Sterilization indicators, biological

List of parameters (Shelf-to-Shelf Temperature Uniformity, Steam Sterilization Study — Shelf Monitoring, Steam Sterilization Study — Chamber Monitoring — Last 15 minutes of Exposure, Steam Sterilization Study — Eg Minutes, Steam Sterilization — Biological Indicator Test Results, Leak Rate Test, Vacuum Pump Down Test) Acceptable Limits, and Study Results... 

Let us do an actual problem, Example 3.1, the data for which are from an actual D value computation for steam sterilization. Biological indicators (strips of paper containing approximately 1 x 10 bacterial spores per strip) were affixed to stainless steel hip joints. In order to calculate a D value, or the time required to reduce the initial population by 1 logio, the adequacy of the regression model must be evaluated. Because bo and bi are unbiased estimators, even when serial correlation is present, the model y = bo + biX + et may still be useful. However, recall that e, is now composed of e,- i + di, where the 4s are N 0, 1). 

Hospital sterilizer loads vary in composition, thus the challenge presented to the test organism can vary considerably, depending on the type and contents of packages in which they are placed. The benefits of a standardized test-pack constmction and test protocol are obvious, and such recommendation is made by AAMI for steam and ethylene oxide sterilizers (11). More recentiy in European (CEN) and International (ISO) standards, biological indicators are considered as additional information supplemental to the measurement of physical parameters. Indeed, for sterilization using moist heat (steam), the biological indicator information is not considered to be relevant. 

Biological indicators for steam sterilization utilize bacillus stearothermophilus. In monitoring industrial cycles, a sufficient number of preparations each having a known degree of resistance are added to the load and retrieved after exposure, and cultured. 

Table 20.1 Inactivation factors (IF) for selected sterilization protocols and their corresponding biological indicator (Bl) organisms... 

Monitoring of the sterilization process canbe achieved by the rrse of physical, chemical or biological indicators of sterilizer performance. Such indicators are frequently employed in combination. 

Biological indicators (Bis) for use in thermal, chemical or radiation sterilization processes consist of standardized bacterial spore preparations which are usually in the form either of suspensions in water or culture medium or of spores dried on paper, aluminium or plastic carriers. As with chentical indicators, they are usually placed in dummy packs located at strategic sites in the sterilizer. Alternatively, for gaseous sterihzation these may also be placed within a tubular hehx (Line-Pickerill) device. After the sterilization process, the aqueous suspensions or spores on carriers are aseptically transferred to an appropriate nutrient medium which is then incubated and periodically examined for signs of growth. Spores of Bacillus stearothermophilus in sealed ampoules of cultrrre medium are used for steam sterilization morritoring, and these may be incubated directly at 55°C this eliminates the need for an aseptic transfer. 

Table 23.2 Biological indicators for monitoring sterilization processes ...

The USP also recommends the use of biological indicators, whenever possible, to monitor all sterilization methods except sterile filtration. Biological indicators are generally of two types. If a product to be sterilized is a liquid, microorganisms are added directly to carefully identified representative samples of the product. When this is not practical, as with solids or equipment to be sterilized, the culture is added to strips of filter paper. The organism chosen varies with the method of sterilization. 

A typical ETO sterilization cycle is shown in Fig. 10. As discussed at the beginning of this section, it is important to determine and monitor the bioburden level of the product entering the sterilizer. Also, the load configuration in the sterilizer is important in achieving uniform and reliable sterilization. Unfortunately, commercially available biological indicators used in ETO sterilization are often unreliable. Hopefully, progress will be made in this field in the years ahead. 

Dry heat is used to sterihze and depyrogenate components and drug products. The definition of dry heat sterilization is 170 °C for at least 2 hours and a depyrogenation cycle at 250 °C for more than 30 minutes. Typical equipment includes tunnel sterilizers (force convection, infrared, fiame) and microwave sterilizers. An important aspect is the need to ensure air supply is filtered through HEPA filters. Biological indicators such as Bacillus subtilis can be used to gauge the performance of sterilization. 

Aseptic BPS machines are subject to steam-in-place sterilization following standard CIP cycles. The SIP cycles are routinely measured by thermocouples located in fixed positions along the product pathway. Validation of SIP cycles should be carried out to demonstrate that consistent sterilization temperatures are achieved throughout the equipment to prove that the system can be effectively sterilized. Validation should also identify suitable positions for routine use, or justify the fixed probe positions already in place. The SIP validation is generally carried out with the help of additional thermocouples and should include the use of biological indicators (appropriate for moist heat sterilization). Test locations should include areas which may be prone to air or condensate entrapment. An accurate engineering line drawing of the system to aid identification of suitable test locations and document test locations selected should be available. 

Biological castles are employed during heat penetration situations in order to demonstrate the degree of process lethality provided by the sterilization cycle. Calibrated biological indicators utilized for this purpose function as bioburden models providing data that can be utilized to calculate Fq or substantiate and supplement physical temperature measurements obtained from thermocouples. 

The most frequently utilized to challenge moist heat sterilization cycles are Bacillus stearothermophilus and Clostridium sporogenes, spore-forming bacteria are selected because of their relatively high heat resistance. In addition to the selection of an appropriate organism for use as a biological indicator, the concentration and resistance of the indigenous microbial population is established. 

The biological indicator can be prepared to adequately challenge a sterilization cycle designed to provide a 10 6 probability of microbial survival with respect to indigenous bioburden. The concentration of spores utilized as the biological indicator can be determined from the following formula ... 

Nj = the load of microorganisms on the product to be sterilized Dg = D value of the most resistant isolate Nq = number of organisms on the biological indicator Dy = D value of biological indicator. 

The heating regimen is developed using a KAYE validator or equivalent and copper-constantan thermocouples. The determination of the load cold spot is achieved by actual experiments. A microbiological challenge test was performed to verify the sterilization conditions. Biological indicators... 

Perform microbiological challenge studies to determine the degree of process lethality provided by the sterilization cycle. The microorganisms most frequently utilized to challenge steam sterilizer cycles are Bacillus stearothermophilus and ATCC 7953. The Kaye validator equipped with 12 (minimum) thermocouples and biological indicators (10 ) shall be positioned in the detected cool points of the chamber and condenser. After the sterilization cycle is complete, the B.I s are recovered and subjected to microbiological test procedures. 

List of operation parameters (cycle events/times, TC distribution probes, biological indicator test, installation qualification review, sterilizer SOP review) acceptable limits, and study results... 

It is a specific kinetic expression for each micro-organism in a specific environment subjected to a specific sterilization agent or condition. In other words, the D value will be affected by a. The type of microorganism used as the biological indicator. ... 

The goal of the biological validation procedure depends on the nature of the process. If the process is intended to sterilize only, the probability of survival approach is used. In this case, validation studies must determine a dry-heat cycle that will assure that the probability of survival of the microbial indicator is not greater than 10 If the process is intended to sterilize and depyrogenate, which occurs when the materials can withstand excessive heat, the overkill approach is used. The goal here is to validate a heating cycle that can produce a 12-log reduction in the biological indicator population. 

Institute a documented monitoring system primarily relying on biological indicators, with lesser reliance on end-product sterility testing. 

Determine whether or not the I) value for the biological indicator varies as a function of the dose rate. With cobalt-60, dose rate differences are not of much concern (variance of 0.1-0.5 Mrad/hr), whereas electron beam sterilization might produce dose rate variances of several Mrads per min ... 

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