VDMA-Documents

Food Processing Machinery

and Packaging Machinery

Code of Practice
Filling Machines of VDMA Hygiene Class V:

Testing the Effectiveness of Packaging

Sterilization Devices

No. 6/July 2002

Revised edition July 2008 / English edition: September 2008
Lyoner Strae 18
Verband Deutscher Maschinen- Fachverband
Nahrungsmittelmaschinen und D-60528 Frankfurt am Main
und Anlagenbau e.V.
Tel.: +49 69 66 03-14 31
Verpackungsmaschinen
(German Engineering
Fax
+49 69 66 03-12 11
(Food Processing and
Federation)
e-mail nuv@vdma.org
Packaging Machinery
Internet www.vdma.org/packtech
Association)
Chairman:
Volker Kronseder
Managing Director:
Richard Clemens

VDMA
Representing the capital
goods industry

Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Contents
1. INTRODUCTION ........................................................................................................................ 3
2. TERMS ....................................................................................................................................... 4
3. SPECIFICATION OF TEST MICROORGANISMS FOR CHECKING PACKAGING
STERILIZATION DEVICES IN ASEPTIC FILLING MACHINES....................................................... 4
3.1
STERILIZATION BY MEANS OF HYDROGEN PEROXIDE ................................................................. 4
3.2
STERILIZATION BY MEANS OF STEAM ........................................................................................ 5
3.1
STERILIZATION BY MEANS OF PARACETIC ACID ......................................................................... 5
3.3
MEDIA FOR SUSPENDING SPORES ............................................................................................ 5
4. METHODS FOR INOCULATING THE PACKAGING................................................................ 5
4.1
SPRAYING .............................................................................................................................. 5
4.2
APPLICATION BY PIPETTE ........................................................................................................ 5
5. COUNT REDUCTION TEST ...................................................................................................... 6
5.1
GENERAL PROCEDURE ........................................................................................................... 6
5.2
TEST METHOD ........................................................................................................................ 6
6. END-POINT TEST...................................................................................................................... 7
6.1
GENERAL PROCEDURE ........................................................................................................... 7
6.2
TEST METHOD ........................................................................................................................ 7
7. TEST REPORT........................................................................................................................... 8
8. USE OF THE CODE OF PRACTICE FOR CHECKING NONASEPTIC FILLING MACHINES
FITTED WITH DEVICES FOR PACKAGING STERILIZATION........................................................ 8
9. REFERENCES ........................................................................................................................... 9
10. STANDARDS CITED ................................................................................................................ 9
APPENDIX I TEST MICROORGANISMS FOR ASEPTIC PLANTS............................................... 11
APPENDIX II COUNT REDUCTION TEST WORKED EXAMPLE .............................................. 12
APPENDIX III END-POINT TEST WORKED EXAMPLE ............................................................. 13
APPENDIX IV CULTURE CONDITIONS FOR THE TEST STRAINS BACILLUS SUBTILIS SA 22
AND BACILLUS ATROPHAEUS AND FOR PREPARATION OF THE SPORE SUSPENSION... 14
APPENDIX V SOURCES OF SUPPLY OF READY MADE SPORE SUSPENSIONS ................... 15

This code of practice is the English translation of a publication which was drawn up in collaboration
with the Industrievereinigung fr Lebensmitteltechnologie und Verpackung e.V (IVLV) in the VDMA
Working Party for Interface Problems in Aseptic Plants. The following people have contributed to
the preparation of the first edition of the working paper:
Mr. Rainer Ammann
Dr. Peter Friedrich
Mr. Willibald Weber
Mrs. Regina Zschaler

Dr. Hong-An Duong

Dr. Peter Golz
Mr. Helmut Weber

Mr. Rudolf Flrke

Dr. Ingo Sabotka
Mr. Joachim Wunderlich

In Spring 2008 this code of practice was revised by the above mentioned working party. Besides
updating the references made in the document in paragraph 5 (count reduction test) the formula to
calculate the mean logarithmic count reduction was simplified by way of mathematical
transformation. Further more the title of the document was changed to mirror the classification of
filling machines worked out by this working party.
Suggestions concerning the contents of the code of practice may be sent to the Verband Deutscher
Maschinen- und Anlagenbau e.V. (VDMA), Fachabteilung Verpackungsmaschinen, Lyoner Strae
18, 60528 Frankfurt/M. (Fax: +49 69 6603-1211).

