PRODUCT CATALOGUE

Biological Indicators and Accessories

for sterilization processes

Spore Strips, Ampoules, Suspensions

Self-contained Mini-Bio-Plus BI
to monitor steam, formaldehyde, hydrogen peroxide,
ethylene oxide and dry heat sterilization processes
as well as room disinfection processes
2
CONTENT

General Information about biological indicators ........................................................ 4

Self-contained biological indicators............................................................................... 6
For steam sterilization processes ..............................................................................................6
For formaldehyde sterilization processes ................................................................................7
For hydrogen peroxide sterilization processes .......................................................................8
For ethylene oxide sterilization processes ...............................................................................8
PCDs and accessories ....................................................................................................... 9
Bio-PCDs and replacement parts...............................................................................................9
Crusher for self-contained biological indicators .....................................................................9
Incubator and accessories ............................................................................................ 10
Stearo-Ampoules ............................................................................................................ 10
Suspensions..................................................................................................................... 11
Geob. stearothermophilus ......................................................................................................... 11
B. atrophaeus .............................................................................................................................. 11
Direct Inoculation Syringe ........................................................................................................ 11
Growth medium ............................................................................................................. 12
Spore strips ..................................................................................................................... 12
For steam and formaldehyde sterilization processes ......................................................... 12
For ethylene oxide and dry heat sterilization processes .................................................... 12
For hydrogen peroxide sterilization processes .................................................................... 13
Spore discs ....................................................................................................................... 13
For hydrogen peroxide sterilization processes .................................................................... 13
Basic principles in kill kinetics...................................................................................... 14

3
GENERAL INFORMATION
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 opera-
tions.

International and local standards and directives. e.g. Medical Device Regulation (MDR) require valida-
tion, batch monitoring and documentation of sterilization processes.

Besides industry, healthcare facilities must follow the same validation, monitoring and documenta-
tion 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 tempera-
ture 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 stop-
pers)
• 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. Af-
ter 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 condi-
tions in such critical internal areas.

Biological indicators are defined in the European and International Standards EN ISO 11138 parts 1
to 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 perox-
ide sterilization processes, Bacillus atrophaeus for ethylene oxide and dry heat sterilization processes
and Bacillus pumilus for radiation sterilization processes.

Depending on the type of sterilization process, a special resistance characteristic of biological indica-
tors 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 reac-
tion 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 activ-
ity, 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 moni-
tored. The kill kinetics is monitored by the right type of bacteria with the total resistance of a biologi-
cal indicator.

4
RESISTANCE OF BIOLOGICAL INDICATORS
The total resistance of a biological indicator depends on the population and resistance of each indi-
vidual 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 pop-
ulation. 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]

6
1 10 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 indica-
tor.

GKE offers its Steri-Record® biological indicators according to EN ISO 11138 series. All biological idni-
cators contain a certificate with all necessary information mentioned above.

After the biological indicator has passed the sterilization process, all treated spore strips have to re-
main in the glassine envelopes. They should be sent with one marked untreated spore strip to a mi-
crobiological lab. All strips should be aseptically transferred into Tryptic Soja broth (TSB) and devel-
oped for at least 7 days. If there is any doubt about the spore type, 1 ml of solution may be devel-
oped 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 TI 730-067). GKE offers growth me-
dium 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 de-
tail, 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
differs from the BI available in stock, it is advisable to check if the BI can be produced with the parti-
cular characteristics.

5
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 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-PCD®s), 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.

Three versions are available:

1.1.1. Standard SCBIs with incubation time of 24 hours

Art.-No. Quantity Product Code Colour of cap Population

324-501 10
324-505 50 B-S-MBP-10-5 Light blue 105
324-510 100
324-601 10
324-605 50 B-S-MBP-10-6 Dark blue 106
324-610 100

1.1.2. Instant-SCBIs for immediate release

The 121°C and 134 °C Instant-Mini-Bio-Plus SCBI contain 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
121-124°C or 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 accord-
ing to EN ISO 11140-1. The Instant-Mini-Bio-Plus SCBI has to be selected according to the sterilization tempera-
ture.

Colour of Popula- Sterilization

Art.-No. Quantity Product Code
cap tion Temperature
324-521 10
B-S-I-MBP-10-5-SV5 Light green 105
324-525 50
121-124 °C
324-621 10 6
B-S-I-MBP-10-6-SV5 10
324-625 50
324-551 10
Light
324-555 50 B-S-I-MBP-10-5-SV4 105
orange
324-550 100
132-137 °C
324-651 10
Dark
324-655 50 B-S-I-MBP-10-6-SV4 106
orange
324-650 100

6
SELF-CONTAINED BIOLOGICAL INDICATORS

1.1.3. Activation Control SCBI (AC-SCBI®)

The Activation Control SCBI contains a glass vial with colourless growth medium which only becomes coloured
if being activated.

Art.-No. Quantity Product Code Colour of cap Population

324-595 50
B-S-AC-MBP-10-5 Light blue 105
324-590 100
324-695 50
B-S-AC-MBP-10-6 Dark blue 106
324-690 100

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.

