A variety of methods are routinely used to monitor & validate sterilization protocols used in the production of sterile products.
Sterilisation protocols include steam, dry heat, ethylene oxide, hydrogen peroxide vapour and gamma irradiation have been developed to deal with the diverse range of materials now used for the preparation and storage of sterile products.
Steam sterilization is routinely carried out at 121°C or 135°C and is used predominantly for sterilisation of paper & fabric wrapped products, bottles liquids, glass & stainless steel equipment. Dry heat sterilization at 180°C is suitable for stainless steel equipment but not for plastics or liquid-containing products. Ethylene oxide and gamma irradiation are ideal for treatment of bulky packs and paper or plastic products which may be sensitive to heat and cannot be sterilized with steam or dry heat. More recently hydrogen peroxide vapour has proved well suited suited to the sterilization of isolator cabinets used in the production and packaging of many pharmaceutical products.
Chemical indicators which give an irreversible colour change are used to provide a visible indication of exposure to a sterilization cycle - the indicators themselves however do not provide a realistic demonstration of cycle lethality. but simply show that the product has been exposed to conditions expected to lead to sterilization. In order to demonstrate sterilization cycle lethality it is necessary to use a biological indicator, (BI).
A biological indicator (BI) is a device containing a known population of a viable organism exhibiting a significant and quantified resistance to the sterilization conditions to be validated.
The challenge organism is usually selected to offer a significantly greater resistance than the organisms normally encountered in order to provide the greatest level of confidence in the procedure. The spores from spore forming bacteria are ideally suited to use as indicator organisms - the organism selected according to the type of sterilization protocol to be tested:
Geobacillus stearothermophilus ATCC 7953 for steam
Bacillus atrophaeus ATCC 9372 for dry heat & ethylene oxide
Bacillus pumilus ATCC 27142 for gamma irradiation
Geobacillus stearothermophilus ATCC 12980 for hydrogen peroxide
Biological indicators exist in a variety of formats designed to facilitate handling, ease of use and to ensure they remain free from post-cycle contamination. Spores can be presented on a variety of carriers (paper strip, stainless steel, cotton thread, stainless steel disc or wire) each individually packaged in glassine or Tyvek envelopes.
After exposure to the sterilization cycle they must be transferred aseptically to individual tubes of Tyryptone soy broth (TSB) for incubation in order to check for presence of viable spores. Self-contained indicators Pro Spore and Pro Spore 2 ampoules) containing both spores and recovery medium have been developed to remove the requirement for aseptic transfer following the sterilization cycle thus making the technique quicker and simpler to carry out and reducing the need to make the transfer under laboratory conditions. .
Validation of sterilization cycles is relatively straight forward for loads comprised of bottles, flasks, tubes, cloth packs etc. where the introduction of BI's into the load at various locations, (centre load, close to drain point etc.) may be accomplished quickly and without problem. Validation of more complex items of equipment (fermenter vessels, filtration sets etc.) may however prove much more difficult due to the complex geometry & bulk of the equipment itself and the restricted access to any entry & exit tubing attached to it.
Tubing has always presented a particularly difficult challenge for process validation, requiring the placement of a BI at the centre point of the tubing run. The positioning and recovery of the indicators is labour intensive and fraught with potential problems Care must be taken when positioning indicators to avoid occluding the tube bore thus preventing easy steam or gas access - any restriction in gas flow may itself contribute to failure of the BI & require lengthy investigation and further test re-validation.
A novel development by Raven Biological Laboratories Inc of Omaha now allows tubing validation to be made as simply and quickly as routine load validation using spore strips.
ProLine, is an in-line Process Challenge Device designed specifically to assist in validating the sterilization of various lengths and diameters of tubing. Available with either B.atrophaeus or G.stearothermophilus ATCC 7953 spores ProLine can be used to validate either ethylene oxide or steam or sterilization cycles with tubing ranging in diameter from 1/8" to 5/8" ID.
Inside the two conical halves of the ProLine device, a paper disc impregnated with bacterial spores is packaged in a glassine pouch and sandwiched between two sets of "O" rings to form a gas-tight seal. The length of tubing to be validated is cut in half and the ProLine device used to attach the two pieces together. The sterilant (steam or Ethylene oxide) must enter both ends of the tubing and penetrate to the mid-point of the tubing where the spore disc is housed within the ProLine device.
When the sterilization cycle is completed, the ProLine and attached lengths of tubing are removed from the sterilizer chamber and transferred to an area where an aseptic transfer of the spore disc to TSB can be carried out. This is accomplished by breaking the two section of the ProLine device apart ( Insert figures 2 and 3) to expose the glassine packaged spore disc. The glassine envelope can be handled without fear of contaminating the exposed spore disc within. The envelope can then be opened aseptically and the spore disc transferred to TSB for incubation (7 days) in order to develop the test result.
Results for validation of steam sterilization using Pro Spore 2 self contained indicators or Raven G.stearothermophilus ATCC 7953 spore strips and modified TSB with Bromocresol Purple indicator are available in only 24 hours. These products and BI combinations have been demonstrated to give reliable and accurate detection of spores surviving the sterilization cycle - A clearly visible colour change from purple to yellow along with turbidity in the broth signals germination and growth of the spores and a failure of the cycle to inactivate the BI.
For further details of the full range of BI's available for sterilizer validation visit the web site at www.m-techmicro.com or contact M-Tech Diagnostics Ltd. directly on Free phone 08-0800-68324 (08-0800-MTECH)