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

1.

Introduction

This code of practice replaces the code of practice Testing the Effectiveness of H2O2 Packaging
Material Sterilization Devices Fitted in Aseptic Plants which was published by the Working Party
for Test Methods for Packaging Sterilization in Aseptic Plants in the Packaging Materials
Microbiology Working Group of the Industrievereinigung fr Lebensmitteltechnologie und
Verpackung e.V. (Industry Association for Food Technology and Packaging) and appeared in the
journal Verpackungsrundschau, Technisch-wissenschaftliche Beilage (Technology Supplement),
Volume 38 (1987), Issue No. 6, pages 45 47.
The present code of practice describes test methods by which the microbiological effectiveness of
filling machines of VDMA hygiene class V (aseptic plants) containing packaging sterilization
devices can be determined. The methods described here count reduction test and end-point test
are suitable for testing the packaging sterilization methods by means of H2O2,, steam and
peracetic acid introduced at the time the code of practice went to press. These so-called challenge
tests which require artifical inoculation of packaging materials with microorganisms under
controlled conditions in order to obtain statistically significant test results allowing objective
comparisons. 1
The choice of test microorganism suitable for the sterilization process in question is particularly
important. The selection criterion is the resistance of the microorganisms and their spores to the
sterilization medium. Accordingly, in this code of practice suitable test microorganisms are
identified for each sterilization method and a method for determining the resistance of the test
microorganism to the sterilization process is described.
The sterilization performance of an aseptic filling machine is dependent on a large number of
machine parameters, such as, inter alia, the concentration and temperature of the hydrogen
peroxide, moisture content and temperature of the steam, if need be dwell time of the film in the
H2O2 bath (as a function of machine performance) and the shape of the container to be sterilized.
These boundary conditions have to be laid down prior to testing. The degree of effectiveness
stated in the test report always relates to the boundary conditions set out in advance. Accordingly,
these must be recorded in the test report.
The degree of microbiological effectiveness found for the aseptic plants is further dependent on a
series of other limiting conditions, including the initial level of contamination of the packaging, the
way the packaging is inoculated and the method of redetermining the microorganism levels. These
limiting conditions are also to be set out in the test report.
Finally, the degree of microbiological effectiveness found is also dependent on the packaging
material investigated. If different packaging materials are used on an aseptic filling machine the test
should be carried out for each of these packaging materials. Thus, independent tests have to be
carried out for cups and lids by way of example.
Extensive specialist knowledge is required for carrying out the test methods described here. They
should, therefore, be undertaken only by institutes or laboratories that are well acquainted with
them. 2 It is recommended that the manufacturer of the machine to be checked be involved. It may
be noted at this point that as a general principle only specialist microbiology staff should be
entrusted with the procurement, storage and handling of the highly concentrated microbiological
test suspensions needed for the test methods. The relevant safety regulations should be drawn to
their attention.
The test methods described here may also be applied with appropriate modifications to checking
other sterilization processes. See Section 8 with regard to this.
1

See VDMA Document No. 12/2007 for distinguishing microbiological challenge tests from other checks on the
microbiological safety of hygienic filling machines of VDMA hygiene classes IV and V.
2

A reference list of establishments well acquainted with the test methods may be requested from the
publisher of the code of practice.

