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General Information about Biological Indicators

Sterilization procedures in hospitals have reached a high standard of quality. Necessary monitoring procedures are costly, however important, to ensure long-term asepsis in all fields of surgical operations.

International and local standards and directives like the European Medical Device Directive (MDD) require validation, batch monitoring and documentation of sterilization processes.Besides industry, healthcare facilities must follow the same validation, monitoring and documentation procedures. Validation and monitoring of sterilization processes is carried out by parametric, chemical and/or biological tests. The validation using biological indicators is necessary if:

The structure of the goods to be sterilized is such that physical sensors cannot be applied (e.g.: small holes, gaps, sealed areas, coatings with oils etc.) lumens of hollow devices are so tiny that the temperature difference between non-condensable gases (inside) and steam (outside) is not detectable. Gases in such small lumens of several 100 µl heat up very quickly to the steam-temperature-level.

The presence of water condensate cannot be detected by physical means (e.g.: If the temperature gradient in the process is so slow that encapsulated non-condensable gases have time to heat up and do not show a detectable temperature difference.)
The surface structure of the medical devices requires specific testing (e.g.: porous rubber stoppers)
The sterilizing agent, the goods to be sterilized and/ or packaging contain salts. The salts may get dissolved in the condensate film and cause big changes of the resistance characteristics.

The condensate contains substances changing the pH-value (e.g.: corrosion-inhibitors) or the material of medical instruments (e.g.: aluminum surfaces) may react with water creating basic hydroxides.

In above cases all surfaces or liquids have to be inoculated with biological indicator suspensions. After a validated population determination, reduced process cycles have to be carried out to achieve survivor curves to determine the kill kinetics on/ in those critical areas. For porous loads and hollow process challenge devices (PCDs) biological indicators may be used to monitor the process conditions in such critical internal areas.Biological indicators are defined in the European and International Standards EN ISO 11138 parts 1-5. For most of the commonly used sterilization processes special reference biological germs have been selected, such as Geobacillus stearothermophilus for steam, formaldehyde and hydrogen peroxide sterilisation processes, Bacillus atrophaeus for ethylene oxide and dry heat sterilisation processes and Bacillus pumilus for radiation sterilisation processes.

Depending on the type of sterilization process, a special resistance characteristic of biological indicators is required, to prove the success of a defined sterilization process. During such a sterilization process the spore population always decreases due to the exponential kill characteristic called reaction kinetics first order. The population however, will never reach an absolute 0-value. Therefore modern definitions of goods declared “sterile” do not specify the absolute absence of biological activity, but determine aseptic conditions with the certain probability, called Sterility Assurance Level (SAL).

According to the European Standard EN 556 the minimum SAL has to be 10-6 CFU/per part or better. This means that out of 1 million units, no more than 1 unit may show growth.Both the kill kinetics and the penetration characteristics of a sterilization process have to be monitored. The kill kinetics is monitored by the right type of bacteria with the total resistance of a biological indicator.

The total resistance of a biological indicator depends on the population and resistance of each individual germ. The resistance of each individual germ is defined by the decimal reduction value which is the time needed to reduce the population of a biological indicator to one tenth of the original population. The total resistance of a biological indicator is expressed by the FBIO value:

FBIO = D121°C value x log (population)

This fact may be demonstrated by the 2 examples below in the table.

As seen above, the D-value of a given strain is never constant and depends on growth and process condition. Therefore, for each batch of biological indicators certificates must be associated to the product indicating the population, individual resistance and the total resistance of a biological indicator.
gke offers its Steri-Record biological indicators according to EN ISO 11138 series. All packs contain a certificate with all necessary information mentioned above. More information is available from our technical data sheet. Each individual package contains a direction for use.

After the biological indicator has passed the sterilization process, all treated spore strips have to remain in the glassine envelopes. They should be sent with one marked untreated spore strip to a microbiological lab. All strips should be aseptically transferred into Tryptic Soja broth (TSB) and developed for at least 7 days. If there is any doubt about the spore type, 1 ml of solution may be developed on TS agar plates (TSA) to determine the spore type. TSA vials without a spore strip should not show any growth, the untreated spore strip should show vital growth. Growth of treated spore strips have to be determined individually (see our technical information). gke offers growth medium test tubes with pH-indicator for faster evaluation.

Self-contained biological indicators contain growth media in a separate vial and may be developed directly at the user’s site. They must not be used in dry heat or formaldehyde sterilization processes. For information in more detail, please see our enclosed data sheet “self-contained biological indicators”.The penetration characteristics are monitored using Process Challenge Devices (PCDs) representing the “worst-case” penetration characteristics of a load. PCDs as described in EN 867-5 “Hollow Load Test” and in DIN 58921 may be used. Biological indicators are used inside to check the penetration of the sterilization agent.

1.gke Steri-Record® Self-contained biological indicators

Mini-Bio-Plus self-contained biological indicators (SCBI) uses a plastic vial containing a spore plate and glass ampoule with a growth medium and pH-indicator inside. It is used for validation and routine monitoring of most sterilization processes without using a microbiological laboratory.
For a better differentiation of the SCBI versions all have different coloured caps. They can also be used inside gke process challenge devices (Bio-C-PCDs), see 1.5. All SCBI fulfill the requirements according to EN ISO 11138-1.

