What are Biological Indicators, Examples, Applications.

Bioindicators are living organisms like plants, plankton, animals, and microbes that help monitor the health of natural ecosystems. They are used to assess environmental health and track changes in the environment.

Biological indicator vials contain spores from B. stearothermophilus, a microorganism that is killed when exposed to steam at 121.1°C for at least 20 minutes. Environmental Health and Safety (EHS) will use biological indicators to test autoclaves treating biological waste every three months.

Definition of Biological Indicators 

  • Biological Indicators are the type of microorganisms that indicate or monitor the health of the environment. 
  • Bioindicators are the type of microorganisms that indicate or monitor the contamination in the sterilization process.

What are Biological Indicators

  • Biological indicators are the special type of live microorganisms which has resistance power to a specific sterilization process.
  • Biological Indicators are also known as Bioindicators.
  • Bioindicators are mostly living organisms such as microorganisms, plants, and animals.
  • Bioindicators are used to confirm sterilization performance, verify the sterilization procedure, monitor the sterilization effect of the production process, etc.
  • For the preparation of biological indicators, it is necessary to determine the characteristics of the microorganisms used.
  • Preparation should be carried out according to certain procedures. 
  • Biological indicators contain a certain number of one or more spores, which attached to carriers, such as filter paper strips, glass slides, culture medium, stainless steel, plastic products, etc.
  • To avoid contamination in bioindicators it should be packaged with appropriate materials, sealed in ampoules, and set validity period. 
  • In the sterilization process, biological indicators are placed in different parts of the sterilization cabinet. And avoid direct contact with the indicator to be sterilized. 
  • A good bioindicator will indicate the presence of the contamination and also attempt to provide additional information about the amount and intensity of the contamination in the sterilization process.

Basic Characteristics of microorganisms for the preparation of biological indicators

Different sterilization methods use different biological indicators, and the microorganisms selected for the preparation of biological indicators must have the following characteristics:
  • The sterilization tolerance of the interested microorganism should be greater than that of all possible contaminating microorganisms in the product to be sterilized.
  • The selected microbial strains should be non-pathogenic.
  • The strain should be stable. 
  • It is easy to store.
  • It should be a long shelf life
  • Easy to cultivate. 

Commonly used biological indicators for the Sterilization Process

(1) Moist heat sterilization method
  • The most commonly used biological indicator for the moist heat sterilization method is Bacillus stearothermophilus spores.
  • Spores of Bacillus stearothermophilus, such as ATCC 7953, NCTC 10 007, NCIMB 8157). 
  • The heat resistance capacity of this bacteria is very high.
  • It completely killed at 121℃ for 19 min. 
  • Clostridium sporogenes spores (Spores of Clostridium sporogenes such as NCTC 8594, NCIMB 8053, ATCC 7955) can also be used as a biological indicator for moist heat sterilization method.
(2) Dry heat sterilization Method
  • The most commonly used biological indicator for the dry heat sterilization method is Bacillus subtilis spores.
  • Spores of Bacillus subtilis, such as NCIMB 8058, ATCC 9372).
  • Escherichia coli endotoxin was used for depyrogenation verification, and the dosage should not be less than 1000 bacterial endotoxin units.
(3) Radiation sterilization method
  • The most commonly used biological indicator for radiation sterilization method is Bacillus pumilus spores.
  • Spores of Bacillus pumilus, such as NCTC 10 327, NCIMB 10 692, ATCC 27 142). 
  • The number of viable spores per slice was 107-108, 
  • The microorganisms used for the sterilization process must exhibit greater resistance to radiation than Bacillus pumilus spores.
  • Therefore, Bacillus pumilus spores can be used to monitor the sterilization process, but not to establish the sterilization radiation dose.
(4) Filtration sterilization method
  • The most commonly used biological indicator for filtration sterilization method is Pseudomonas diminuta 
  • Pseudomonas diminuta such as ATCC19 146, which is used for filters with a pore size of 0.22 μm.
  • ATCC 14 756 for filters with a pore size of 0.45 μm.
(5) Gas Sterilization:
  •  It is a method of killing microorganisms by using the gas formed by chemical disinfectants. 
  • Commonly used chemical disinfectants are formaldehyde fumigation, ethylene oxide, gaseous hydrogen peroxide, atomized hydrogen peroxide, and ozone (O3). 
  • We know that no matter what gas sterilization method is used, the corresponding biological indicator can be used to evaluate this sterilization method.

(a) Ethylene oxide Sterilization Method:

  • For ethylene oxide gas sterilization the biological indicator id Bacillus subtilis spores (ATCC9372) that were sterilized with dry heat was used.

(b) Hydrogen peroxide sterilization method

  • As we know hydrogen peroxide sterilization method is one of the effective methods to remove or kill microorganisms quickly and efficiently.
  • The biological indicator used in the verification of the gaseous hydrogen peroxide sterilization method is the same as the moist heat sterilization method.
  • Bacillus stearothermophilus spores are most commonly used as a bioindicator for hydrogen peroxide sterilization method (Spores of Bacillus stearothermophilus, ATCC 7953).
  • Some commonly used biological indicators for gaseous hydrogen peroxide sterilization are Bacillus stearothermophilus spores, such as ATCC 7953, NCTC 10007, NCIMB 8157.

Biological Indicators Table and Uses

Spores of Bacillus
ATCC7953Moist heat sterilization validation and
gaseous hydrogen peroxide validation
Spores of Bacillus
ATCC9372Dry heat sterilization method validation
Ethylene oxide gas sterilization method validation
Ozone gas sterilization method validation
Spores of Bacillus
ATCC27142Validation of 60Co-γ-ray radiation sterilization method
of Bacillus subtilis
ATCC12980Hydrogen Peroxide Verification
PseudomonasdiminutaATCC19146Filter sterilization validation
CMCC(B)44112Validation of Ozone for Cleanroom Gas Sterilization
Escherichia coliCMCC(B)44102Validation of dry heat sterilization
CMCC(B)63501Aseptic process simulation test
CMCC(F)98003Aseptic process simulation test
CMCC(B)64941Aseptic process simulation test
CMCC(B)26003Aseptic process simulation test

Application of biological indicators

  • Biological indicators are used to check the sterilization process, equipment in pharmaceutical processes.
  • It is also used for verification of sterilization procedures by gas sterilization and filter sterilization and other physical and chemical methods of sterilization.
  • Biological indicators are very important Fermentation and pharmaceutical process verifications, such as Culture Media Sterilization, Bioreactor sterilization, aseptic process simulation test, sterile preparation sealing test, etc.
  • The performance of the sterilization process can be monitored by some parameters of the sterilization process, the killing degree of the biological indicator.
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