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

2. Terms
Aseptic filling machines (aseptic plants) 3
Filling machines operating in aseptic manner (aseptic plants) are packaging machines which
charge a sterile product to be filled (e.g. food) without recontamination into a sterilized pack, the
latter sterilization usually being done on the machine. In order to achieve this, high requirements
are imposed on the effectiveness of the devices for sterilizing the packaging, the interior of the
machine and the parts conveying product (see VDMA Documents No. 11/2006)). Thus, in
packaging sterilization a count reduction of test microorganisms suitable for the sterilization method
in question of at least four powers of ten is considered necessary.
Aseptic filling machines are typically employed in the filling of low acid products (pH 4.6) which
should be imperishable without refrigeration for a relatively long period.
Packaging
A manufactured unit made from packaging material which is destined to envelope or hold together
the product to be packed so that it is in a form suitable for shipping, storage and sale. 4
Packaging sterilization device
A packaging sterilization device is an essential component of an aseptic filling machine. Its purpose
is to reduce the level of microorganisms in the packaging to be processed to such an extent that
recontamination of the filled product by the packaging may be ruled out. The performance of
packaging sterilization devices is usually aimed at a specified, usually low level of microorganisms.
By means of appropriate specifications for the supplier of the packaging, storage of the packaging
under adequate hygiene conditions and compliance with hygiene regulations in the filling plant can
be ensured. 5 An elevated level of microorganisms in the packaging carries the risk of
recontamination of the filled product even though the packaging sterilization device is functioning
properly!
Test microorganism
Test microorganisms are used to check the performance of sterilization devices. They should
exhibit a high and as far as possible defined resistance to the sterilization method in question. They
should also be easy to detect and present no hazard to health. The description of a test
microorganism should contain the following characteristics: name, D value, precise designation of
strain (ATTC No. or DSM No.) and batch number (in the case of ready-made spore suspensions).
Inoculation
Artificial infection of the packaging with test microorganisms.

3. Specification of test microorganisms for checking packaging

sterilization devices in aseptic filling machines
3.1

Sterilization by means of hydrogen peroxide

It is customary to use spores of Bacillus atrophaeus (ATCC 9372, DSM 675, formerly Bacillus
subtilis) and Bacillus subtilis SA 22 (identical to NCA 72-52 and DSM 4181). 6

nd

Filling machines of VDMA hygiene class V. See VDMA documents No. 2/2000 (2 edition 2006).
In accordance with DIN 55405, Part 3
5
Corresponding notes are provided in VDMA Documents No. 11/2006.
6
Since the method of isolation of the spores has an effect on their resistance characteristics the production specification or
the source of supply of the spore suspension should be noted in the test report. Examination of the resistance of the spores
to the sterilizing agent to be investigated is recommended.Sources of supply of ready-made spore suspensions are listed in
appendix V. Instructions for preparing the spore suspension may be found in Appendix IV of this code of practice.
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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

3.2

Sterilization by means of steam

It is customary to use spores of the strain Geobacillus stearothermophilus NCA 1518, ATTC 7953
(identical to DSM 5934) 7 . 8

3.1

Sterilization by means of paracetic acid

It is customary to use spores of Bacillus atrophaeus (ATCC 9372, DSM 675, formerly Bacillus
subtilis) and Bacillus subtilis SA 22 (identical to NCA 72-52 and DSM 4181). 9

3.3

Media for suspending spores

Ethanolic solution ( e.g. 70 %) or distilled water 10 . The concentration of the ethanolic solution as
well as other additives are to be specified in the test report.

4. Methods for inoculating the packaging

4.1

Spraying

Application by pipette 11

4.2

Inoculation in germ-free environment

Uniform, fine application
Drying at room temperature in germ-free environment until completely dry
Advantage: Spore distribution mimics the natural dissemination of microorganisms
Disadvantages: Possible contamination of the environment; relatively costly operation

Inoculation in germ-free environment

Note point of application and the total count in the test report 12 13
Drying at room temperature in germ-free environment until completely dry
Advantages: Method is less costly than the spraying method and allows selective
application of the spore suspension at points of the packaging critical for sterilization
Disadvantage: Method very probably results in application of several layers of
microorganisms.

