample it will yield approximately 30-300 colony forming units (CFU). The number of dilutions necessary to accomplish this may vary considerably depending upon the contamination level of the sample. This level may be the result of the material source (natural or synthetic), the vendor, the manufacturing process of the material, etc. 
如果是固体样品，先溶解样品，如果是液体的，取样并放在足够量的大豆消化酪素培养基中或者其他合适的稀释液。如果有必要，准备10倍系列稀释液，以便当电解法分离样品时能产生大约30-300个菌落形成单位（CFUs）。稀释梯度个数根据样品的污染水平变化。污染水平由材料来源（天然的或合成的）、供应商、材料的生产工艺等决定。

Raw materials of unknown quality or of animal origin should be diluted to 1:100,000 or further. Typically, duplicate plates are prepared for each dilution. Add approximately 15-20 mL of Soybean-Casein Digest agar, which has been prepared and cooled to approximately 45 ℃ , to each plate. Cover and mix the sample by swirling the plates to assure an even distribution of organisms within the agar. Allow all plates to solidify at room temperature, invert, and incubate. (Typically incubation at 30-35 ℃ for 48-72 hours is used. Reference Section 1I.C for additional information.)
质量未知或动物来源的原材料，稀释到1:100,000倍或者更甚。为每个稀释度样品准备平板。在每个平板中添加大约15-20ml，温度大约为45℃的大豆消化酪素培养基。盖好平皿，旋转平板以混合样品，确保微生物在琼脂里的分布。平板在室温下凝固，反转平板在30-35℃培养48-72小时。（其他信息参考1I.C）

Remove plates from incubation and count the total number of colony forming units (CFU) per plate. Perform the colony count on the plates which contain between 30-300 CFU. If the count is less than 30 CFU, use the count from the lowest dilution.
从培养箱中取出平板并计数每个平板上的菌落数。包含30-300个菌落的平板用于实验。如果菌落数少于30，这个数来自最低稀释度。

c. Quantitative Recovery-Spread Plate
定量回收—涂布平板法

A solid sample must be dissolved to initiate this test. A liquid sample may be tested without further dilution. Place a sufficient volume (0.1 or 1.0 mL) on the surface of a Soybean-Casein Digest agar plate or other appropriate growth medium. (The volume should be such that all liquid can be absorbed into the agar.) With agitation, or by the use of a sterile bent glass rod, assure that the liquid is evenly dispersed over the entire surface of the agar. Allow the liquid to absorb into the agar, invert, and incubate. (Typically incubation at 30-35℃ for 48-72 hours is used. Reference Section 1I.C for additional information.) Remove plates from incubation and count the CFU.
在这个测试中，固体样本必须溶解。测定液体样本不需要进一步稀释。将足量的（0.1或1.0ml）样本放在大豆消化酪素琼脂培养基表面或其他合适的培养基。（滴加的量应该是全部能被琼脂吸收）。震荡，或用弯玻璃棒搅动，确保液体均匀分散在平皿的整个表面。等琼脂吸收液体后，反转并在30-35℃培养48-72小时。（其他信息参考1I.C）。从培养箱中取出平板并计数。

d. Quantitative Recovery-Automated Procedures
定量回收—自动化操作程序

Automated dilution/plating equipment, such as the spiral plater, may also be employed. The spiral plater inoculates a rotating agar plate with a liquid sample. The volume dispensed decreases as the dispensing stylus moves from the center to the edge of the rotating agar plate. A detailed description of this procedure can be found in the Bacteriological Analytical Manual (1).
可以使用自动稀释/电解设备，例如接种仪。接种仪接种液体样本在旋转琼脂平板上。分配的量减少因为滴加样本的笔尖从旋转琼脂平板的中心移到边缘。细菌学分析手册中有这个过程的详细描述。

e. Quantitative Recovery-Most Probable Number (MPN)
定量回收—最大可能数

The Most Probable Number (MPN) method should be used when the sample cannot be tested by one of the quantitative methods described. The mathematics of the MPN method are based on probability statistics. The results are directly related to the frequency of occurrence of a series of positive results most likely to occur when a given number of bacteria are present in a sample. As the number of tubes inoculated for each dilution increases, the confidence limits for MPN are narrowed.
当样品不能通过以上所述的定量方法检测时，使用最大可能数法。最大可能数法的原理是概率统计。结果直接与一系列阳性结果的出现率相关，当细菌的给定数量出现在样品中时，阳性结果最可能出现。随着接种试管数量的增加，对最大可能数的限制在缩小。

Tubes of Soybean-Casein Digest Media, or an appropriate recovery media, are inoculated in sets (3 or 5) with serial tenfold dilutions of the sample. (Typically, incubation at 30-35℃ for 48-72 hours is used. Reference Section 1I.C for additional information.) Based on the number of positive tubes per set, within 3 sequential dilutions, the "combination of positive tubes" is derived. The Table I is an example of the selection criteria employed when working with the raw data.
大豆消化酪素琼脂培养基试管，或其他合适的回收培养基，按组（3或5个一组）接种样品的10倍系列稀释液（在30-35℃培养48-72小时，其他信息参考1I.C）。基于每组3个连续稀释度里阳性试管的数量，出现了“阳性试管组合”。表格I是使用原始数据时，所用选择标准的例子。

Select the highest dilution in which all tubes are positive (3) and the next 2
highest dilutions (example a, b, c, and e). If only 3 dilutions are made (example a and d), use those 3 dilutions to obtain an MPN. Where there are skips, as in example e and f, add the highest dilution to the next lowest dilution and derive the combination number from 3 dilutions as before.
选择最高稀释度和下两个稀释液，最高稀释度里所有的试管都是阳性对照（例如a、b、c、e）。如果只做了3个稀释度，就用这3个稀释度获得最可能数。数据出现跳跃，如例子中的e和f，添加最高稀释度到下一个的最低稀释度并从3个稀释度得到组合数据。

Using the "combination of positive tubes" and the dilution factors, the MPN can be located on established tables which are found in multiple publications (1-3). These tables may or may not have resulting data calculated with confidence levels. The MPN can also be calculated using the Thomas equation (3):
用“阳性试管组合”和稀释因子，通过确定的表格得到MPN数值，在出版刊物中可以找到这些表格。这些表格不一定能得到结果数据，可以通过托马斯方程计算得到MPN。
（ Number of positive tubes X 100 M.P.N. = Per 100 ,L* (Number or
mL* aisample in negarivc tuk) x (Number or mL* of sample In all tubes) *
(g) can be substituted if sample is being measured by weight as opposed to
volume. ）

       文章来源：药品微生物检测

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