Biological Indicators

What are biological indicators


Biological indicators are currently the foremost method of assessing the sterility of any medical or pharmaceutical environment through the use of some of the most resistant microorganism strains (eg. Bacillus or Geobacillus), rather than simply relying on meeting the requirements of both physical and chemical sterilization.

Microorganisms used as biological indicators present a higher concentration and are more resistant than the common biological contaminants found in pharmaceutical or medical environments, tested environments found with inactive biological indicators can be of assured sterility.


Unlike biological indicators, enzyme indicators uses thermostable Adenylate Kinase, an enzyme isolated from thermophilic Sulpholobus acidocaldarius bacteria, which are commonly found in thermal vents and hot springs.


Geobacillus stearothermophilus and biological indicators

Geobacillus stearothermophilus is a type of thermophile found widely distributed in hot springs, ocean heat vents, ocean sediment, and soil, and is characterized by its rod-like shape and gram-positive thick cell wall (a member of firmicutes).  These heat loving bacteria will thrive within a temperature range of 30-75 degrees Celsius, with some strains capable of aerobically oxidizing carbon monoxide.


Up until 2001 these bacteria fell under the genus Bacillus (group five) until their fairly recent reclassification under the new genus Geobacillus. Though previously thought to be a single species (Bacillus stearothermophilus), it became obvious there were differences in phenotypic characteristics from group to group, such as preferred temperature ranges, and now it’s more than clear that Geobacillus comprises several unique species.


Do biological indicators work

When biological indicators are used correctly with recommended frequency, they can be an accurate determinant of any given medical or pharmaceutical environment’s sterility.  As biological indicators are highly resistant microorganisms, you can be assured that any sterilization process that renders them inactive will have also killed off more common, weaker pathogens.


Biological indicator tests require a seven day incubation period before results can be determined, which is where enzyme indicators are overtaking the market with instant results of decontamination cycle performance.  Not only are the results of enzyme indicators instant, but enzyme indicators are manufactured to assure one-hundred percent accurate results to eliminate the lengthy and expensive false positive investigations that can often result with biological indicator tests.


How are biological indicators used

Biological indicators introduce highly resistant microorganisms to a given environment before sterilization, and then are measured afterwards to test the effectiveness of current sterilization processes.  Much like biological indicators, enzyme indicators achieve the same purpose, only with more accurate, quantifiable results delivered instantly and for a fraction of the costs associated with biological indicators.


Why are biological indicators important

Sterility is of the upmost importance when it comes to the health and safety of medical environments.  By using biological indicators to test the sterility of such environments regularly, environmental protection from more common, less resistant pathogens can be assured.  When it comes to making sure a medical environment is free of potentially harmful or disruptive pathogens, however, enzyme indicators outperform their biological counterparts by providing instant, quantifiable data that is 100% performance assured.


Types of biological indicators

Biological Indicators are preparations of known bacteria with a stable, defined resistance thresholds and a predictable response to various sterilisation processes.  In the lab or aseptic cleanroom environment, Biological Indicators are used to periodically revalidate sterilisation protocol and confirm inactivation of pathogens.


There are at least three different types of Biological Indicators used in sterilisation monitoring, with each type using bacterial spores of known sterilisation and sterilisation procedure resistance.  It is also possible for a Biological Indicator to incorporate two distinct species of microorganisms.


Biological Indicators – Carriers

A carrier is a vessel (plastic, glass, filter paper, etc) which contains spores and is sealed so as to preserve the viability of the microorganisms. The carriers are well made and designed not to be degraded by the sterilisation process the Biological Indicator is meant to monitor, as it would compromise Biological Indicator performance. The packaging itself also undergoes aseptic manufacturing procedures to ensure it does not contain any contaminants which would affect the stability or the performance of the Biological Indicator.


Biological Indicators – Spore Suspension

Biological Indicators can also come in a spore suspension, which is then directly applied to the product, packaging, or surface that will undergo the sterilisation procedure.  Simulations of inoculated products are sometimes used in sterility monitoring, but it is usually a requirement to show the final product, which will receive the inoculation, does not alter the effectiveness of the Biological Indicator, thereby invalidation results.