Art.-No. Quantity Product Code Colour of cap

325-601 10
B-F-MBP-10-6 Yellow
325-605 50

7
SELF-CONTAINED BIOLOGICAL INDICATORS

1.3. for hydrogen peroxide/plasma sterilization processes

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

Art.-No. Quantity Product Code Carrier Colour of cap

327-601 10
327-605 50 B-V-G-MBP-10-6* Glass fiber Light grey
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 Purple
357-605 50

(*) 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.

1.4. for ethylene oxide sterilization processes

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

Art.-No. Quantity Product Code Colour of cap

326-605 50
B-E-MBP-10-6** Red
326-610 100

(**) 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.

8
PCDS AND ACCESSORIES
1.5. Process Challenge Devices (PCD) for self-contained biological indicators

Bio-C-PCD®s, 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 system according to EN ISO 11140-1.

Each PCD comes along with 5 seal rings in addition for replacement in the screw cap.

PCD- Penetration
Art.-No. Product Code
Version Characteristics
300-031 B-PM-OCPCD-0 oval Very low requirements
300-032 B-PM-RCPCD-0 round for air removal

300-033 B-PM-OCPCD-1 oval Minimal requirements for air

300-034 B-PM-RCPCD-1 round removal

300-035 B-PM-OCPCD-2 oval Low requirements

300-036 B-PM-RCPCD-2 round for air removal

300-037 B-PM-OCPCD-3 oval Air removal less difficult than

Hollow Load Test
300-038 B-PM-RCPCD-3 round according to EN 867-5
300-039 B-PM-OCPCD-4 oval Air removal equal to
Hollow Load Test
300-040 B-PM-RCPCD-4 round according to EN 867-5
300-041 B-PM-RCPCD-5 round Air removal more difficult than
Hollow Load Test
300-042 B-PM-RCPCD-6 round according to EN 867-5
Type test according to Line/
300-028 B-E-PM-HPCD Helix Pickerill (EN 1422 EO monitoring)

1.6. Accessories
1.6.1. Replacement parts for PCDs

Art.-No. Product Code Quantity

Replacement screw cap
300-005 5
(M14x1 thread)
Replacement seal kit for all PCDs
300-006 5
listed above

1.6.2. Crusher for SCBIs

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

Art.-No. Product Code Material Quantity

224-002 I-C Stainless steel 1
224-004 I-PC Plastic 10

9
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 with-
out 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

Art.-No. Product Code Temperature Application
With aluminium block for 12 SCBIs (hole diameter 10 mm)
to incubate
610-119 I-37-AB-MBP 37 B. atrophaeus biological indicators
to incubate G. stearothermophilus
610-120 I-57-AB-MBP 57 biological indicators
variable
610-121 I-V-AB-MBP temperature selection
30 - 60 variable temperature selection
610-122 I-V-T-AB-MBP and programming of the
incubation time

Without aluminium block

to incubate
610-109 I-37 37 B. atrophaeus biological indicators
to incubate G. stearothermophilus
610-110 I-57 57 biological indicators
variable
610-111 I-V temperature selection
30 - 60 variable temperature selection
610-112 I-V-T and programming of the
incubation time
2.2. Accessories
Aluminium blocks to insert SCBIs, Stearo-Ampoules or growth medium tubes (12 pcs each).

Art.-No. Product Code Diameter Application

610-113 I-AB-MBP 10 mm for all GKE Mini-Bio-Plus SCBIs

for all GKE Standard

610-114 I-AB-AMP 11 mm Stearo-Ampoules (1.5 ml)

610-115 I-AB-CM 16.5 mm for all GKE growth medium tubes

3. GKE Steri-Record® Stearo-Ampoules

to monitor extreme wet steam or liquid sterilization processes. The ampoules are available in two different siz-
es. It contains a 1.5 or 0.2 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.

Population Diameter/
Art.-No. Quantity Product Code
[CFU/Amp.] Height
225-550 B-S-AMP-10-5 105 11 / 45 mm
50
225-650 B-S-AMP-10-6 10 6 (1.5 ml)

235-510 B-S-MAMP-10-5 105 5 / 25 mm

100
235-610 B-S-MAMP-10-6 10 6 (0.2 ml)

10
SUSPENSIONS AND AMPOULES
4. 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.

4.1. for dry heat and ethylene oxide sterilization processes

The suspension B. atrophaeus is delivered with certificate which states nominal population and D-value for eth-
ylene oxide EN ISO 11138-2 EP and USP. If the suspension shall be used in dry heat or hydrogen peroxide steri-
lization processes, this must be already stated with the order, since a special manufacturing procedure is nec-
essary. The D-value for dry heat (art.-no. 226-999) and hydrogen peroxide (art.-no. 226-997) can be determined
at extra cost.

Population
Art.-No. Product Code Population/bottle
[CFU/ml]
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

4.2. 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 or hydrogen peroxide. A standard for H2O2 does not yet
exist. If the suspension shall be used in hydrogen peroxide sterilization processes, this must be already stated
with the order, since a special manufacturing procedure is necessary. The D-value for formaldehyde according
EN ISO 11138-5 (art.-no. 228-998) can be determined at extra cost.

