TSB液体培养基有没有可能出现浑浊，但不是染菌

莎瓦娜

今年因为变更，计划连续灌装3批培养基，但是前几天在进行第二批培养基20~25℃7天培养之后的目检，发现有6瓶培养基在灯检仪下观察有那种类似浑浊的情况，这6瓶培养基在普通光线下很难看出跟其他培养基有不一样，只有在灯检仪下才能看到培养基呈现出不一样的折射光，培养基仍然还是透明的，没有发现悬浊物。后来将这6瓶培养基分别都抽出一些到新鲜的TSB中培养，同时也做了镜检，现在培养3天了，新鲜培养基没有东西生长。现在我们有两拨人有不同的观点，一拨人认为实验到现在为止了，已经可以确定这6瓶不是染菌，第二批培养基没有问题；另一拨人认为有些微生物生长需要更长的时间，还要继续培养更长的时间。现在纠结我们究竟要不要再灌装3批培养基？

莎瓦娜

zhaosc8326 发表于 2019-1-22 19:16
3. A firm has multiple media fill failures. They conducted their media fills using TSB (tryptic soy  ...

完整的原文有吗，或者是哪一个报告号

18503871991

假阳性和假阴性

jixia

支原体？不是微生物就是TSB的原因了，更换厂家再执行无菌模拟很难么

window

3楼观点支持
FDA的案例  

stupid22273

dingkaiwang 发表于 2019-1-22 16:25
支原体？不是微生物就是TSB的原因了，更换厂家再执行无菌模拟很难么

仅仅是其中6瓶的话，不太可能时TSB的问题

zhaosc8326

window 发表于 2019-1-22 17:26
3楼观点支持
FDA的案例

FDA案例原文在哪？学习下

zhaosc8326

3. A firm has multiple media fill failures. They conducted their media fills using TSB (tryptic soy broth) prepared by filtration through 0.2 micron sterilizing filter. Investigation did not show any obvious causes. What could be the source of contamination?
A firm recently had multiple media fill failures. The media fill runs, simulating the filling process during production, were conducted inside an isolator. The firm used TSB (non-sterile bulk powder) from a commercial source, and prepared the sterile solution by filtering through a 0.2 micron sterilizing filter. An investigation was launched to trace the source of contamination. The investigation was not successful in isolating or recovering the contaminating organism using conventional microbiological techniques, including the use of selective (e.g., blood agar) and nonselective (e.g., TSB and tryptic soy agar) media, and examination under a microscope. The contaminant was eventually identified to be Acholeplasma laidlawii by using 16S rRNA gene sequence. The firm subsequently conducted studies to confirm the presence of Acholeplasma laidlawii in the lot of TSB used. Therefore, it was not a contaminant from the process, but from the media source.

Acholeplasma laidlawii belongs to an order of mycoplasma. Mycoplasma contain only a cell membrane and have no cell wall. They are not susceptible to beta-lactams and do not take up Gram stain. Individual organisms are pleomorphic (assume various shape from cocci to rods to filaments), varying in size from 0.2 to 0.3 microns or smaller. It has been shown that Acholeplasma laidlawii is capable of penetrating a 0.2 micron filter, but is retained by a 0.1 micron filter (see Sundaram, et al.). Acholeplasma laidlawii is known to be associated with animal-derived material, and microbiological media is often from animal sources. Environmental monitoring of mycoplasma requires selective media (PPLO broth or agar).

Resolution:

For now, this firm has decided to filter prepared TSB, for use in media fills, through a 0.1 micron filter (note: we do not expect or require firms to routinely use 0.1 micron filters for media preparation). In the future, the firm will use sterile, irradiated TSB when it becomes available from a commercial supplier. (Firm's autoclave is too small to permit processing of TSB for media fills, so this was not a viable option.) The firm will continue monitoring for mycoplasma and has revalidated their cleaning procedure to verify its removal. In this case, a thorough investigation by the firm led to a determination of the cause of the failure and an appropriate corrective action.

zhaosc8326

window 发表于 2019-1-22 17:26
3楼观点支持
FDA的案例

找到了原文

jaysun

我只能说抚育条件不同，不是所有微生物都能被鉴别出的

zhaosc8326

莎瓦娜 发表于 2019-1-23 14:04
完整的原文有吗，或者是哪一个报告号

这是FDA问与答中的内容

莎瓦娜

dingkaiwang 发表于 2019-1-22 16:25
支原体？不是微生物就是TSB的原因了，更换厂家再执行无菌模拟很难么

那是不是还需要进行连续3批灌装呢

jixia

莎瓦娜 发表于 2019-1-30 11:34
那是不是还需要进行连续3批灌装呢

有充分证据表明是培养基导致的浑浊，不是无菌工艺本身的问题，可以不用重复3次。如果你证明不了，建议还是3批。