Spore Suspension Rubber Stopper Autoclave Validation


Just wondering if anyone has any experiences of validating the steam sterilisation of rubber stoppers using B.I spore suspension innoculation?

Has it led to the need for an increase in the cycle sterilisation exposure time?

What BI etc do you commonly use to innoculate Geobacillus stearothermophilus or Bacillus subtilis?

Any feedback would be very much appreciated.


Hi !
The Bacillus stearothermophilus is loaded as the BI in the bung steriliser, However the FDA expects it to be on the bung. i.e. you have to load the bung ( Not bung steriliser) with the 10 X6 suspension of the B. Stearothermophilus organism, Dry in Laminar and use it as BI.
Your cycle development will itself consider this parameter,hence the time of exposure may not chnage.



Thanks for your response. Is there a need to verify independently the D value of BI suspension innoculated on the bung?

Not required ,

The D value for the organism need not determine, since we consider the D value of the suspension and we follow the overkill approach.


Actually, it is better if you have an understanding of how the rubber affects the resistance of the organism. Rubber can increase resistance enough to fail half cycle validation. Without characterizing the load and just saying we do overkill sterilization means that you have no idea of your edge of failure and therefore investigations into failures are difficult if not impossible to complete.

You are right,
The haf cycle may fail and rubber can increase the resistance of bacteria.
Surprisingly, thats what we want to interprete. Whether my cycle can kill the organisms of located on the rubber stopper and I may have to increase my cycle, if I have to kil the organism from rubber stopper.
See our intention is to kill the organisnm not pass the cycle. If the resistance of the organism increase by the rubber , thenwe should increase the cycle time.

You can take inoculated stoppers and send them to be evaluated in a bier vessel. And the rubber does increase the resistance. One other issue with inoculating stoppers is the recovery method and population counts. The other thing that will affect your sterilization is the load configuration, the resistance of a single stopper is nice to have but also how is resistance changed when you have 20,000 stoppers in a container.

Several companies send product stoppers to Labs for D-Value testing to characterize the spores on the stopper as a true BI for resistance. Yes, type of rubber does make a tremendous difference on resistance. Once the D-Value is known, now cycle development starts to assure lethality of BI. Then, each month or so, a group of color coded stoppers can be sent to the Lab and using the same Spore Batch as used to determine D-Value can inoculate the group and send them back to the facility to use in future load monitoring. When supply of inoculated stoppers gets low, more are sent out to be inoculated and so on. Russ

Load monitoring? What do you mean by load monitoring? Would that be placing inoculated product into the autoclave and then using absence of growth as a criteria for sterilization? IMHO You should just do parametric release on a validated cycle. Also, studies I have seen don’t support that rubber affects D values greatly, but shape, mass, and load density are better attributes to look at. (penetration)

Two additional questions along this same line:

  1. Our stopper sterilization validation has been criticized by one regulatory body. That Agency has indicated we need to inoculate five (5) stoppers per bag of stoppers as opposed to our current requirement of one (1) inoculated stopper per bag. The bags monitored are distributed throughout the load. In all 18 bags in the load are monitored using 1 inoculated stopper per bag. Has anyone else run into this requirement of five (5) stoppers per bag or perhaps a statistical approach to monitoring the x number of stoppers in a bag containing y number? Can someone help me locate a reference for this? I am interested in reviewing the reference(s) to make certain we are not missing anything else.

  2. For periodic requalification of the autoclave…do you think inoculating stoppers is necessary or is it enough to monitor the loads selected for the requalifications with thermocouples and spore strips, and ensuring no changes? How are you approaching this?

Thanks very much for your input…