Harishchandra was a legendary Indian king. He gave away his kingdom, sold his family and agreed to be a slave to fulfill a promise he had made to the sage Vishwamitra. While employed in a cremation ground, Harishchandra told her wife that he would cremate his dead son only after getting the cremation fee. He was honest and never lied even during his difficult times. Just a simile to spores that they don’t lie and are true indicators. Yes, SPORES DON’T LIE
What are bacterial spores?
Bacterial spores are highly resistant, dormant structures formed in response to adverse environmental conditions. Once the environmental conditions are favourable, they revert to their vegetative state. They help in the survival of the organisms during adverse environmental conditions. They are generally resistant to high degree of heat. The oldest preserved live bacterial spore is 250 million years old [Isolated from salt crystal-salt mine in Russia]. Bacterial spores are used as Biological indicators to evaluate the sterilization process.
What is a biological indicator [BI]?
A biological indicator provides information on whether necessary conditions were met to kill a specified number of microorganisms for a given sterilization process, providing a level of confidence in the process. A biological indicator is a microbiological test system consisting of a standardized viable population of specific microorganisms inoculated on a carrier contained within its primary pack ready for use and providing a defined resistance to a specified sterilization process. Bacterial spores are used in BIs.
Use of BI’s
- Sterilizer cycle development.
- Validation of the sterilizer.
- Routine monitoring of the sterilizer.
Forms of biological indicators
| 1 | Spore strips | Strips of paper inoculated with spores |  |
| 2 | Ampoules | Spores in liquid media and spore suspension |  |
| 3 | Self-contained | Strips of paper inoculated with spores in a package that contains a separate media ampoule |  |
Types of BI’s
These spores are recognized to be the most resistant to the various sterilization processes.
| 1 | Moist Heat sterilization-High temperature > 1210C | Geobacillus stearothermophilus ATCC* 7953 |
| 2 | Moist Heat sterilization-Low temperature < 1210C | Bacillus subtilis ATCC* 5230 |
| 3 | Dry Heat sterilization | Bacillus atrophaeus ATCC* 9372 |
| 4 | Ethylene Oxide sterilization | Bacillus atrophaeus ATCC* 9372 |
| 5 | Radiation sterilization | Bacillus pumilus ATCC* 27142 |
| 6 | Hydrogen Peroxide sterilization | Geobacillus stearothermophilus ATCC* 7953 |
*ATCC-American Type Culture Collection. It is the premier global biological materials resource and standards organization whose mission focuses on the acquisition, authentication, production, preservation, development, and distribution of standard reference microorganisms.
Microscopic view of Spores
Performance evaluation-Manufacturer’s responsibility
- COA of the validity of the label claim for each lot with the sterilization process.
- Provide D value, spore population, storage conditions [Temperature and RH%].
- Media for the recovery after the exposure cycle and the incubation conditions.
Performance evaluation- User’s responsibility
- Verify the purity up to the genus level and the morphology of the received lot.
- Perform the spore count test as per USP <55> and the recovery should be -50 to +300% of the stated spore value in the COA.
- Perform evaluation tests, if the BI’s are stored more than 12 months at the specified storage conditions.
Recommended minimum requirement as per USP
| Sterilization cycle | Viable spore population | D value | Z value |
| Steam 121°C | 105 | 1.5 minutes | 10°C |
| Steam 132°C | 105 | 10 Seconds | 10°C |
| Steam 134°C or 135°C | 105 | 8 Seconds | 10°C |
| Ethylene Oxide 600 mg/L, 60% RH, 54°C | 106 | 40 Seconds | NA |
| Dry Heat 160°C | 106 | 3 minutes | NA |
Terms in BI’s
D-value: The time it takes at a specified set of condition to reduce the population by one log or 90%.
Z-value: The changes in temperature that it takes change the D value by one log or 90%.
Fo value: Equivalent process time to 1 minute at 121C based on a Z of 10oC.
Temperature F value
121 1.00
122 1.259
123 1.585
124 1.995
125 2.512
Biological Indicator testing procedure
The BI’s are exposed to the sterilization process and then incubated under defined growth conditions to determine whether any spores survived the process. If no spores survive, none grow and the test is a pass. If growth is detected, the test is a fail.
Positive BI = sterilization failure.
Negative BI = adequate sterilization.
Recovery time:
The exposed BI’s should be inoculated into the medium and the incubation to be started NMT 4 hours after the completion of the exposure cycle as per USP<55>.
Placement inside the sterilizer
Biological Indicator should preferably placed alongside a thermocouple
Self-contained BI Inside IV Drip Chamber
Inside a liquid container
Standards for BI’s
| AAMI/ISO – Sterilization of health care products- Biological Indicators | |
| 11138-1 | General |
| 11138-2 | EO |
| 11138-3 | Moist Heat |
| 11138-4 | Dry Heat |
| 11138-5 | Low-temperature steam and formaldehyde |
| EN 866 | European Norm adopted ISO 11138 series |
| AAMI/ISO 14161 | Sterilization of health care products – Biological indicators – Guidance for the selection, use and interpretation of results |
| AAMI/ISO 18472 | Sterilization of health care products – Biological and chemical indicator – test equipment |
| USP | United States Pharmacopeia <1035> |
| EP | European Pharmacopeia |
| IP | Indian Pharmacopeia |
Conclusion
Biological indicator is a true indicator of the sterilization process. When coupled with chemical indicators and when the sterilization cycle meets the established cycle parameters, the Quality program becomes robust and gives sterility assurance. The articles / aids / product sterilized in the sterilizer gives a very high degree of assurance in sterility. The 6 log reduction i.e. the complete killing of all the challenged heat resistant spores [Having a population of 106] ensures high degree of assurance of the sterilizer. Most importantly are the storage conditions of the BI’s, recovery time of the exposed spores in the carrier after the sterilization cycle, positive and negative controls during the BI incubation.
Future scope
Microbiological testing takes it own time due to the different generation time [Tg] of the Micro organisms. The scope for the future would be devising or correlating a method to identify the Micro organisms in a very shorter time and technology would definitely aid, which will help to reduce the incubation time.
“Man needs to explore a lot, the usefulness of Microorganisms for the benefit of mankind. The world needs not only one Newton or Pasteur but many Newton’s and Pasteur’s in the future”.
Muthu
Note-The images given for representation in this blog are taken from Google Images. Many thanks for Google.
Ultimate article , keep it up sir , it’s required.
Thank you Rohit for the comments!