Biological Indicators – Self-Contained Indicators

Self-contained indicators have packaging which contains the recovery growth medium for the microorganisms’ incubation period following sterilisation exposure.  Self-contained Biological Indicators containing both the bacteria spores and the growth medium are an enclosed system, which provides sterilisation resistance.


Sterilisation resistance of the self-contained indicator’s system depends on the length of time it takes for the sterilant to penetrate the package.  Ease of penetration is controlled through composition and design at the manufacturing level.  These types of indicators often contain a dye to indicate negative or positive growth in bacteria spores following incubation, and the sample is ready for incubation directly following the sterilisation process.


Key Characteristics of biological indicators

Biological indicators are classed by the bacteria name of the microorganism used, the D-value, and the total amount of viable spores per carrier.  One of the most important characteristics of a biological indicator sample is that the bacteria must be ready for sporulation and it must occur on the selected medium, followed by germination should any of the spores survive the decontamination protocol.


Here are a few key characteristics of biological indicators:


  • The biological indicator must contain only the microorganism(s) specified on the label, and this microorganism should be verified to be correct by at least phenotypic identification.
  • Biological indicator populations are traditionally < 1 x 106 as equipment, devices, and spaces purported to be sterile are designed to achieve 10-6 microbial survival probability


The D-value, or decimal reduction value, is the level of resistance presented by a microorganism when exposed to defined sterilisation parameters.

Spore criteria for use as biological indicators

The requirements a spore must meet in order to be considered for use as a biological indicator are as follows, as laid out by both the Public Health Service of the United States and published in British Pharmocopeia:


  • The spore must not be pathogenic in nature.
  • The spore must not produce toxins or pyrogen.
  • The spores must not germinate below 37o
  • The spores show unique natural resistance to specific sterilisation procedures.
  • The spores should be able to be kept in storage for long periods.

Resistance and biological indicators

A whole host of factors can influence the resistance to sterilisation procedures displayed by microorganisms in the endospore state.   Some bacterial endospores are able to survive a number of different sterilisation protocols, such as radiation, chemical, pressure, desiccation, and heat treatments.  The bacteria’s ability to survive is due to a number of different factors which can include genetic components, low water content within the spore itself, and a thick proteinaceous spore coating.


Microbial genetics play an important role in bacterial resistance.  For example, Geobacillus stearothermophilus spores are far more resistant than those of Bacillus subtilis when it comes to steam sterilisation, due to differences in genetic material.  To make matters more complicated, not all genotypes of any particular microbial species, which is why only specific culture collection numbers can be used as biological indicators, as they have undergone strict cultural growth that adheres to an unchanging, stable genotype.


Biological indicators and types of resistance

Often for steam sterilisation the Geobacillus genus is selected due to its genetic characteristics.  Endospore forming thermophiles comprise the genus Geobacillus.  Found in cold ocean sediment and cool soil samples, these bacteria exist in unexpected high numbers even though the temperatures of their habitat fall well below that required for bacterial growth.


On the other hand, Bacillus atrophaeus is a better miscroorganism for evaluating dry heat sterilisation.  Much like G. stearothermophilus, it is down to a number of genetic mechanisms that create a resistance in Bacillus spores to chemicals, heat, and even radiation.  Moist heat resistance in a microorganism, as example, is thought to be due to the presence of dipocolinic acid within the spore casing and its chelating abilities.   Additionally, spores with low core water content are oftentimes resistant to wet heat, as are spores with specific levels of certain mineral ions found in the core.  Spore DNA containing alpha / beta small acid soluble spore proteins (SASP) help protect against wet heat related damage to the DNA.  It’s not well understood how wet heat kills spores, though it appears to not be through DNA damage, such as that caused by other forms of sterilisation such as gamma radiation and ultraviolet light.




Some bacterial spores have resistance to ultraviolet radiation, caused by alpha / beta SASP binding to the microorganism’s DNA, as well as to the photosensitising process of dipicolinic acid.  Gamma radiation is also able to destroy a microorganism’s DNA, though it is not well understood the mechanism by which allows microorganisms to possess gamma radiation resistance.


