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Geobacillus stearothermophilus spores Photo courtesy of 3M.

Incubation time

This incubation time was established in the early days of biological indicators, and was based on the technology available at that time. An incubation period of seven days is not at all practical or useful in today’s healthcare environment. So, for biological indicators, there was a need for speed.

The incubation time for a biological indica- tor is the amount of time that the BI must be incubated before a decision can be made that the BI is negative (i.e., the spores are all dead) and the test is complete. This concept takes a little more explanation ... if a biological indica- tor turns positive, it has completed its “task” of providing information on the quality of the biological indicator system (in the case of a positive control) or of the sterilization process itself (a positive BI test indicates a sterilization process failure). If a biological indicator turns positive you will end the BI test at that point and take appropriate action. But, how long must you incubate a BI before you can decide that it is truly negative and end the test? This time frame is called the incubation time. The international biological indicator per- formance standards state that the reference incubation time for a biological indicator is 7 days.3

incubated at the proper temperature for up to seven days. So, how could you tell if the BI was positive or negative? The user needed to look for a “signal” from the spores that they were alive (positive BI), or dead (negative BI). The original signal used to determine a posi- tive or negative result was the development of turbidity, or cloudiness, in the test tube. If the BI placed in the medium had viable spores (either a positive control or a sterilization process failure), the spores would convert to vegetative cells, and begin to grow or repli- cate. Over time, the number of cells in the test tube would increase to the point where the density of cells in the tube was high enough to scatter light passing though the test tube, making the medium appear cloudy. While effective, this process required a significant amount of incubation time (up to 7 days) to allow the spores to germinate and the cells to grow for enough generations to be able to scatter light.

Biological indicator evolution The first generations of biological indicators consisted of spores applied to some sort of car- rier, such as a piece of suture material. These early BIs evolved into spore strips, where the spores were applied to a small paper strip that was enclosed in a glassine envelope which al- lowed sterilant penetration while protecting the spore strip from outside contamination. After the sterilization process the indicators were transferred to a test tube containing the growth medium using a process called aseptic transfer. This was typically done in a micro- biology laboratory equipped with special laminar flow hoods to try and prevent any environmental organisms from contaminat- ing the spore strip or the media, which would create a false positive result. (Note: This process is not required with self-contained biological indicators). The test tubes were then

The next advance in technology introduced a color-based pH indicator into the growth media, to make the biological indicator signal a color change rather than cloudiness. A pH indicator is a chemical that responds to the acidity of the solution, and will typically be one color at an alkaline pH and change to another color as the solution becomes more acidic. Biological indicators utilizing the pH color change system have growth media that is specially formulated so that bacteria grow- ing in the medium will produce acidic by- products. As the bacteria continue to grow, the growth medium will continue to become more acidic until the pH indicator changes color. This technology enabled the development of self-contained biological indicators in the 1970s. The glass media ampoule in the SCBI was too small to see development of turbidity, but a color change was readily apparent. The optimization of the media in SCBIs and the user’s ability to detect the color change signal faster than the cloudiness signal reduced the incubation time from 7 days to 2 days. This was much faster and easier than spore strips, but still required incubation times that were not optimal for healthcare.

The next major leap in reduction of biologi- cal indicator incubation time came from new technology that enabled detection of biologi- cal signals from viable spores much earlier in their germination and outgrowth process. To understand this, we need to understand a little more microbiology. The spore activation and germination processes may sound simple but they are actually complicated, multi-step processes. A good analogy is the steps that occur when a computer powers up. Once the


power button is pushed the computer goes through a series of actions that turn on many programs and sub-systems in the computer in a specific order, until the computer is fully operational and ready for use. In the spore, the cell’s “sub-systems” are created and activated by many biochemical reactions. Specialized proteins called enzymes act as catalysts that make these complicated reactions happen much more quickly. The first rapid readout biological indicators used the actions of some of these “boot up” enzymes to produce a sig- nal that could be detected earlier in the spore outgrowth process, reducing the required BI incubation time from days to hours. The enzymes used to produce a signal for rapid readout biological indicators are enzymes that become active early in the activation and germination processes. These enzymes have a specific natural role for the cell, but their catalytic actions can also be uti- lized to produce a signal that can be detected and analyzed as a positive response. Rapid readout biological indicator technology uses a special indicator in the growth medium that can interact with the enzyme. This chemical is like the pH indicator discussed earlier, ex- cept that instead of turning color based on a change in acidity this indicator changes from a non-fluorescent molecule to a fluorescent molecule when it is acted on by the enzyme. Fluorescence means that it will “glow” or emit light at a certain wavelength (say, Wavelength B), if it is first exposed to light of a different wavelength (Wavelength A). So, rapid read- out BIs use a biological indicator reader that shines Wavelength A light onto the incubating biological indicators, and has a detector that is sensitive to Wavelength B light to look for a fluorescent signal. If the enzyme is active in the biological indicator (i.e., a positive control BI or a positive BI from a sterilization process failure), the sensors will detect the fluorescent signal and the reader analyzes this signal and indicates a positive BI result.

Rapid readout biological indicator technol- ogy has reduced incubation times from days to hours. Continued improvements of the physical design of these biological indicators concentrated the fluorescent signal to make it easier to detect. These changes, coupled with improved sensors and electronics in the read- ers, have now reduced biological indicator incubation times to less than an hour, and in some cases, less than 30 minutes. This dra- matic improvement in incubation time, from 7 days to less than 30 minutes, means that this important quality control information regard- ing the efficacy of the sterilization process is now available in a timeframe that fits with the healthcare facilities’ workflow.

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Self-Study Test Answers: 1. A, 2. A, 3. A, 4. A, 5. B, 6. B, 7. B, 8. B, 9. A, 10. A

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