1.1. for steam sterilization processes according to EN ISO 11138-3

G. Stearthermophilus, available with population of 105 and 106, paper carrier
Two versions are available:

1. Standard version with incubation time of 24 hours

2.Instant version for immediate release

The Instant-Mini-Bio-Plus SCBI contains a class 5 chemical indicator allowing that the result of steam sterilization processes can be instantly evaluated at the end of the steam sterilization process at 132-137°C. Therefore, it is not necessary to wait for the result of the SCBI incubation since the class 5 indicator provides equivalent or better information about the result of the sterilization process according to EN ISO 11140-1.

1.2. For formaldehyde (LTSF) sterilization processes according to EN ISO 11138-5
G. stearothermophilus available with population of 106, paper carrier

The growth medium also contains a neutralization agent for remaining formaldehyde, so that the pretreatment with Na2SO3 is not required as described in EN ISO 11138-5.

1.3. For all hydrogen peroxide / plasma sterilization processes

G. stearothermophilus, available with population of 106 , free of cellulose carrier

1.4. for ethylene oxide sterilization processes
according to EN ISO 11138-2B. atrophaeus, available with population of 106, paper carrier

1.5. Process Challenge Device for self-contained biological indicators

Bio-C-PCDs, colour: green, to be used with all Mini-Bio-Plus SCBIs decribed before, for validation and routine monitoring of steam, ethylene oxide, formaldehyde and hydrogen peroxide sterilization processes.It is recommended to use the round versions in large and the oval versions in small sterilizers. A PCD with SCBI placed inside is called a class 2 indicator according to EN ISO 11140-1.

Each PCD contains 10 seal rings in addition for replacement in the screw cap.

1.6 Accessories
1.6.1 Replacement parts for PCD

1.6.2 Crusher for SCBIsD

To activate all gke SCBIs. The gke incubator already includes a crusher.

2. gke Steri-Record® Stearo-Ampoules with G. stearothermophilus

suspension and growth medium according to EN ISO 11138-1 + 3to monitor extreme wet steam or liquid sterilization processes. The ampoule contains a 1.5 ml
Geob. Stearothermophilus suspension with growth medium and pH-indicator, available with populations of 105 and 106

.

3. gke Steri-Record® Incubator and accessories
3.1. Incubator and corresponding aluminium blocks

The incubator is available in four versions with different temperatures. The incubation temperature is visible in the display. The incubator will be delivered with an aluminium block to incubate SCBIs. An aluminium block to incubate Stearo-Ampoules can be ordered separately. The plug contains a CE conformity for the low voltage directive

3. 4. gke Steri-Record® Suspensions and inoculation kit

The spore suspensions (10 ml) are available in 40% ethanol/water, packaged in glass bottles with
rubber septum and comply with EN ISO 11138-1.
4.1. For steam, formaldehyde and hydrogen peroxide sterilization processes
Geob. Stearothermophilus (ATCC No. 7953)

The suspension will be delivered with certificate which states population and D121°Cvalue for steam according to EN ISO 11138-3. G. Stearothermophilus is also used for formaldehyde and hydrogen peroxide sterilization processes. A standard for H2O2 does not yet exist. The D-value for formaldehyde and hydrogen peroxide* can be determined at extra cost.

4.2. For dry heat and ethylene oxide sterilization processes
B. atrophaeus (ATCC No. 9372)
The suspension is delivered with certificate which states population and D-value for ethylene oxide and dry heat according to EN ISO 11138-2 + 4.

4.3. Direct Inoculation Kit with Geob. Stearothermophilus suspension

To test complex instruments using steam, formaldehyde or hydrogen peroxide sterilization processes. The precision sysringe can also be used for ethylene oxide and dry heat sterilization processes if a B. atrophaeus suspension is purchased

5. gke Steri-Record® Biological indicator spore strips

The biological indicators consist of bacteria spores inoculated on filter paper strips with the size of 6 x 38 mm and packaged in glassine envelopes. Packages of 100, 500 and 1.000 strips are available. They also contain a certificate which states population and D-value.All spore strips can be also used inside of process challenge devices (PCD). After the spore strips is aseptically removed from the glassine envelope and placed into the PCD like a chemical indicator. At the end of the sterilization process the PCD must not be opened but transferred into a microbiological laboratory where the strip is aseptically removed and incubated. For further information see
separate product catalogue.

5.1 For steam and formaldehyde sterilization processes
Geob. stearothermophilus (ATCC No. 7953)

For Art.-No. 330-501 both resistance values DFA and D121°C are determined according to EN ISO 11138-3 + 5 and are specified in the certificate.

5.2 For hydrogen peroxide sterilization processes
Geob. stearothermophilus (ATCC No. 7953)

Spore strips on PET plastic carrier. For resistance determination there is no standard availalbe. The D-values are determined in a resistometer and are specified in the certificate

5.3 For ethylene oxide and dry heat sterilization processes

B. atrophaeus (ATCC-No. 9372) according to EN ISO 11138-2 + 4

6. Growth media in test tubes

Test tubes with CASO-Boullion (TSB) with pH-indicator and plastic screw cap.The test tubes have optimized dimensions and volume to fit all kind of spore strips. If germs are growing the pH-indicator changes its colour and allows a quick evaluation of the result.

6.1 For steam and hydrogen peroxide sterilization processes
G. stearothermophilus
.

6.2 For formaldehyde sterilization processes
G. stearothermophilus

The growth medium contains a neutralization agent for remaining formaldehyde, so that the pretreatment with Na2SO3 is not required as described in EN ISO 11138-5

6.3 For ethylene oxide and dry heat sterilization processes

B. atrophaeus