Population Population per

Art.-No. Product Code Sterilization Process
[CFU/ml] bottle
228-107 B-S-F-SUS-10-7 107 108 Steam,
228-108 B-S-F-SUS-10-8 108 109 Formaldehyde

229-107 B-V-SUS-10-7 107 108

Hydrogen Peroxide
229-108 B-V-SUS-10-8 108 109

4.3. Direct inoculation syringe

to test complex instruments in sterilization processes. To be used with G. stearothermophilus or B. atrophaeus
suspension (see 4.1 and 4.2).

Art.-No. Quantity Product Code Content

Precision syringe with a needle
228-111 1 I-SYRINGE of 20 cm lengths

11
GROWTH MEDIA AND SPORE STRIPS
5. GKE Steri-Record® Growth media
Test tubes with aluminium screw cap (diameter: 16.1 mm) filled with TSB and pH-indicator.
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.

Art.-No. Quantity Product Code Process Germ

221-010 10 ethylene oxide,
B-E-H-CM B. atrophaeus
221-100 100 dry heat

223-010 10 steam,
B-S-V-CM
223-100 100 hydrogen peroxide
G. stearo-
330-010* 10 thermophilus
B-F-CM formaldehyde
330-100* 100
* on demand only

6. GKE Steri-Record® Spore strips

The biological indicators (6 x 38 mm) inoculated with bacteria spores contain a certificate which states nominal
population and D-value. All spore strips can be also used inside of process challenge devices (PCD). Alternative-
ly the bacteria spores are inoculated on discs with 7 mm diameter and are packaged individually or together in
a blister bag.

6.1. for dry heat and ethylene oxide sterilization processes

Spore strips B. atrophaeus, packaged in glassine envelopes, 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.

Art.-No. Quantity Carrier Product Code Population

221-601 100
221-605 500 Paper B-E-H-SS-10-6 106
221-610 1,000

6.2. for steam and formaldehyde sterilization processes

Spore strips G. stearothermophilus, packaged in glassine envelopes, 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).

Art.-No. Quantity Carrier Product Code Population

223-501 100
223-505 500 B-S-SS-10-5 105
223-510 1,000
Paper
223-601 100
223-605 500 B-S-SS-10-6 106
223-610 1,000

12
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 biologi-
cal 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. Spore suspensions analyzed in this way are
used to inoculate carriers. Therefore, the influence of the carrier material on the resistance of the BI suspen-
sion remains unconsidered.

6.3.1. as strip (6 x 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 of 94 x 65 mm) or in bulk in a blister box.
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).

Art.-No. Quantity Packaged Carrier Product Code Population

332-407 100 individually B-V-ST-SS-10-4 104
332-507 100 individually Stainless B-V-ST-SS-10-5 105
332-607 100 individually Steel
B-V-ST-SS-10-6
332-608 40 bulk
332-601 100 individually
PET B-V-P-SS-10-6
332-604 40 bulk*
106
332-602 100 individually
Glas 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*
* not in stock, available on request.

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 of 60 x 65 mm) or in bulk in a blister box.

Art.-No. Quantity Packaged Carrier Product Code Population

332-417 100 individually B-V-ST-DIS-SP-10-4 104
332-517 100 individually Stainless B-V-ST-DIS-SP-10-5 105
332-617 100 individually Steel
B-V-ST-DIS-SP-10-6
332-615 110 bulk
332-612 100 individually
PET B-V-P-DIS-SP-10-6
332-614 110 bulk*
106
332-616 100 individually
Glas 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*
* not in stock, available on request.
13
 Basic Principles in Kill Kinetics and
Design of Sterilization Processes
Author: Dr. Ulrich Kaiser

Content
1. Kill kinetics in sterilization processes
1.1. Definition of reaction kinetics first order
1.2. Inactivation factor
1.3 Decimal reduction factor (D-value)
1.4 Experimental determination of the D-value
1.4.1. MPN-Method (most probable number)
1.4.2 Determination using the survivor curve
1.4.3. Survival-Kill-Window

2. Definition of the sterility assurance level (SAL)

2.1. Definition of a sterile product according to the European standard EN 556

3. Temperature dependence of sterilization processes

3.1. Arrhenius equation
3.2. Definition of the z-value

4. Sterilization equivalence value (F(T,z)-Wert)

4.1. F0-value
4.2. Other F(T,z)-values

5. Process Design
5.1. Process definition with known bioburden values
5.2. Process definition with unknown bioburden values

6. Requirements and selection of biological indicators for validation and routine

monitoring
6.1. Selection of strain
6.2. Resistance of biological indicators
6.3. Selection of biological indicators for routine monitoring
6.4. Positioning of biological indicators

