DOC 7

A METHOD FOR THE ASSESSMENT OF BACTERIA

TIGHTNESS OF FOOD PROCESSING EQUIPMENT
Second Edition, July 2004
European Hygienic Engineering and Design Group
EHEDG Secretariat
Ms. Susanne Flenner
Lyoner Str. 18
60528 Frankfurt, Germany

Tel.: +49-69-66 03-12 17

Fax: +49-69-66 03-22 17
E-Mail: [email protected]
Website: www.ehedg.org

Developed with support from the European Commission and in co-operation with 3-A and NSF
International.

THE ENGLISH VERSION OF THIS EHEDG DOCUMENT IS THE OFFICIAL VERSION. THE EUROPEAN
COMMISSINON SUPPORTS THE DEVELOPMENT OF THE EHEDG GUIDELINES. THE RESPONSIBILITY
FOR THE PREPARATION, DEVELOPMENT AND ISSUANCE OF SUCH GUIDELINES LIES WITH EHEDG.
DUE TO THE TECHNICAL AND GENERAL NATURE OF THE GUIDELINES, NEITHER THE EC NOR EHEDG
MAY ASSUME ANY LIABILITY RESULTING FORM THE INTERPRETATION, APPLICATION OR USE OF SUCH
GUIDELINES.

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Contents Page

Introduction.........................................................................................................................................................5
1 Definitions ..............................................................................................................................................5
2 Materials .................................................................................................................................................5
2.1 Indicator micro-organisms ...................................................................................................................5
2.2 Tripticase soy broth ..............................................................................................................................5
2.3 Test equipment ......................................................................................................................................6
3 Test procedure.......................................................................................................................................6
3.1 Test circuit .............................................................................................................................................6
3.2 Equipment soiling .................................................................................................................................6
3.3 Detection of penetrating bacteria ........................................................................................................7
3.4 Interpretation of results ........................................................................................................................7
3.5 Positive controls....................................................................................................................................7
4 Discussion .............................................................................................................................................7
5 References .............................................................................................................................................9

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 A METHOD FOR THE ASSESSMENT OF BACTERIA TIGHTNESS OF
FOOD PROCESSING EQUIPMENT*
(Second Edition)

T. Bénézech** (1), F. Bourion (2), B. Carpentier (3), G.J. Curiel (4), C. Faille (1), P. Gustavsson (5),
C. Hermon (6), J. Hofmann (7), J. Kastelein (8), J. Kold (9), A.W. Timperley (10), G. Wirtanen (11)

©EHEDG

(1) Laboratoire de Génie des Procédés et Technologie Alimentaires, INRA, 369 Rue Jules Guesde, B.P.
39, F-59651 Villeneuve D'Ascq Cedex, France.

(2) ASEPT, Rue des Docteurs Calmette et Guérin, BP49/53020 Lavel Cedex, France.

(3) Laboratoire d'Etudes et de Recherches sur la qualité des aliments et sur les procédés agro-
alimentaires, AFSSA, 22 Rue Pierre Curie, BP332, F-94709 Maisons-Alfort Cedex, France.

(4) Unilever R&D Vlaardingen, PO Box 114, 3130 AC Vlaardingen, the Netherlands.

(5) SIK, Swedish Institute for Food and Biotechnology, P.O. Box 5401, S-402 29 Gothenborg, Sweden.

(6) Centre Technique des Industries Mécaniques, CETIM, 74 Route de la Jonelière BP 82617, 44326
Nantes Cedex, France.

(7) Technische Univesität München, Lehrstuhl für Maschinen und Apparatekunde, Am Forum 2, 85350
Freising, Germany.

(8) TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, the Netherlands.

(9) Biotechnological Institute, Holbergsvej 10, P.O. Box 818, DK-6000 Kolding, Denmark.

(10) Campden & Chorleywood Food Research Association Group, Chipping Campden, Gloucestershire
GL55 6LD, United Kingdom.

(11) VTT Biotechnology, Tietotie 2, Espoo, P.O. Box 1500, FIN-02044, Finland.

* Update prepared by the “Test Methods” Subgroup of the European Hygienic Engineering and Design Group
(EHEDG), July 2004.

** Chairman

The production of EHEDG Guidelines is supported by the European Commission under the Quality of Life
Programme, Project HYFOMA (QLK1-CT-2000-01359).

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Introduction
The ‘Test Methods’ subgroup of the European Hygienic Engineering & Design Group (EHEDG) is responsible
for producing standardised test methods for assessing the hygienic and aseptic capability of food processing
equipment. Methods for assessing in-place cleanability (ref.1), in-line pasteurisability (ref. 2) and in-line steam
sterilisability (ref. 3) have been published.