Sources of supply of ready-made suspensions are listed in appendix V.:

Since the resistance of the spores to moist heat may vary from batch to batch the D121 value and the method of calculating it
should be specified in the test report. (For description of biological indicators of resistance to moist heat see DIN EN ISO
14161.)
8
Clostridium sporogenes PA (SC-218) is also applied.
9
Since the method of isolation of the spores has an effect on their resistance characteristics the production specification or
the source of supply of the spore suspension should be noted in the test report. Examination of the resistance of the spores
to the sterilizing agent to be investigated is recommended.Sources of supply of ready-made spore suspensions are listed in
appendix V. Instructions for preparing the spore suspension may be found in Appendix IV of this code of practice.
10
The test microorganisms should as far as possible be applied in distilled water or ethanolic solution since when using
buffers or common salt solution high concentrations of salt can arise in the course of drying and as a result any protective
layers formed may give a false indication, in this case an undervaluation of killing rates.
11
This paragraph also applies for inoculation by swab or by glass scoop.
12
Determination of count density from the spore suspension by repeat determination. Stating the mean value in colony
forming units per ml.
13
Applied quantity recommended: 0,01ml. Total counts of the starting suspension have to be verified.
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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

5. Count reduction test

5.1

General procedure

In the count reduction test the items of packaging infected with the test microorganism are passed
through the aseptic plant. In doing so the number of viable spores is determined before and after
passage of the sterilization device and from the difference in microorganism counts the killing rate
is determined.

5.2

Test method

i)
Investigation of the critical process parameters prior to the test run is recommended (e.g.
concentration of the hydrogen peroxide, temperatures)
ii)
Provision of at least 25 packaging units 14 which for the test have been inoculated under the
same conditions. Each packaging unit has an initial microorganism count of at least 105 spores for
the test. 15
iii)
Determination of the initial count IC for 5 artificially infected packaging units from ii)
(Transportkontrolle). To get out the microorganisms from the inner surface of the containers, they
are rinsed with a test medium. On sheet packaging the microorganisms are removed by swabs in
accordance with DIN 10113-2.
iv)
Setting of the specified machine parameters. It is advisable for the machine manufacturer
and the machine operator to jointly agree the parameters.
v)
Introduction of at least 20 infected packaging units into the filling machine. In multiline filling
machines the infected packaging units have to be uniformly distributed across the individual lines
with the filling line to be mentioned on the individual packaging units. When there are more than 2
lines it has to be ensured that at least 10 packaging units can be investigated on each line. The
total number of packaging units to be introduced has to be correspondingly increased.
vi)
Carrying out the test run. If possible the packaging units should be filled during the test run
to 25 % of the nominal filling volume with sterile skimmed milk cooled to room temperature or a
sterile, pipettable or filterable liquid. The packaging units are to be cooled immediately after filling.
The test data are then documented.
vii)
If during the test run the packs are not filled with a test medium the sterilized packaging
units are to be passed on as quickly as possible after the test run for microbiological analysis in
order to avoid falsification of the test results. 16
viii)
Determination of the survivor count (SC) for each of the artificially infected packaging units
and determination of the initial count (IC) of retain sample (see 5.2 (iii)).
ix)

Calculation of the microorganism count reduction (for worked example see Appendix II).
MLKCR (Mean logarithmic count reduction) = log[(1/5)*ICj)] log[(1/20)*SCi]
(1/5)*ICj :
(1/20)*SCi :

14

mean initial count

mean survivor count

In multiline filling machines it may be necessary to increase the number of packaging units provided as set out in v) .
If possible the initial count should be raised to the power of 1 to the mean logarithmic reduction contractually agreed on.
Corresponding deviations from 5(ii) should be noted in the test report.
16
Wetting the sterilized packaging with a test medium prevents any secondary action by residues of the sterilizing agent (in
the case of H2O2 sterilization) and serves to revive sublethally harmed microorganisms.
VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,
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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

i = 1, , 20 17
j = 1, , 5
In the case of multiline filling machines analysis of the results from the individual lines is
recommended.
x)
The test yields a positive result when for each line investigated at least the mean count
reduction previously set is achieved.