7. Glossary of symbols used within the text

14
1. Kill kinetics in sterilization processes If equation 1 is integrated and the natural logarithm is
exchanged against the decade logarithm the new reac-
The mathematical laws for the inactivation of microor- tion kinetics constant k is defined:
ganisms are very similar in most sterilization processes
under the condition that the physical and/or chemical
parameters remain constant during the sterilization pro- N0
cedure. Even if the sterilization conditions are constant, lg = k t (2)
the resistance of the same strain may be quite different NF
depending on the vegetative growth and sporulation con-
ditions. Even spores of identical strain with the same ref- t = Sterilization time [min]
erence number (i.e. G. Stearothermophilus) may be quite N0 = Number of germs when starting the process [CFU]
different and may vary by a factor up to 10. NF = Number of germs after sterilization [CFU]
IF = Inactivation Factor [number]
Under the condition using identical germs and steriliza- k = Reaction kinetics constant [min-1]
tion processes the velocity of kill is only dependent on (valid for the decade logarithm)
the existing amount of alive germs measured in colony
forming unit (CFU). The kill kinetics equation has been 1.2 Inactivation factor
proven valid for dry heat, steam, formaldehyde, ethylene
oxide and hydrogen peroxide sterilization processes. In diagram 1 the colony forming units [CFU] are plotted
1100000 on a linear scale showing e-function curves. If the same
1000000 diagram is plotted on a half logarithmic scale, the curves
900000 become a straight line for the same type of germs if
colony forming units [CFU/part]

800000 steam, ethylene oxide, dry heat and LTSF sterilization

700000 processes are used. If the line is not straight, the same
600000 population may contain germs of the same strain but
500000
with different resistance. Due to their complex chemical
400000
110 °C
process hydrogen peroxide sterilization processes do not
300000
121 °C form a straight line.
200000 134 °C
100000
Equation 2 can be changed to:
0
0 2 4 6 8 10 12 14 16

lg N0 − lg NF = k  t = IF
time [min]
(3)
Diagram 1: Survival curve for steam sterilization at different
temperatures The term inactivation factor (IF) describes the efficacy of a
sterilization process. If a sterilization starts with 106 [CFU]
1.1. Definition of reaction kinetics first order and finishes with 102 [CFU], there is a population reduc-
tion of the power of 4 or has an inactivation factor IF = 4.
The kill velocity in sterilization processes is described by
equation 1 and describes the reduction of the germ 1.3 Decimal reduction factor (D-value)
amount N over the time t and is called reaction velocity.
The decimal reduction factor, quite often called D-value,
represents the resistance characteristic of an individual
dN germ for a defined sterilization process. The D-value de-
= −k 'N (1)
termines how long a germ must be inside of a steriliza-
dt tion process to reduce the starting population by 90 % of
the starting bioburden. In steam, ethylene oxide, formal-
t= Sterilization time [min] dehyde, dry heat and hydrogen peroxide sterilization
N= Nominal population on a medical device [CFU] processes the D-value is expressed in a time scale [min].
k’ = Reaction kinetics constant using the natural If a radiation sterilization process is used, it is expressed
logarithm [min-1] in the radiation dose [Mrad]. The D-value may be experi-
mentally determined by plotting the logarithm of the still
The reaction velocity [dN/dt] is always proportional to the remaining population in the sterilization process against
current existing amount of alive germs in the process. the time, the reciprocal slope of the straight line is the
The proportional constant k’ is called reaction kinetics definition of the D-value. The D-value is only valid for a
constant. k’ describes the kind of sterilization process. defined sterilization process and a defined germ. In a
The constant is dependant in thermal processes on tem- steam sterilization process the D-value contains in the
perature, in chemical processes also on the gas concen- index the sterilization temperature. The certificate of a
tration. biological indicator must always specify under which con-
ditions the D-value is tested. The D-value is very tempera-
ture dependent as shown in diagram 2.

15
 1.4 Experimental determination of the resistance (DT)
of a biological indicator
1 (4)
DT = The resistance (D-value) may be determined with two
k methods according to EN ISO 11138-1 (see Annex C and
D) or with the determination of the survival kill window
DT = Decimal Reduction Factor [min] or [Mrad] (see Annex E of the standard).
at a tested temperature [t]
k = Reaction kinetics constant of the decimal 1.4.1 D-value determination using the MPN-Method
logarithm [min-1] (most probable number)

Biological indicators with a defined population are put in

several steam sterilization processes with modified steri-
lization times where all other process variables remain
constant except the time. For each sterilization time a
minimum of 20 biological indicators is required. After
sterilization the biological indicators are checked for
growth. A minimum of 7 different sterilization times is to
be tested.

a) minimum 1 sterilization time where all biological

indicators are growing
b) minimum 4 sterilization times where at least
some biological indicators are growing
c) minimum 2 sterilization times where no growth
of biological indictors is detected.
Diagram Using the above results the D-value will be calculated
2: Definition of the D-value at different temperatures using the equations below.
If equation 4 is put into equation 3, the result is: Number of
Sterilization Number of
trials without
time trials
growth
t (5)
lg N0 − lg NF = = IF [ti] [ni] [ri]
DT r1 = 0
t1 n1
N0 = Starting bioburden [CFU] t2 n2 r2
NF = Number of germs after sterilization [CFU]
DT = Decimal reduction factor [min] or [Mrad] t3 n3 r3
(DT-value)
t = Sterilization time [min] t4 n4 r4
IF = Inactivation factor [number]
(Decimal reduction level) t5 n5 r5
The coefficient of sterilization time divided by the D-value
provides also the inactivation factor equivalent to the t6 n6 r6
number of decimal reduction steps.
t7 n7 r7
A D-value time equivalent reduces the population by
90 % or one decimal reduction step.
If the D-value is known, it is possible to calculate the steri- t1 is the shortest sterilization time where all BIs should
lization time to reduce the population by defined amount grow. The sterilization times t1 – t7 are increasing steriliza-
of decimal reduction steps independent from the starting tion times using the result of t6 and t7 should not show
population. alive BIs.

