GKE Australia 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. e.g. the European Medical Device Directive (MDD) re-quire 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 gas-es (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.: aluminium 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 chal-lenge 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.

Example Population [CFU/strip] D121-Value [min] FBio-Value [min]
1 106 1,0 6
2 105 2,0 10

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.
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 un-treated 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 processes. For information in more detail, please see our 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.
gke biological indicators are available with different D-values. If a particular D-value is required, that differes from the BI available in stock, it is advisable to check if the BI can be produced with the particular characteristics.

SELF-CONTAINED BIOLOGICAL INDICATORS

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

Mini-Bio-Plus self-contained biological indicator (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 sterili-zation 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-PCDs), see 1.5. All SCBI fulfil the requirements according to EN ISO 11138-1.

1.1. for steam sterilization processes

G. Stearothermophilus available with population of 105 and 106, on paper carrier according to EN ISO 11138-3.
Two versions are available:

1.1.1. Standard SCBIs with incubation time of 24 hours

Standard SCBIs | Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Colour of cap Population
324-501 10 B-S-MBP-10-5 Light blue 105
324-505 50
324-510 100
324-601 10 B-S-MBP-10-6 Dark blue 106
324-605 50
324-610 100

1.1.2. Instant-SCBIs for immediate release

The Instant-Mini-Bio-Plus SCBI contains a type 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 type 5 indicator provides equivalent or better information about the result of the sterilization process according to EN ISO 11140-1.

Instant-SCBIs | Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Colour of cap Population
324-551 10 B-S-I-MBP-10-5-SV4 Light orange 105
324-555 50
324-550 100
324-651 10 B-S-I-MBP-10-6-SV4 Dark orange 106
324-655 50
324-650 100

1.2. for formaldehyde (LTSF) sterilization processes

G. stearothermophilus available with population of 106, paper carrier according to EN ISO 11138-5
The growth medium also contains a neutralization agent for remaining formaldehyde, so that the pre-treatment with Na2SO3 is not required as described in EN ISO 11138-5.

formaldehyde sterilization processes |Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Colour of cap
325-601 10 B-F-MBP-10-6 Yellow
325-605 50

1.3. for hydrogen peroxide (plasma) sterilization processes

G. stearothermophilus available with population 106, on different carrier materials

hydrogen peroxide sterilization processes | Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Carrier Colour of cap
327-601 10 B-V-G-MBP-10-6* Glass fiber Light Grey
327-605 50
327-610 100
337-601 10 B-V-T-MBP-10-6* Tyvek Colourless
337-605 50
347-601 10 B-V-ST-MBP-10-6* Stainless steel Dark grey
347-605 50
357-601 10 B-V-P-MBP-10-6* PET Light green
357-605 50

1.4. for ethylene oxide sterilization processes

B. atrophaeus available with population 106, on paper carrier according to EN ISO 11138-2

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Colour of cap
326-605 50 B-E-MBP-10-6** Red
326-610 100

(*) For hydrogen peroxide sterilization processes four different versions are available using exactly the same germ and population. It shows that the resistance of biological indicators in hydrogen peroxide sterilization processes depends not only on population and spore type but also extremely on the carrier material used.
(**) Additional the following resistance determinations according to European Pharmacopeia (EP) are carried out:
Kill 25 min 54°C, 600 mg/l EO, 60% rel. humidity
Survival 50 min 30°C, 600 mg/l EO, 60% rel. humidity
The resistance determination according to EP is available (art.-no. 326-999) at extra cost.