6. End-point test
6.1

General procedure

In the end-point test the packaging is also artificially inoculated with test microorganisms as in the
count reduction test but in this case in three graduated infection stages each being greater by a
power of ten than the one before.
The main difference with respect to the count reduction test is that in the end-point test the
artificially infected packaging is filled with a sterile culture medium matched to the test
microorganism and after an incubation phase only the number of unsterile packaging units is
determined. Beyond the effectiveness of the packaging material sterilization the end-point test
provides information about the entire process from supplying the product and filling through
recontamination-free closure of the packs.

6.2

Test method

i)
Performance test using packaging units which have not been artificially infected 18
(optional).
ii)
Provision in each case of at least 100 packaging units selected for the test each unit having
an initial microorganism count of 102, 103 or 104 spores of the test microorganism. 19,
iii)
Uniform distribution of the packaging units over the packaging lines in the case of multiline
filling machines.
iv)
Setting of the predetermined machine parameters. It is advisable for the machine
manufacturer and the machine operator to jointly agree the parameters.
v)
Test run using the test medium (e.g. sterile skimmed milk or a culture medium matched to
the test microorganism). Incubation of the closed packs (at least one week at 30 C) 20 . The number
of unsterile packs is then determined . Unsterile packs are to be investigated with regard to the
microorganisms which occur. 21
vi)
Determination of the mean logarithmic count reduction for each of the three levels of
contamination according to the following formula 22 (for worked example see Appendix III):

17

If more than twenty packaging units are inoculated the formula is to be adjusted accordingly
This test series serves to check the general functions of the machine and the sterility of the filled product.
When higher initial infection levels are used this should be noted in the test report.
20
When Geobacillus. Stearothermophilus is used as test microorganism incubation takes place at 55 C.
21
The cause of the occurrence of extraneous microorganisms has to be investigated.
22
This formula is also used by the NFPA (National Food Processors Association, USA).
If all packs evaluated are sterile the solution of this formula is not defined. In this case the formula is to be calculated with
one packaging unit assumed to be unsterile or the test is to be considered to yield a positive result.
As an alternative MLK can also be calculated according to the MPN-method. (MPN: Most probable number, see Moruzzi et
al. (2000)). When the MPN-method is used this should be noted in the test report.
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19

Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

MLKEP (Mean logarithmic count reduction) =

log (Initial count per pack) (log ln (Number of packs tested/Number of sterile
packs))
vi)
The test yields a positive result when at least the previously established value for the mean
count reduction is achieved for each test series.

7. Test report
Points to be recorded in the test report giving reference to this code of practice are:
Name of institute conducting the test
Brief description of the aseptic plant tested (manufacturer, exact model designation, type of
packaging material sterilization device)
Settings of machine parameters relevant to sterilization in accordance with the machine
manufacturer (e.g. machine efficiency applied (strokes per minute), ....)
Required count reduction capability of the aseptic plant
Type and concentration of the sterilizing agent
Date of test runs
Precise designation of the test microorganism including values from resistance testing if
available
Description of spore suspension (concentration, production specification or source of
supply)
Method of packaging inoculation
Identity of test medium, if applicable
Residual quantity of sterilizing agent, if applicable (does not apply in case of sterilization by
means of steam)
Method of determining survival count (count reduction test)
Method of determining unsterile packs (end-point test)
Statement of mean logarithmic count reduction with identification of the type of test carried
out
Deviation, if any, from the test specification
Test staff involved
Signature of person responsible for the test

8. Use of the code of practice for checking nonaseptic filling

machines fitted with devices for packaging sterilization
The test specifications set out in this code of practice may also be applied with appropriate
modifications to the checking of packaging sterilization devices in hygienic filling machines which
do not meet the strict requirements of VDMA document No, 11/2006. 23 For this test microorganism
and filling medium must be adapted to the sterilization process and the field of application. In the
case of acid products, for example, the efficiency of the packaging sterilization device is usually
tested using Aspergillus niger spores. 24 Coordination with the machine manufacturer is
recommended in every case.