This method details the test procedure for assessing whether an item of food processing equipment, intended
for aseptic operation, is impermeable to micro-organisms. Small motile bacteria penetrate far more easily
through microscopic passages than (non-motile) moulds and yeasts. The facultative anaerobic bacterium
Serratia marcescens (CBS 291.93) is therefore used to test the impermeability of equipment to micro-
organisms. This impermeability to micro-organisms is usually called ‘bacteria tightness’. The method is based
on a Unilever Research Laboratorium procedure and is designed to determine whether bacteria are able to
penetrate from the environment into the product line via the piece of test equipment. The method described is
suitable for equipment, which is already known to be in-line steam sterilisable.

1 Definitions
The definitions in the EHEDG Glossary (see www.ehedg.org/glossary.pdf) apply to this guideline. The most
relevant definitions specific to this test method are:

Microbial impermeability
The ability of equipment to prevent the ingress of bacteria, yeasts and moulds from the outside (environment)
to the inside (the product area).

Sterilisability
The suitability of clean equipment to be freed from viable micro-organisms including relevant bacterial spores
(i.e. sterilised) by, for instance, a treatment with pressurised water or saturated steam at 121°C for 30 minutes,
or another sterilising fluid. (Alternative conditions can be used depending on local circumstances).

Sterilisation
The removal or destruction of micro-organisms, including all relevant bacterial spores.

2 Materials

2.1 Indicator micro-organisms

To test the bacteria-tightness of equipment, Serratia marcescens is used. The test strain is a small, strongly
motile rod shaped micro-organism. It is able to penetrate through small holes and crevices that are very
difficult to detect by physical methods.

The indicator micro-organism is easily recognised by a strong red pigment, which colours the growth medium
(see 3.2 below) pink on incubation.

2.2 Tripticase soy broth

The indicator micro-organism is cultivated in sterile trypticase soy broth (TSB; concentration 15g 1-1 trypticase
soy) at 30°C for 24 hours prior to each experiment. TSB of the same concentration is also pumped through
the test circuit to provide a growth medium for any indicator micro-organisms able to penetrate the test
equipment.

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2.3 Test equipment

Prior to testing, the equipment to be investigated is dismantled and thoroughly degreased (using a solvent
such as alcohol), cleaned by hand (using a neutral detergent solution) and, if necessary, descaled (using a
1% w/w aqueous acetic acid solution). The dismantled equipment (if the components are relatively small)
should then be sterilised in an autoclave at 121°C for 30 minutes before reassembly under aseptic conditions.
Alternatively, the equipment may be reassembled and sterilised in-line by pressurised steam at 121°C for 30
minutes.

Notes:

1. All construction materials, gaskets, etc. must be capable of withstanding the cleaning and sterilisation
procedures.

2. Equipment with shaft passages should be equipped with double seals and provision made for flushing
the space between the two seals with a sterilising fluid.

3. Occasionally, gasket materials have antimicrobial properties, which may influence the test results.
Therefore, controls should be undertaken in which gasket samples are placed in petri dishes and just
submerged in TSA (BBL) pre-inoculated with S. marcescens. After solidification of the agar the petri
dishes are incubated at 30°C for 24 hours. If after 24 hours no growth is observed in the agar
immediately surrounding the sample, the gasket material must be regarded as unsuitable for this
method. If no growth is observed on the petri dish at all, a second TSA plate, pre-inoculated with S.
marcescens is incubated at 30°C for 24 hours to demonstrate positive growth of the test strain.

3 Test procedure

3.1 Test circuit

An example of a test circuit used for conducting in-line steam sterilisability testing is shown in Figure 1. An
aseptic vessel fitted with two aseptic flow-through valves (Figure 2) and containing an appropriate volume of
TSB, is sterilised in an autoclave at 121°C for 30 minutes.

Both side connections of the flow-through valves are short-circuited during autoclaving (Figure 2) and the
valves are left in the open position. After autoclaving the valves are closed and the vessel is incorporated into
the test circuit by means of the valves’ side connections. Once the test circuit has been assembled, the item of
equipment to be evaluated is treated with steam at 121°C for 30 minutes. The steam must be saturated and
the required back pressure, 0.2Mpa (2 bar) absolute, controlled by means of the throttle valve. Temperature
and pressure within the system must be in agreement with those expected for saturated steam. If this is not
the case, the test is invalid (the steam may contain gases, such as air). To ensure that no cold spots are
formed in the system, care must be taken to ensure that no condensate can accumulate during the steam
treatment. When the sterilisation procedure is completed, the two one-way valves either side of the flow-
through valves are closed. The flow-through valves are then opened, thus effecting an aseptic connection with
the TSB vessel. All test circuit components designated ‘aseptic’ must have been proven to be sterilisable and
bacteria tight, otherwise they may adversely influence the test results. In the case of small equipment, flexible
tubing and tube clamps may be used. All components (including any flexible tubing) must be connected such
that there are no places where solids or air can be trapped.

