Method for determining lethality in sterilization process - biological indicator / bioburden method
A.1 General This method combines the knowledge of biological indicators against resistance and bioburden and resistance for a given period to establish periodic parameters (time of action). The use of this method requires proof that the bioburden level of the product remains relatively stable for a certain period of time and that the resistance of the bioburden is less than or equal to the resistance of the biological indicator. The resistance of the biological indicator is demonstrated by gradually increasing the duration of action and determining the inactivation rate of the cycle. This ratio and knowledge of the number of bacteria and relative resistance of the bioburden can be used to determine the sterilization time so that the SAL can be predicted.
A guide to this method can be found in ISO 14161.
A.2 procedure
A.2.1 Determine where the most difficult to achieve sterility in the product.
A.2.2 Place the biological indicator in the location of the product where it is most difficult to achieve sterilization conditions, creating a monitoring of the sterilization process, including the known number of microorganisms and known resistance to EO. If the location of the monitoring is not the most difficult to sterilize, determine its relationship to the most difficult to sterilize position.
This requirement can be met by using a PCD that demonstrates resistance to the sterilization process that is greater than the resistance of the product to the sterilization process. Pay attention to the effects of packaging and removal of sterilant from the PCD.
A.2.3 The above PCD is packaged in the same packaging as the conventional product and placed in the sterilized article.
A.2.4 Selecting a condition that is less lethal than conventional conditions exposes the sterilized item to EO (see Section 8) so that the reference microorganism is not completely inactivated.
A.2.5 After the EO action time increases and the other parameters remain unchanged, use one of the following methods to determine the lethality of the process:
a) direct enumeration (see A.3.1) or
b) partial negative method (see A.3.2 or A.3.3) or
c) The combined method of a) and b).
Note: Partially negative method uses the method of bacterial growth in the PCD reactivation test after partial gas action time. Based on this result, the inactivation rate of the reference microorganism can be calculated.
A.2.6 Based on the knowledge of the product bioburden (see ISO 11737-1), the resistance of the bioburden to the sterilization process and the inactivation rate of the reference microorganisms determine the range of treatment required to achieve the specified SAL .
A.3 Determining the process lethality
A.3.1 Direct enumeration
A.3.1.1 Determine the lethality of the sterilization cycle by directly enumerating the survival microbes to create a survival curve.
A.3.1.2 ISO 14161 and ISO 11138-1: 2006 C.3 give details of the method.
ISO11138-1:2006 C.3 requires at least five effects to cover the following aspects:
a) One time, the sample is not exposed to the sterilizing agent (eg, the action time is 0);
Note: There is no sterilizing agent or it is replaced by inert gas or media.
b) at least once, the number of viable cells decreased to 0.01% of the initial number of bacteria (reduced by 4 log 10);
c) Act at least three times, covering the period of time between a) and b).
A.3.2 Partial negative method using the Hocomb-Spearman Karber program (HSKP)
Biological indicators for EO sterilization should be exposed to ethylene oxide with increasing duration of action while other parameters remain unchanged. After the action, the method of directly immersed in an appropriate culture medium for a test sample to carry out testing. The ratio of the aseptically grown samples after the culture to the total number of samples was calculated. Details of the method are given in ISO 14161 and ISO 11138-1:2006 D.3.1.
ISO11135-1:2007
ISO11138-1:2006 D.3.1 requires at least five conditions to cover the following:
a) at least one set of samples, all test samples showing bacterial growth;
b) at least two sets of samples, some of which show bacterial growth (quantum zone);
c) At least two sets of samples showing sterile growth.
If the number of samples at each time point is the same and the time interval is constant, the modified HSKP method can be used: the Limited Hocomb-Spearman Karber program (LHSKP). See ISO 11138-1:2006 D.3.2 for further information . A.3.3 Partially negative method using the Stumbo Murphy Cochran program (SMCP) The Stumbo Murphy Cochran program (SMCP) requires that the result of a period of time in a partial negative range includes the time t, the number of units in which the growth of the bacteria is negative, and the number of repetitions n and each time the initial number N0 contaminating bacteria. To obtain valid data using the SMCP program, ISO 11138-1:2006 D.3.3 requires the D value to be calculated from the average of at least three partial negative runs to confirm reproducibility.
See ISO14161 for further guidance.
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