23

A classification of hygienic filling machines for the food industry is presented in the VDMA publication Hygienic Filling
Machines for Liquid and Paste-Form Foods Classification and Typical Fields of Application (in German)
nd
(VDMADocuments No. 2/2000 (2 edition 2006)).
24
For strain designation and production specification for obtaining the spore suspension see DIN EN 1650.
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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

9. References
Industrievereinigung fr Lebensmitteltechnologie und Verpackung e.V. (publishers), 1987
Merkblatt "Prfung von Aseptikanlagen mit H2O2-Packstoffsterilisationsanlagen auf deren
Wirkungsgrad (Code of Practice Testing the Effectiveness of Aseptic Plants Fitted with H2O2
packaging sterilization units)
Verpackungsrundschau Vol. 38 (1987) No. 6, pp. 45-47
D.T. Bernard et al.: Validation of Aseptic Processing and Packaging, Food Technology, December
1990, pp. 119-122
Guido Moruzzi, Wallace E. Garthright, John D. Floros, 2000
Aseptic packaging machine pre-sterilisation and packaging sterilisation: statistical aspects of
microbiological validation
in Food Control 11 (2000) pp 57-66
VDMA FV NuV, 1997 (English version: October 2000)
VDMA Documents Food Processing Machinery and Packaging Machinery No. 3/2000 (2nd edition
2008)
VDMA Checklist for Quality Assurance and Maintenance
VDMA FV NuV, Frankfurt 2000
VDMA Food Processing Machinery and Packaging Machinery Publications 2000/No. 2 (2nd edition
2006)
Hygienic Filling Machines for Liquid and Viscous Foods - Classification and Typical Fields of
Application, obtainable as downloadable file from www.vdma.org/packtech
VDMA FV NuV, Frankfurt 2005
VDMA Documents Food Processing Machinery and Packaging Machinery No. 8/2005
Code of practice- Testing aseptic systems: Sterilizing the sterile section in the interior of the
machine
VDMA FV NuV, Frankfurt 2006
VDMA Documents Food Processing Machinery and Packaging Machinery No. 10/2006
Hygienic filling machines of VDMA class IV for liquid and pasty foods Minimum requirements and
boundary conditions for a operations in line with regulations
VDMA FV NuV; Frankfurt, 2006
VDMA Documents Food Processing Machinery and Packaging Machinery No. 11/2006
Aseptic packaging machines for the food industry:
Minimum requirements and boundary conditions for operations in line with regulations, as
replacement for VDMA 8742
VDMA FV NuV, Frankfurt 2007
VDMA Documents Food Processing Machinery and Packaging Machinery No. 12/2007
Guide to testing the microbiological safety of filling machines of VDMA hygiene classes IV and V
VDMA FV NuV, Frankfurt 2006
VDMA Documents Food Processing Machinery and Packaging Machinery No. 14/2006
Code of practice Testing hygienic filling machines of VDMA class V (aseptic filling machines)
External sterilization of packaging units
All VDMA documents are available as download in German and English from
www.vdma.org/packtech

10. Standards cited

DIN EN ISO 14161,
VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,
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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Norm, 2001-02
Sterilisation von Produkten fr die Gesundheitsfrsorge - Biologische Indikatoren - Leitfaden fr die
Auswahl, Verwendung und Interpretation von Ergebnissen (ISO 14161:2000); Deutsche Fassung
EN ISO 14161:2000
Sterilization of health care products - Biological indicators - Guidance for the selection, use and
interpretation of results. (ISO14161:2000)
DIN EN 1650
Norm , 2008-08
Chemische Desinfektionsmittel und Antiseptika- Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und
Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und ffentliche Einrichtungen Prfverfahren und Anforderungen (Phase 2, Stufe 1); Deutsche Fassung EN 1650:2008
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of
fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food, industrial,
domestic and institutional areas- Test method and requirements
DIN 10113-2
Norm , 1997-07
Bestimmung des Oberflchenkeimgehaltes auf Einrichtungs- und Bedarfsgegenstnden im
Lebenmittelbereich Teil 2: Semiquantitatives Tupferverfahren
Determination of surface colony count on fitment and utensils in foodareas Part 2:
Semiquantitative swab method (in German)
DIN 55405, Part 3
Terms in the packaging industry Packaging (in German)

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Appendix I
Test microorganisms for aseptic plants
Survey by Bernard et al. (1990)
Sterilization method

Test microrganism

Superheated steam

Bacillus stearothermophilus*
B. polymyxa**

Dry heat
H2O2 + heat

Notes on survey by Bernard et al.