If the starting population N0 is changed, the final popula-

tion is changing accordingly, if the same sterilization pro-
cess is used. Therefore the starting population, also
called bioburden, determines the result of the final num-
ber of germs NF. To get the necessary sterilization time,
equation 5 can be changed accordingly:

(6)
t = (lg N 0 − lg N F )  DT = IF  DT

16
Using these data the factors x and y are calculated for the A minimum of 2 consecutive tests shall be carried out.
sterilization times t1 – t7. For each test a minimum of 4 biological indicators shall
be used. After sterilization the population of the biologi-
cal indicator is determined using the method provided by
t i + t( i +1) the manufacturer. The logarithm of the remaining germ
xi = (7) population is plotted against the sterilization time. The
2 reciprocal slope provides the D-value in minutes provid-
ed that the reaction kinetics is first order.
r( i +1) ri (8)
yi = −
n( i +1) ni
For t1 where all samples show growth r1 = 0. In this case yi
is determined by:

r( i +1)
yi = (9)
n( i +1)

Using the calculated values of xi and yi the sterilization

time µi may be calculated:

i = xi  y i [min] (10) Diagram 3: Time to reduce the amount of germs in a steam

sterilization process at 121°C with a D-value of 2 min
The mean sterilization time  which does not show
growth may be calculated summarizing all µi:
1.4.3. Survival/kill window
i =6 The survivor/kill window is defined with the guaranteed
 =  i [min] (11) survival of a biological indicator. A biological indicator
i =0 shall contain a minimum of 100 germs, if it has been in-
side of a sterilization process with the initial population
N0 for the following sterilization time:
If the interval d between the sterilization times is con-
stant, and the same number of tests for each sterilization Survival time = (log N0 - 2) x D [min]
time is used, the mean value for no growth  may be cal-
culated using the following equation: The guaranteed kill of a biological indicator occurs after
the following sterilization time at 121°C:
d d i =6
 = t6 − −  ri [min] (12)
Kill time = (log N0 + 4) x D [min]
2 n i =1
This sterilization time determines a sterility assurance
level of SAL = 10-4 (every 10.000th germ may remain alive).
The mean D-value will be calculated using the equation:
Number of micro-biological
Survivors [CFU/part]
― Bio-Burden = Various germs with different population 107


10000000 and resistance
106

D= [min] (13)
1000000
― Biological indicators (BI) = Germs with defined
105

0,2507 + lg N0
100000 population and resistance
104
10000
103
1000
102
where N0 is the starting population CFU/test.
100

10 101

1 100

1.4.2 D-Value Determination using the survivor curve 0,1 Survival – 10-1
Kill-Window
0,01 Sterility Assurance

Biological indicators have to be sterilized with different 0,001

Level (SAL)
10-3

sterilization times where all process variables have to 0,0001

10-4

remain constant except the time. 0,00001

10-5
0,000001
-6
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 10 = SAL
5 different sterilization times should be used: Dxx-Value FBio-Value
Time [min]
FO-Value (according EN 556)

a) One exposure in which the sample is not sub- Diagram 4: Definition of: Bio-Burden, SAL, Biological indica-
jected to the sterilant (e.g. 0 time exposure) tor, FBio-Value, F0-Value
b) At least one exposure in which the viable popu-
lation is reduced to 0.01 % of the original inocula
(4 log10 reduction)
c) A minimum of three exposures covering the in-
tervals between exposure a) und exposure b)
above.
d) Not less than four test samples shall be used for
each exposure in each determination.
e) The same number of replicates shall be used for
each exposure.

17
2. Definition of the sterility assurance level (SAL) 3. Temperature dependence of sterilization processes