Chemical resistance can oftentimes vary chemical to chemical, but generally speaking there are still a few factors that are important for chemical resistance in spores:

  1. Alpha / beta SASP saturation of the DNA for protection.
  2. Spore core protected by a semi impermeable membrane for protection.
  3. The outer coating of the spore contains proteins which will work to detoxify chemicals through chemical reactions.
  4. DNA repair takes place.

Intrinsic physiological factors also play a role in spore resistance in addition to the genetic components. As example, biological indicators containing G. stearothermophilus for steam sterility monitoring are prepared in such a way that places numerous sub-cultures between the working culture and the master culture.   This is oftentimes why biological indicators may vary from manufacturer to manufacturer, or even change over time from the same supplier.   In addition cultivation and conditions for G. stearothermophilus spores for steam sterility monitoring and the steps undertaken to prepare the spores in a suspension have all been shown to affect spore resistance.


Extrinsic Resistance and biological indicators

Carriers can have a big impact on spore resistance when undergoing sterility monitoring.  To complicate matters, the resistance is affected by both the environment being sterilised as well as the type of carrier selected and the specific culture of the biological indicator it houses.   Unfortunately carriers will always be unavoidable when it comes to biological indicators as it’s simply what facilitates the biological indicator’s practical use, which means the biological indicator culture will always come in a carrier.  Commercially available strip carriers, as example, oftentimes are packaged in in an exterior plastic envelope, which allows the carrier to be penetrable by the sterilisation procedure yet not undergo harm before use.  Biological indicators with a paper carrier are often sealed in a container or envelope made of paper, Tyvek, glassine, or Mylar to prevent culture contamination.


The response of a biological indicator can also be greatly affected by not only the carrier itself, but also in the way that the spores are laid out within the carrier.  Many commercially available biological indicators use carriers of metal strips, paper, or coupons and the biological indicator spores respond differently to each individual type, which will affect positively or negatively the resistance of the spores to the sterilisation procedure.   All of this is taken into account when manufacturing biological indicators, and also should be taken into account when developing new sterilisation cycles.


Biological indicator preparation

Though research shows exactly how different types of carriers affect biological indicator resistance, it’s significantly harder to judge the ways in which inconsistent mounting procedure of the microorganism to the carrier medium affects resistance.   Testing shows that microorganisms can often be found among the cell debris of the parent culture, which might then help protect the microorganism from the sterilisation procedure it is meant to test. As well, concentrating spores in a large group on the carrier can add extrinsic resistance to the microorganism against gas sterilisation methods.


Spore resistance can also be affected by culture production, which may include the temperature and growth medium used, as well as the medium which houses the spores themselves.  Some suspensions, like saline, can leave a residue on the spore, giving the spore an abnormally high resistant challenge.  Alcohol is commonly used as a suspension agent due to the fact it dries rapidly, leaving no time for spore clumping.


Storage of Biological Indicators

Both the behaviour of a biological indicator and spore resistance can be affected by environmental conditions such as humidity.  Suspensions and ampoules should be refrigerated at 2-8 ° C.   In suspensions, the spores are in constant contact with the growth medium, so they are refrigerated to keep from reaching their optimal growth temperature and germinating.   Biological indicators should also be stored away from direct sunlight, chemicals, and sterilizing agents as it can compromise the biological indicator and invalidate sterility testing results.


Handling of biological indicators

Much like when considering the sterility of a cleanroom, humans are the biggest source of contaminates in the sterile environment, which is why it’s an important factor to consider when handling biological indicators.  As mentioned previously, biological indicator carriers will come packaged in a material that protects the carrier from contamination until it is ready for use.   However, when going to handle the culture for sterility procedure testing its important for personnel to follow gowning, de-gowning, and hygiene procedures outlined for the clean environment, as such practices also protect against contamination of the biological indicator culture.   Dressing for the clean environment can include wearing any of the following, according to proper protocol and procedure: bouffant caps, shoes, face masks, beard covers, boots, gowns, aprons, gloves, frocks, hoods, and shoe covers.