(1990)
* New denomination: Geobacillus
stearothermophilus
Its customary to use spores of the
strain Geobacillus
stearothermophilus NCA 1518,
ATCC 7953 (identical to DSM 5934)

** New denomination: Paenibacillus

polymyxa
B. stearothermophilus*
* New denomination: Geobacillus
stearothermophilus
B. subtilis A or B. subtilis var. B. subtilis A could not be traced in
globigii
commercial reference stocks.
There is no exact equivalent in
DSMZ for B. subtilis var. globigii. A
close match is B. atropheus DSM
2277 (B. globgii; B. subtilis subsp.
niger)
In section 3.1 this VDMA document
refers to spores of Bacillus
atropheus (ATCC 9372, DSM 675,
frher Bacillus subtilis) and Bacillus
subtilis SA 22 (identical to NCA 7252 and to DSM 4181) for this
application.

H2O2 +UV

B. subtilis A.

B. subtilis A could not be traced in

commercial reference stocks.
In section 3.1 this VDMA document
refers to spores of Bacillus
atropheus (ATCC 9372, DSM 675,
frher Bacillus subtilis) and Bacillus
subtilis SA 22 (identical to NCA 7252 and to DSM 4181) for this
application.

Heat of formation

B. stearothermophilus*

Gamma radiation
Wet heat

B. pumilus
B. stearothermophilus*
Clostridium sporogenes

* New denomination: Geobacillus

stearothermophiluss
* New denomination: Geobacillus
stearothermophilus

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Appendix II
Count reduction test Worked example
MLKCR (Mean logarithmic count reduction) = log[(1/5)*ICj)] log[(1/20)*SCi]
(1/5)*ICj :
(1/20)*SCi :

mean initial count

mean survivor count

i = 1, , 20 25
j = 1, , 5
Zahlenbeispiel:
(1/5)*ICj :
(1/20)*SCi :

500 000
12

MLKCR = log[(1/5)*ICj)] log[(1/20)*SCi]

= log (500 000) log (12)
= 5,699 1,079
= 4,62

25

If more than twenty packaging units are inoculated the formula is to be adjusted accordingly.
VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,
Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

Page 12/16

Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Appendix III
End-point test Worked example
MLKEP (Mean logarithmic count reduction) =
log (Initial count per pack) (log ln (Number of packs tested/Number of sterile
packs))
Test series 1
2

Initial count 10 :
Number of packs tested:
Number of sterile packs:

4.35*102
100
99

log Count reduction

= log (4.35*102) log (ln (100/99))

= 2.638 log (ln 1.0101)
= 2.638 log (0.0105)
= 2.638 (- 1.997)
= 4.635

Test series 2
3

Initial count 10 :
Number of packs tested:
Number of sterile packs:

3.83*103
100
90

log Count reduction

= log (3.83*103) log (ln (100/90))

= 3.583 log (ln 1.111)
= 3.583 log (0.105)
= 3.583 (- 0.978)
= 4.561

Test series 3
4

Initial count 10 :
Number of packs tested:
Number of sterile packs:

5.37*104
100
63

log Count reduction

= log (5.37*104) log (ln (100/63))