The number of germs decreases during a sterilization 3.1 Arrhenius equation

process first order with each D-value time unit by a pow-
er of ten or 90 % of the previous value. After the number As reported in part 1 the constant k’ and k and also the D-
of germs has reached 1 CFU with each D-value steriliza- values are temperature-dependent. This dependency is
tion time unit the population is decreased by another described by the Arrhenius equation:
power of ten reaching 0.1 CFU. Values below 1 do not
determine the number of alive germs on a part but deter- − Ea
mine the probability how many parts still have alive (14)
germs. If 10 parts which contain one germ each are steri- k = k0  e RT
lized for another D-value time unit, again 90 % of the
germs are inactivated. Therefore the value 0.1 CFU ex-
R = General gas constant [8,314 J/mol K]
presses that 9 out of 10 parts become sterile and one
T = Temperature [K]
part is still non-sterile. The value 0.01 or 10-2 means ac-
k = Reaction kinetics constant of the
cordingly that out of 100 parts 99 parts are sterile and
decimal logarithm [min-1]
one part is non-sterile. Population values < 1 do not de-
k0 = Reaction kinetics constant defines a
termine the number of germs but the sterility assurance
sterilization process [min-1]
level. This is the ratio between non-sterile and sterile
Ea = Activation energy of the process
products in one process.
[J/mol]
2.1 Definition of a sterile product according to the
The constant k0 depends only on the type of sterilization
European standard EN 556-1
process, is independent from the temperature and may
be experimentally achieved. The activation energy Ea is
The classic definition of sterility determines that non-
the energy amount to start the kill reaction. Using the
viable germs are inside a sterile product. The kill kinetics
Arrhenius equation the experimental change of the D-
law first order however demonstrates that the SAL-level
value versus the temperature may be derived. This de-
may be reduced as longer the sterilization process is car-
pendence is expressed with the z-value (see diagram 4).
ried out, however the SAL will never reach zero. That
means the probability of sterility may be increased as
3.2 Definition of the z-value (Temperature coefficient
longer the sterilization is carried out, however the abso-
of the D-value)
lute sterility cannot be achieved. Since absolute sterility
cannot be achieved, goods may be labelled sterile accord-
The z-value describes the dependence of the kill velocity
ing EN 556-1 if the SAL ≤ 10-6 is reached for terminal steri-
of microorganisms with changing temperature. Mathe-
lized products. For sterile liquid fillings in part 2 of EN 556
matically the z-value is the necessary temperature differ-
an SAL of ≤ 10-3 is accepted since the production process-
ence to change the D-value by a factor of 10 keeping all
es cannot achieve better results. If a sterility assurance
other sterilization conditions constant. If D-values are
level of ≤ 10-6 is achieved, those products according to
achieved at different temperatures and are put in a half-
EN 556-1 may be labelled sterile in Europe. In other coun-
logarithmic D-value scale against the temperature a
tries outside of Europe the accepted SAL-level is different
straight line is achieved where the reciprocal slope deter-
depending on the application and defined by local regula-
mines the z-value.
tions. The direct biological proof for such values cannot
be achieved by experimental tests but is available by ex-
trapolation of the straight line of the kill kinetic equation.

Diagram 5: Determination of the z-value

If the z-value is known, D-values at given temperatures

may be converted to D-values with another temperature.

1 lg DT 1 − lg DT 2
=−
z T1 − T2
(15)

18
4. Sterilization equivalence value (F(T,z)-value)

Referring to equation 6 the sterilization time can be

achieved multiplying the decimal reduction and inactiva-
tion factor. Since the D-value is valid only for one temper-
ature, the sterilization time at different temperatures
during the come-up time has to be adjusted to one de-
fined temperature. This sterilization time at one tempera-
ture is defined as the equivalent time (FT,z) using the in-
dex of the temperature and the z-value of the steriliza-
tion process. This F-value determines the sterilization
time at a constant temperature. The F0-value expresses
the sterilization power of a defined sterilization process
and is usually expressed in minutes at a given tempera-
ture. The inactivation factor alone is no value for the ster-
ilization power, since germs with low resistance are killed
much quicker in comparison to germs with high re-
sistance or D-values. Diagram 7: Illustration of the F-value

As shown above the sterilization time at a given tempera- 4.1. Definition of F0-value
ture may be calculated, if the starting bioburden (N0) is
known to achieve a defined final SAL. In reality a sterilizer The F0-value is defined at a sterilization temperature of
is heating up over a certain period until the sterilization 121°C and a z-value of 10°C and is used in industry as a
plateau temperature of for example 121°C is reached. reference for sterilization processes.
During the come-up and go-down time between 100 and
121°C germs are killed already . 4.2. Other FT,z-values

Other F-values may be defined with other temperatures

and z-values. In the metric system the Fc-value is defined
at 120°C and z = 10°C.

5. Design of a sterilization process

Before the validation of a sterilization process can be

carried out, the sterilization starting conditions have to
be known (sterilization type, goods to be sterilized, pack-
aging, etc.). Hydro- and thermo-stable products may be
sterilized in steam sterilization processes. Non tempera-
ture stable products are sterilized in low temperature
sterilization processes in industry with EO or radiation
sterilization processes, in health care with Formaldehyde
sterilization processes. After the sterilization process is
Diagram 6: F0-value integral of all time above 100 °C defined, the sterilization process parameters have to be
defined that the SAL ≤ 106 has to be achieved at the end.
This inactivation has to be added to the plateau steriliza- If the starting bioburden including other constant starting
tion time. Is the z-value known, the additional sterilization conditions which are available in industry sterilizing new
times outside the plateau period may be recalculated to products, the F0-values can be determined using the
the temperature at the plateau period. The summary of starting bioburden and the SAL which has to be achieved.
all time integrals may be added to the total sterilization If constant starting conditions cannot be guaranteed like
time of 121°C and is a definition of the equivalence time. in health care units, a so-called overkill-process is used.

