Principle
The Sterikon^® plus Bioindicator consists of an ampoule that contains nutrient broth, sugar, a pH indicator and spores of an apothogenic organism, Bacillus stearothermophilus ATCC 7953 (sporulation optimized).
The thermal resistance is such that the spores are totally killed after 15 minutes, when heated in compressed steam at a temperature of 121 ° ± 0,5 °C (1 bar). At lower temperatures or lower exposure time the spores can survive at least partly.
The ampoules are placed into the autoclave along with the batch to be sterilized. After autoclaving, the success ot the sterilization process is checked by incubation of the ampoules.
No growth of Bacillus stearothermophilus indicates adequate sterilization, whereas growth shows inadequate sterilization.


Procedure
An appropriate number of ampoules are included in the batch to be sterilized. Autoclaves with a capacity of up to 250 liters should be checked with at least 2 ampoules, while those with a greater capacity should have at least 6 ampoules. In order to avoid contamination by accidental breakage, it is advised to place the ampoules in a glass beaker.
The ampoules are placed in the autoclave at sites where the most unfavourable conditions for sterilization are thought to exist, i.e. at the bottom and in the middle of the autoclave. If a single large volume of material is to be sterilized (e.g. flasks containing a liquid), a test using the bioindicator is possible only, when the ampoule is placed in the centre of the vessel in question (e.g. suspended in a flask or immersed in the contents of a metal container). The Sterikon^® plus Bioindicator can also be used to monitor the functional efficiency, i.e. to test whether the prescribed temperature of 121 °C is reached within the entire autoclave and whether the temperature remains constant over the entire prescribed period of 15 minutes.
After sterilization, the ampoules are removed and incubated up to 48 hours at 60 ° ± 2 °C. A non-sterilized ampoule should also be incubated to serve as a control. Use of the ampoules at temperatures exceeding 125 °C sterilization temperature should be avoided to prevent the possibility of damaging the bioindicator. Damage to the bioindicator by over-heating may result in color changes without increased turbidity.

Evaluation
If sterilization is adequate, the Bacillus stearothermophilus spores are killed off. The contents of the ampoule(s) remain(s) opalescent and red-violet in color.
If sterilization is inadequate, the Bacillus stearothermophilus spores survive. The contents of the ampoule(s) then usually turn(s) yellow within 24 hours due to the formation of acid as a result of sugar fermentation and also become(s) turbid due to microbial growth. In cases in which the spores are partially damaged, the reaction may be delayed to 48 hours.
The contents of the control ampoule also turns yellow. If this does not occur, consider the test invalid.

Stability
When stored at the prescribed temperature (+2 ° to +8 °C) in the refrigerator, the bioindicator is stable at least up to the expiration date printed on the pack.

Storage

The ampoules should be stored in the refrigerator at +2 ° to
+8 °C. Storage at room temperature (up to approx. 25 °C) is possible for a limited period of about 1-2 weeks. Storage at temperatures exceeding +30 °C affects the stability.

Specification
The specifications of Sterikon^® plus Bioindicator are as follows:

n =   > 5 x 10⁵  spores per ampoule
D*121 =   1.5 - 3.0 minutes
F121 =   15 minutes

According to USP, the heat resistance and the number of spores are optimized, so that after a sterilization time of 5 minutes at 121 ° ± 0.5 °C in all ampoules living spores are present, whereas after 15 minutes of sterilization at 121 ° ± 0.5 °C all spores are dead. For the period in between ampoules can be found with either living or dead spores.
The spores are already in nutrient broth.

*Determined at time of manufacture, using fraction negative analysis. The D-value is reproducible at the stated temperature and only under the exact conditions under which it was determined. The user would not necessarily obtain the same results.