PCDS AND ACCESSORIES

1.5. Process Challenge Devices (PCD) for self-contained biological indicators

Bio-C-PCDs, colour: green, to be used with all Mini-Bio-Plus SCBIs described before, for validation and routine monitoring of steam, ethylene oxide, formaldehyde and hydrogen peroxide sterilization processes or Helix-PCD according to EN 1422 for ethylene oxide 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 type 2 indicator according to EN ISO 11140-1.
Each PCD contains 5 seal rings in addition for replacement in the screw cap.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code PCD-Version Penetration Characteristics
300-009 B-PM-OCPCD-0 oval Very low requirements for air removal
300-010 B-PM-RCPCD-0 round
300-011 B-PM-OCPCD-1 oval Minimal requirements for air removal
300-016 B-PM-RCPCD-1 round
300-012 B-PM-OCPCD-2 oval Low requirements for air removal
300-017 B-PM-RCPCD-2 round
300-013 B-PM-OCPCD-3 oval Air removal less difficult than Hollow Load Test according to EN 867-5
300-018 B-PM-RCPCD-3 round
300-014 B-PM-OCPCD-4 oval Air removal equal to Hollow Load Test according to EN 867-5
300-019 B-PM-RCPCD-4 round
300-015 B-PM-RCPCD-5 round Air removal more difficult than Hollow Load Test according to EN 867-5
300-020 B-PM-RCPCD-6 round
300-028 B-E-PM-HPCD Helix Type test according to Line/Pickerill (EN 1422 EO monitoring)

1.6. Accessories

1.6.1. Replacement parts for PCDs

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Quantity
300-005 Replacement-screw cap (M14x1 thread) 5
300-006 Replacement seal kit for all PCDs listed above 5

1.6.2. Crusher for SCBIs

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

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Material Quantity
224-002 I-C Stainless steel 1
224-004 I-PC Plastic 10

INCUBATORS AND ACCESSORIES

2. gke Steri-Record® Incubators and accessories

The incubator is available in four versions with different temperatures. The incubation temperature is visible in the display. All incubators are either available with an aluminium block to incubate SCBIs or alternatively without aluminium block. In this case an aluminium block available for different applications (see 2.2 accessories) has to be ordered separately. The plug contains a CE conformity for the low voltage directive.

2.1. Dry Bath Incubators

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Temperature Application
With aluminium block for SCBIs
610-119 I-37-AB-MBP 37 to incubate B. atrophaeus biological indicators
610-120 I-57-AB-MBP 57 to incubate G. stearothermophilus biological indicators
610-121 I-V-AB-MBP 30 – 60 variable temperature selection
610-122 I-V-T-AB-MBP variable temperature selection and programming of the incubation time
Without aluminium block
610-109 I-37 37 to incubate B. atrophaeus biological indicators
610-110 I-57 57 to incubate G. stearothermophilus biological indicators
610-111 I-V 30 – 60 variable temperature selection
610-112 I-V-T variable temperature selection and programming of the incubation time

2.2. Accessories

Aluminium blocks to insert SCBIs, Stearo-Ampoules or growth medium tubes (12 pcs each).

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Diameter Application
610-113 I-AB-MBP 10 mm for all gke Steri-Record® Mini-Bio-Plus SCBIs
610-114 I-AB-AMP 11 mm for all gke Steri-Record® Stearo-Ampoules
610-115 I-AB-CM 16,5 mm for all gke Steri-Record® growth medium tubes

SUSPENSIONS AND AMPOULES

3. gke Steri-Record® Suspensions

All spore suspensions are delivered in 10 ml glass bottles with a septum, suspended in 40% ethanol/water and comply with EN ISO 11138-1.

3.1. for dry heat and ethylene oxide sterilization processes

The suspension B. atrophaeus is delivered with certificate which states nominal population and D-value for ethylene oxide and dry heat according to EN ISO 11138-2 + 4, EP and USP.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Population[CFU/ml] Population per bottle
226-107 B-E-H-SUS-10-7 107 108
226-108 B-E-H-SUS-10-8 108 109
226-109 B-E-H-SUS-10-9 109 1010

3.2. for steam, formaldehyde and hydrogen peroxide sterilization processes

The suspension G. stearothermophilus will be delivered with certificate which states nominal population and D121°C-value for steam according to EN ISO 11138-3 + 5. A standard for H2O2 does not yet exist. The D-value for formaldehyde (art.-no. 228-998) and hydrogen peroxide (art.-no. 228-997) can be determined at extra cost.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Product Code Population[CFU/ml] Population per bottle
228-107 B-S-F-V-SUS-10-7 107 108
228-108 B-S-F-V-SUS-10-8 108 109

3.3. Direct inoculation syringe

to test complex instruments in sterilization processes. To be used with G. stearothermophilus or B. atrophaeus suspension (see 3.1 and 3.2).