The F-value is a sterilization time at one defined tempera- To take care of the sterilized goods and to minimize steri-
ture, in radiation sterilization it is defined by a radiation lization times the sterilization time and temperature
dose. should be adapted to the necessary kill values only. To
achieve this goal not only the necessary process parame-
ters need to be calculated but also it is necessary that all
FT ,z = (lg N0 − lg NF )  DT = IF  DT process parameters are kept constant during steriliza-
(16) tion.

19
5.1. Process design with known starting
bioburden values 11C
lg D110C = lg D121C +
If the starting bioburden of the products to be sterilized z
is known (types, population and resistance of all germs),
the most resistant germs including the z-values have to lg D110C = lg 1,5 + 1,1 = 1,276
be determined. If these data are available, the steriliza-
tion parameters may be calculated as demonstrated at
the following example:
D110C = 101,276 = 18,8
Starting conditions for exercises 1-4: [min]

Starting germ number: 2. Calculation of the sterilization time using equation 6:

N0 = 103 CFU
F 110°C,10 = (lg N 0 – lg N F ) • D T
Expected SAL: NF = 10-6 CFU = SAL = 10-6 F 110°C,10 = (3+6) • 18.8 min = 170 min
D121-value = 1,5 min
z-value = 10°C The sterilization time at 110°C is 2 h, 50 min.

Exercise 4:
Exercise 1: Which temperature has to be used that the sterilization
Calculate the inactivation factor necessary: time should not be longer than 3 min? The temperature
may be above 121°C.
IF= lg N0 – lg NF
IF = lg 103 – lg 10-6 = 3 – (-6)=9 1. Determination of the D-value:

The inactivation factor has a value of 9 decimal reduction

steps to reach the sterility assurance level SAL = 10 -6.
t
DT =
lg N 0 − lg N F
Exercise 2:
Which sterilization time at 121°C is required:
3 min
F 0 = (lg N 0 – lg N F ) • D T DT = = 0,33 min
3+6
F 0 = (3+6) • 1.5 min = 13.5 min
2. Determination of the sterilization temperature
The necessary equivalence sterilization time for this pro-
cess at 121°C is 13.5 min. 1 lg DT1 − lg DT2
=−
Exercise 3: z T1 − T2
Since the sterilization goods are not stable at 121°C, a
sterilization temperature of 110°C should be used. How
long is the required sterilization time F110°C, z = 10 K? T1 = z  (lg DT2 − lg DT1 ) + T2
1. Calculation of the D-value at 110°C using equation 16:

T1 = 10C  (lg1,5  lg 0,33) + 121C

1 lg DT 1 − lg DT 2
=−
z T1 − T2
T1 = 127,56C

z  (lg DT 2 − lg DT 1 ) = T1 − T2 For a sterilization time of 3 min a temperature of 127.6°C

is required.
(T1 − T2 )
lg DT 2 = lg DT 1 +
z

20
In diagram 8 all 3 processes are plotted. 6. Requirements and selection of biological indica-
tors for validation and routine monitoring

If there are changing parametric sterilization conditions,

like changing steam quality, a validation using parametric
release is impossible. In this case only the direct inocula-
tion with biological indicator suspension at worst case
conditions can be carried out. In low temperature sterili-
zation processes biological indicators are exclusively used
for validation.

6.1. Selection of strain

Depending on the sterilization process non-pathogenic

germs are selected having a higher resistance in compari-
son to pathogenic germs. The international standard
EN ISO 11138 recommends individual germs for different
sterilization processes. Preferably spore generating
germs with defined populations are produced. They re-
Diagram 8: Illustration of the examples main their population over several years. Only biological
indicators with certificate should be used stating the
germ, population, D-value, manufacturer and expiry date
5.2. Process design with unknown or changing start- and are manufactured according to above standard. The
ing bioburden values (overkill process) certificate also should refer from which culture collection
the strain is coming from.
If the starting conditions like in healthcare units are un-
known because of changing load configurations and Like:
changing bioburdens, the process has to be developed
using worst case conditions. For those conditions mini- DSM = Deutsche Sammlung für Mikroorganismen
mum F0-values are described in the European Pharmaco- (German collection of Microorganisms)
peia (EP) and the US Pharmacopeia (USP). For steam ster- ATCC = American Type Culture Collection,
ilization processes two alternative sterilization times and NCTC = National Collection of Type Culture
temperatures are given, however having two different F 0- (London)
values:
The following table lists most popular strains for different
sterilization processes:
Temperature [°C] Time [min] F0-value [min]

121 15 15 Name ATCC No: Sterilization process

134 3 > 60 Atrophaeus 9372 Ethylene oxide, dry heat

Stearother-
The reason for a much higher F0value at 134°C is that mophilus 7953 Steam, Formaldehyde, H2O2
temperature equilibration times are added. Short sterili-
zation times below 1 min bear the risk that the tempera- Pumilus 27142 γ and β radiation
ture is not achieved at all locations within the load. There-
fore a longer sterilization time is used as required for the
F0-value. 6.2. Resistance of biological indicators:
Comparing the same F0-values at 121°C with 15 min it The total resistance of a biological indicator depends on
would be at 134°C only about 0.75 min using a z-value of the population and resistance of each individual germ.
10°C. The time of 0.75 min is the at 134°C only. In addi- The resistance of each individual germ is defined by the
tion all other times have to be added to reach 134°C. It is decimal reduction value which is the time needed to re-
not absolutely sure if the sterilization time is reached in duce the population of a biological indicator to one tenth
the chamber or reached at all surfaces of the goods to be of the original population. The total resistance of a bio-
sterilized because non-condensable gases (NCG) may logical indicator is expressed by the FBIO value:
hinder the homogenous heat-up of the goods. If extreme
short sterilization times are used this potential problem FBIO = D121°C value x log (population)
may occur. Therefore the sterilization time at 134°C has
been extended as a safety margin.