= 4.729 log (ln 1.5873)
= 4.729 log (0.462)
= 4.729 (-0.335)
= 5.064

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

Page 13/16

Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Appendix IV
Culture conditions for the test strains Bacillus subtilis SA 22 and
Bacillus atrophaeus and for preparation of the spore suspension
Bacillus subtilis SA 22 respective Bacillus atropheus is first of all precultured in tryptone soya broth
for 24 hours at 30 C before samples of 0.1 ml of this preculture in each case are transferred by
means of Drigalski spatula onto plate count agar drugged with 0,001g/l MnSO4 (according to
Ph.EU) and incubated for 7 days at 30 C.
The Petri dishes in which the test microorganism has grown are each covered with a wash of 3 ml
of 0.9 % sterile common salt solution after which the bacterial spore bed is carefully removed using
a Drigalski spatula.
The spores harvested from several Petri dishes are combined and centrifuged at 10,000 g for
approximately 20 minutes. The supernatant liquid is then decanted off and the sedimented spores
are resuspended in sterile 0.14 M Srensen phosphate buffer (K2HPO4/KH2PO4, pH 7). After this,
centrifuging and resuspension are carried out twice in the same way until finally the spore sediment
is finely suspended in sterile phosphate buffer. The spore suspension obtained in this way is
heated for 20 minutes at 80 C in order to kill off vegetative unspored cells (pasteurization). The
spore suspension to be deployed is extracted by repeated centrifugation and absorption in
ethanolic solution (70%). The resultant spore suspension may be kept at 4 C for about 8 weeks.
The spore count should range from 108 to 109/ml.

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

Appendix V
Sources of supply of ready made spore suspensions
Ready made spore suspensions of Bacillus atropheus (ATCC 9372, DSM 675, formerly Bacillus
subtilis) may be sourced from (amongst others):
The National Food Laboratory, Inc.
Process Research & Microbiology Division
2441 Constitution Dr
Livermore, CA 94551
BAG - Biologische Analysensystem GmbH
Amtsgerichtstrae 1-5
35423 Lich
facsimile: 06404/3087
Raven Labs
8607 Park Drive
P.O. Box 27261
Omaha, NE 68127 TEL: 1.800.728.5702 or 1.402.593.0781
FAX: 1.402.593.0921 or 1.402.593.0995
info@ravenlabs.com
http://www.ravenlabs.com/
http://www.ravenlabs.com/page/indusebis#suspensions
Ready made spore suspensions of Bacillus subtilis SA22 (identical to NCA 72-52 and to DSM
4181) may be sourced from (amongst others):
Dr. Frh Control GmbH
Bettina-von-Arnim-Strae 3
61476 Kronberg
Biotecon Diagrnostics
Hermannswerder Haus 17
14473 Potsdam
phone.: 0331/230020
Ready made spore suspensions of Geobacillus stearothermophilus NCA 1518, ATCC 7953
(identical to DSM 5934) may be sourced from (amongst others):
BAG - Biologische Analysensystem GmbH
Amtsgerichtstrae 1-5
35423 Lich
facsimile: 06404/3087
The National Food Laboratory, Inc.
Process Research & Microbiology Division
2441 Constitution Dr
Livermore, CA 94551
Raven Labs
8607 Park Drive
P.O. Box 27261
Omaha, NE 68127 TEL: 1.800.728.5702 or 1.402.593.0781
facsimile: 1.402.593.0921 or 1.402.593.0995
info@ravenlabs.com
http://www.ravenlabs.com/
VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,
Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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Code of Practice
Filling Machines of VDMA Hygiene Class V:
Testing the Effectiveness of Packaging Sterilization Devices

http://www.ravenlabs.com/page/indusebis#suspensions
Ready made spore suspensions of Aspergillus niger may be sourced from (amongst others)::
Biotecon Diagrnostics
Hermannswerder Haus 17
14473 Potsdam
phone: 0331/230020
Raven Labs
8607 Park Drive
P.O. Box 27261
Omaha, NE 68127 TEL: 1.800.728.5702 or 1.402.593.0781
facsimile: 1.402.593.0921 or 1.402.593.0995
info@ravenlabs.com
http://www.ravenlabs.com/
http://www.ravenlabs.com/page/indusebis#suspensions
Ready made spore suspensions of C. sporogenes PA (NFL-Stamm SC220 und SC 218) may be
sourced from (amongst others)::
The National Food Laboratory, Inc.
Process Research & Microbiology Division
2441 Constitution Dr
Livermore, CA 94551

VDMA Fachverband Nahrungsmittelmaschinen und Verpackungsmaschinen,

Lyoner Strasse 18, 60528 Frankfurt, July 2002 (revised edition July 2008/English edition September 2008)

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