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Content
228-111 1 I-SYRINGE Precision syringe with a needle of 20 cm lengths

4. gke Steri-Record® Stearo-Ampoules

to monitor extreme wet steam or liquid sterilization processes. The ampoules are available in two different sizes. It contains a 1.5 or 0,25 ml G. stearothermophilus suspension with growth medium and pH-indicator, available with nominal populations of 105 or 106 CFU. They comply with EN ISO 11138-1 + 3, EP and USP.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Population [CFU/Amp.] Diameter/Height
225-550 50 B-S-AMP-10-5 105 11 / 45 mm (1,5 ml)
225-650 B-S-AMP-10-6 106
235-550 100 B-S-MAMP-10-5 105 5 / 25 mm (0,25 ml)
235-650 B-S-MAMP-10-6 106

GROWTH MEDIA AND SPORE STRIPS

5. gke Steri-Record® Growth media

Test tubes (diameter: 16,1 mm) with CASO-Boullion (TSB) with pH-indicator and aluminium screw cap with seal.
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.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Process Germ
223-010 10 B-S-V-CM steam, hydrogen peroxide G. stearo-thermophilus
223-100 100
221-010 10 B-E-H-CM ethylene oxide, dry heat B. atrophaeus
221-100 100

6. gke Steri-Record® 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 or foil/tyvek envelopes. They also contain a certificate which states nominal population and D-value. All spore strips can be also used inside of process challenge devices (PCD). Alternatively the bacteria spores are inoculated on discs with 7 mm diameter and are packaged individually or together in a resealable bag.

6.1. for dry heat and ethylene oxide sterilization processes

Spore strips B. atrophaeus according to EN ISO 11138-2+4.
The resistance determination according to EP (art.-no. 221-999, see 1.4) is available at extra cost.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Population
221-601 100 B-E-H-SS-10-6 106
221-605 500
221-610 1.000

6.2. for steam and formaldehyde sterilization processes

Spore strips G. stearothermophilus according to EN ISO 11138-3. The resistance determination for LTSF (formaldehyde) according to EN ISO 11138-5 is available at extra cost (art.-No. 223-998).

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Product Code Population
223-501 100 B-S-SS-10-5 105
223-505 500
223-510 1.000
223-601 100 B-S-SS-10-6 106
223-605 500
223-610 1.000

SPORE DISCS AND GROWTH MEDIA

6.3. for hydrogen peroxide decontamination and sterilization processes

To decontaminate isolators and rooms, vaporized hydrogen peroxide processes are used. Unpackaged biological spore discs can be placed in critical locations inside the room or in equipment.
For resistance determinations there is currently no EN or ISO standard available. The D-values are determined by exposure of spore suspensions to hydrogen peroxide solutions. Spores suspensions analyzed in this way are used to inoculate carriers. Therefore, the influence of the carrier material on the resistance of the BI suspension remains unconsidered.

6.3.1. as strip (6×38 mm)

The biological indicators consist of G. Stearothermophilus bacteria spores inoculated on different carriers with the size of 6 x 38 mm and packaged individually (in Tyvek envelope) or in bulk in a blister box. They also contain a certif-icate which states nominal population and D-value. All spore strips can be also used inside of process challenge de-vices (PCD).

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Packaged Carrier Product Code Population
332-601 100 individually PET B-V-P-SS-10-6 106
332-604 40 bulk
332-602 100 individually Glass fiber B-V-G-SS-10-6
332-605 40 bulk
332-603 100 individually Tyvek B-V-T-SS-10-6
332-606 40 bulk
332-607 100 individually Stainless steel B-V-ST-SS-10-6
332-608 40 bulk

6.3.2. as discs (7 mm diameter)

The G. Stearothermophilus bacteria spores are inoculated on discs with 7 mm diameter (available with different carriers) and are packaged individually (in Tyvek envelopes) or in bulk in a blister box.

Biological Indicators for Sterilization | GKE Australia Biological Indicators
Art.-No. Quantity Packaged Carrier Product Code Population
332-612 100 individually PET B-V-P-DIS-SP-10-6 106
332-614 110 bulk
332-616 100 individually Glass fiber B-V-G-DIS-SP-10-6
332-611 110 bulk
332-618 100 individually Tyvek B-V-T-DIS-SP-10-6
332-613 110 bulk
332-617 100 individually Stainless steel B-V-ST-DIS-SP-10-6
332-615 110 bulk