21
This fact may be demonstrated by the 2 examples below 6.4 Positioning of biological indicators
in the table.
Biological indicators should never be placed outside of
packs. In the standards of biological indicators there are
Example Population D121-value FBio-value no recommendations given where to position the biologi-
[CFU/unit] [min] [min] cal indicators inside of a sterilization load. Biological indi-
1 106 1,5 9 cators have to be placed always at the worst case loca-
tion according to the validation standard, e.g. for steam
2 105 2 10 sterilization processes EN ISO 17665-1, which may be
inside of a package with solid goods or inside of instru-
As seen above, the D-value of a given strain is never con- ments containing hollow lumens and/or splits. If biologi-
stant and depends on growth and process condition. cal indicator strips cannot be placed into hollow devices
Therefore, for each batch of biological indicators certifi- or splits, direct inoculation with biological indicator sus-
cates must be associated to the product indicating the pensions have to be made or they have to be placed in-
population, individual resistance and the total resistance side process challenge devices (PCDs).
of a biological indicator.
On top the small load effect and non-condensable gases
6.3. Selection of biological indicators for inside of the sterilizer chamber have to be recognized.
routine monitoring Non-condensable gases (NCG) mixed with steam inside
the sterilization chamber are transferred in a single pack
For routine monitoring biological indicators have to be creating dangerous amounts of NCG inside.
selected according the requirements of the international
standards and need to be adopted to the F0-value of the
sterilization process. To monitor overkill processes in
steam sterilization processes the FBio-value should be
selected that the SAL of the biological indicator at the end
of the sterilization process should reach 10 -4. Therefore
the FBio-value can be calculated:

F0 = FBio + 4 • D121

FBio = F0 – 4 • D121

The sterility test according EN 556 with biological indica-

tors is directly not possible since it is not feasible to make
tests with one million biological indicators. To check if the
SAL ≤ 10-6 is achieved, the FBio-value of the biological indi-
cator should be above the bioburden of the load (see
diagram 9).
1,0E+04

1,0E+03 CFU of the

biological
colony forming units [CFU/part]

1,0E+02 indicator

1,0E+01
bioburden of the
1,0E+00 insterile product
F=2x5=10
1,0E-01 (90% kill rate)
F=2x6=12
1,0E-02 (99% kill rate)
1,0E-03 F=2x7=14
(99,9 % kill rate)
1,0E-04

1,0E-05

1,0E-06 "Sterile product"

0 2 4 6 8 10 12 14 16 18
time [min]

Diagram 9: Selection of biological indicators

22
7. Glossary of symbols used within the text

Symbol Unit Description Unit Abbr. Unit Application Description

Colony Amount of germs on a biological
CFU Number of germs number
forming unit indicator
Describes the resistance of a bio-
logical indicator, describes the
Decimal reduction factor
DT time or dose [min] time necessary to kill 90 % of the
(D-value) at temperature T
starting bioburden or reduces
population by one decade
Activation energy of the Reaction Reaction energy to start chemical
E0 [J/mol]
reaction energy reaction
Correlation of all sterilization
times at different temperatures to
Equivalence time of a a given reference temperature,
F(T,z) time [min]
sterilization process expresses the sterilization effort,
given as a time at a defined tem-
perature
Equivalence time of a
For steam sterilization processes
sterilization process under
F0 time [min] 121°C and a set value of 10°C,
standardized conditions
expresses the sterilization effort
(i.e. steam 121°C)
Population reduction during a
sterilization process, expressed by
IF Inactivation factor amount N the number of decimal reduction
numbers (log difference of popu-
lation)
Reaction kinetics constant of Used if the decade logarithm is
k 1/time [min-1]
the decimal logarithm used
Reaction kinetics constant of Used if the natural logarithm is
k’ 1/time [min-1]
the natural logarithm used
Temperature dependent factor Specific for individual sterilization
k0 1/time [min-1]
of the reaction kinetics constant processes
Nominal population on a number of Number of germs on an instru-
N [CFU/part]
medical device germs ment
Number of germs on a medical
Number of germs on a MD after number of
NF [CFU/part] device after a process with sterili-
a sterilization cycle germs
zation time F has been carried out
Starting bioburden on a medical number of Bioburden of a MD before sterili-
N0 [CFU/part]
device germs zation
PCD Process challenge device
constant val-
R General gas constant [J/mol K] = 8,314 [J/mol K]
ue
SAL Sterility Assurance Level

t Sterilization time time [min] Time elapsed during sterilization

Describes the modification of the
z Temperature coefficient temperature [°C] D-value depending on the
temperature

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23
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