3.2 Equipment soiling

A freshly prepared culture (which will contain approximately 109 bacteria ml-1) is diluted (1ml in 9ml) in sterile
TSB and spread over all critical and suspected parts of the equipment by means of brushes, syringes, etc. All
areas where leakage may occur are treated twice a day, for at least 3 days in succession, or longer if required.
Where applicable, the equipment is operated 10 times after each treatment (e.g. valves may be actuated or
pumps operated manually). It is not necessary to know the exact inoculum level, as the number of organisms
spread over areas of potential leakage can be regarded as a substantial microbiological challenge.

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To obtain sufficient mixing and ensure rapid detection of microbial growth the broth is circulated for 2 hours
every day by means of a peristaltic pump. The flow rate at which the broth is circulated will depend on the
volume contained within the system and should be set to give two volume changes within the vessel during
the two hour circulation period each day. The test circuit is kept at ambient temperature (approximately 20-
25°C) during the soiling procedure. If the ambient temperature fluctuates outside the stated limits, it must be
confirmed experimentally that the growth and motility of S. marcescens are not adversely affected.

3.3 Detection of penetrating bacteria

After the soiling procedure the system is kept at ambient temperature (approximately 20-25°C for 5 more days.
The broth is circulated for 2 hours every day at the same flow rate used during the soiling procedure.

3.4 Interpretation of results

Whilst it is well recognised that ‘no visible growth’ is suggestive of a population of <106 cells ml-1, S.
marcescens grows readily in TSB at ambient temperatures (turbidity within 24 hours) and a relatively long
incubation time has been chosen (5 days). The reason for this is that cells that penetrate into the test item
between equipment parts may become damaged and may have to undergo a period of resuscitation before
they are able to multiply and turn the broth turbid. If the broth still remains clear after the 5-day detection
period, the equipment is classified as bacteria tight for the duration of the soiling procedure. If the broth
becomes turbid a sample is taken and examined for the presence of S. marcescens. The broth sample is
incubated at 30°C for 2 days. A red discoloration of the broth confirms the presence of S. marcescens.

3.5 Positive controls

As a positive control, a small volume of the S. marcescens culture used to inoculate the test item is aseptically
inoculated onto the surface of a suitable test material and allowed to dry for 1 hour (or until visibly dry if this
takes longer than 1 hour). The test material is representative of the material of construction of the test item,
usually stainless steel. The material is then placed in a suitable container containing sterile TSB and
incubated at the same ambient temperature as the test item. Growth of the S. marcescens in the broth is
indicative of the viability of the inoculating culture.

When, after bacteria tightness testing, the TSB is still clear (no growth) a small volume of the S. marcescens
culture used to inoculate the test item is aseptically inoculated into the TSB of the test circuit. Growth of the S.
marcescens in the broth, within 3 days at ambient temperature, is indicative of both the viability of the
inoculating culture and the capacity of the TSB to allow maximum growth (productivity of TSB).

4 Discussion
If Serratia marcescens is present in the system the equipment has failed the test and is, therefore, not
bacteria tight and hence not suitable for aseptic use. The organism is certain to have penetrated from outside,
because its heat resistance is so poor that it could not survive the steam treatment of 121° C for 30 minutes.

Tests should be conducted a minimum of three times. If varying results are obtained, a thorough examination
should be conducted to ascertain whether the tests have been adversely influenced by faults in either the item
of test equipment, the test circuit or the testing conditions and/or analysis. If any faults are discovered, these
should be rectified and the tests repeated. If no obvious faults are discovered and the results still remain
variable, it can be concluded that the item of test equipment is not bacteria tight and hence, not suitable for
aseptic use.

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Notes:

1. If an item of equipment is to be assessed for in-line steam sterilisability the test for bacteria tightness
may be conducted using the same test circuit and batch of broth, providing that the broth has
remained clear for the 5 day duration of the in-line steam sterilisability test.

2. If a piece of equipment passes the test it cannot be assumed that it will remain bacteria tight in the
future unless periodic maintenance is conducted (timely replacement of gaskets and seals).

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5 References

EHEDG Document No.2*), A method for the assessment of in-place cleanability of food processing
equipment, Third Edition (2004).

EHEDG Document No.4, A method for the assessment of in-line pasteurisation of food processing
equipment. (1993) Also as an extended abstract in Trends in Food Science
and Technology, February 1993, 4, 52-55.

EHEDG Document No.5, A method for the assessment of in-line steam sterilisability of food processing
equipment, Second Edition (2004).

Lelieveld, H.L.M. (1985). Hygienic design and test methods. Journal of the Society of Dairy Technology, 38,
No. 1, 14-16.

Order information for all EHEDG Documents can be obtained from the website www.ehedg.org.

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 Figure 1. Test circuit for testing the bacteria tightness of equipment

1 = TSB vessel
3 = Aseptic flow-through valves
10 = Bacteria/air filter

Figure 2. TSB vessel with aseptic flow-through valves during sterilisation in an autoclave.

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