Are Bacterial spores descendants of Legendary Indian King Harishchandra?

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

1Spore stripsStrips of paper inoculated with spores     
2AmpoulesSpores in liquid media and spore suspension       
                           
3Self-containedStrips 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.

1Moist Heat sterilization-High temperature > 1210CGeobacillus stearothermophilus ATCC* 7953
2Moist Heat sterilization-Low temperature < 1210CBacillus subtilis ATCC* 5230
3Dry Heat sterilizationBacillus atrophaeus ATCC* 9372
4Ethylene Oxide sterilizationBacillus atrophaeus ATCC* 9372
5Radiation sterilizationBacillus pumilus ATCC* 27142
6Hydrogen Peroxide sterilizationGeobacillus 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

  1. COA of the validity of the label claim for each lot with the sterilization process.
  2. Provide D value, spore population, storage conditions [Temperature and RH%].
  3. Media for the recovery after the exposure cycle and the incubation conditions.

Performance evaluation- User’s responsibility

  1. Verify the purity up to the genus level and the morphology of the received lot.
  2. 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.
  3. 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 cycleViable spore populationD valueZ value
Steam 121°C1051.5 minutes10°C
Steam 132°C10510 Seconds10°C
Steam 134°C or 135°C1058 Seconds10°C
Ethylene Oxide 600 mg/L, 60% RH, 54°C10640 SecondsNA
Dry Heat 160°C1063 minutesNA

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  
11138-1General  
11138-2EO  
11138-3Moist Heat  
11138-4Dry Heat  
11138-5Low-temperature steam and formaldehyde  
EN 866European Norm adopted ISO 11138 series  
AAMI/ISO 14161Sterilization of health care products – Biological indicators – Guidance for the selection, use    and interpretation of results
AAMI/ISO 18472Sterilization of health care products – Biological and chemical indicator – test equipment
USPUnited States Pharmacopeia <1035>  
EPEuropean Pharmacopeia  
IPIndian 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.

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