Biological Indicators versus Enzyme Indicators

Though Biological Indicators have long held the market in sterilisation monitoring, Enzyme Indicators are set to replace them as a faster, cheaper alternative.  A variety of different industries depend on Biological Indicators to provide sterilisation monitoring, and stopping research or production while waiting on Biological Indicator results to come back and be frustrating, not to mention costly.  Enzyme Indicators are able to provide instant, quantifiable results, which means no longer having to stop cleanroom or aseptic environment operations waiting for spores to incubate.


Sterilisation and Biological Indicators

It is important that cleanroom personnel not only maintain the sterility of the clean environment but also test for the sterilisation process’s effectiveness. Biological indicators have largely been the industry standard for monitoring a sterilisation process as the highly resistant spores used (of the Bacillus or Geobacillus species) can definitively determine whether or not sterilisation has been effective by directly assessing the process’s lethality to microbes.  As the spores in Biological Indicator testing kits are stronger and more resistant than normal pathogens typically found in the lab or clean environment, inactivating BI spores is a quantifiable indicator other pathogens have been cleared successfully as well.


Biological indicators for sterility testing come in microbiological testing kit, which contains a live culture of inoculated spores ready for use.   These highly resistance spores provide a definable resistance to sterilisation procedures, and such procedures should only be considered successful once the desired inactivation of the spores has been achieved in accordance with the clean environment’s sterilisation monitoring programme.




Biological monitoring of the effectiveness of a sterility process should be inexpensive, easy to use, and provide definitive results as quickly as possible so that corrections can be made to sterilisation parameters.  This is why Biological Indicators are the current gold standard in providing an ideal monitoring solution for sterilisation.


How often should biological monitoring be carried out?

Sterilisation cyclers and procedures will be laid out in the cleanroom or clean environment operative, set by the company or institution invested in maintaining the clean space. Biological Indicator manufacturers recommend using BIs at least weekly to test the effectiveness of sterilisation processes.  A control group Biological Indicator (one that has not undergone sterilisation) should also be used and incubated in the same manner as the test Biological Indicator as should produce a positive control result.


Positive Biological Indicator spore tests

A positive result with Biological Indicators shows that a flaw exists within the current sterilisation process.  In this case, the first step is to review proper environment sterilisation procedure as outlined by the institute / employer / lab etc. and review whether all steps were followed.  Then, using the same steps in the previous BI test, test the environment again.  If the second result shows a negative, then it is possible human error lead to improper sterilisation protocol in the first instance. If the second test yields positive results again, then work within the cleanroom or clean environment will need to be suspended until such time as the sterilisation process can be reviewed and a new one implemented.

Enzyme Indicators to overtake Biological Indicators in Sterilisation Monitoring

Biological Indicators have long been accepted by the authorities as the gold standard in sterilisation monitoring as they provide definitive results attesting to the effectiveness of a sterilisation process.  BIs are depended upon in a variety of different industries, cleanrooms, and are crucial to aseptic manufacturing procedure, which is why when a test is positive so many industries feel the dread of halting work or production, waiting on the results of the biological indicator testing to come back.   This is where Enzyme Indicators are going to overtake their slower predecessors by providing reliable, instant testing results. The thermostable enzyme Adenylate Kinase used in Enzyme Indicators boasts high environmental tolerances and a reliable, quantifiable inactivation profile, and is testing to be super in sterilisation monitoring.  As the luciferin reaction Adenylate Kinase undergoes emits light, EIs produce quantifiable data on decontamination, unlike their Biological Indicator counterparts.  This enables EIs to give instant, reliable results in sterilisation monitoring.


Bioindicators versus biological indicators

Biological indicators, the cultures of specific microorganism spores used to test the effectiveness of a sterility cycle, should not be confused with bioindicators.  Bioindicators are any species whose population, function, or status gives a qualitative report on the condition of the environment in which they are found.  Unlike chemical or physical testing of an environment, bioindicator species are able to reflect a problem (as well as show how long the problem has existed) and show the collective effects of pollutants in a particular environment.   However, it’s not entirely unlike biological indicators, as they too are a living species of microorganism able to give us data on the sterility of a given environment.