Reviews in Environmental Science and Bio/Technology 2: 293306, 2003.

293

2004 Kluwer Academic Publishers. Printed in the Netherlands.

Disinfection in food processing ecacy testing of disinfectants

G. Wirtanen* & S. Salo

VTT Biotechnology, Microbiology, Tietotie 2, P.O. Box 1500, FIN 02044, VTT, Finland
(*author for correspondence: e-mail: gun.wirtanen@vtt.)

Key words: biolm, microbes, chemical residues, cleaning, disinfection, food processing

Abstract
The key to eective cleaning and disinfection of food plants is the understanding of the type of the soil to be
removed from the surfaces. An ecient cleaning and disinfection procedure consists of a sequence of rinses
using good quality water with application of detergents and disinfectants. Disinfection is required in food
plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The ecacy
of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces
where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give
adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a
proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the
major sources of variation in the procedure. In response to the need for a relatively realistic, simple and
reliable test for disinfectant ecacy a method for culturing laboratory model biolms has developed. The
use of articial biolms i.e. biolm-constructs inoculated with process contaminants in disinfectant testing
can also be used for screening the activity of various disinfectants on biolm cells. Both biolm carrier tests
showed clearly that the biolm protects the microbes against the disinfectants. The chemical cleanliness is
also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the
microbial load using culturing techniques. These results can give an incorrect picture of the total cleanli-
ness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent
bacteria light inhibition method oers a useful alternative for testing chemical residue left on surfaces after
cleaning and disinfection operations.

1. Introduction are surfaces from which undesirable chemical res-

The general aims for microbial control including
biolm removal are to prevent spoilage of prod-
ucts and to ensure that the quality specications of
the product are met. The most important means
for maintaining ecient microbial control include:
(1) minimizing the microbial load from outside
sources to the process, (2) ecient control of
growth at microbiologically vulnerable sites and
(3) adequate cleaning and disinfection of the pro-
cess lines (Wirtanen et al. 2000). Physical, chemical
and microbiological cleanliness is essential in food
processing plants. Physical cleanliness means that
there is no visible waste, foreign matter or slime on
the equipment surfaces. Chemically clean surfaces
idues have been removed, whereas microbiologi-
cally clean surfaces imply freedom from spoilage
microbes and pathogens (Gould 1994).
Attached microbes or microbes in biolms can
be a problem in food processing, because they
adhere to the surfaces and if the cleaning is
insucient the remaining cells start to grow and
contaminate the product (Hood & Zottola 1995).
The selection of detergents and disinfectants in the
food industry depends on the ecacy, safety and
rinsability of the agent as well as where it is cor-
rosive or aects the sensory values of the products
manufactured. An independent quality control
system to monitor the cleaning results for a food
plant can be integrated in the Hazard Analysis
294
Critical Control Points (HACCP) program. The
key to eective cleaning and disinfection of food
plants is the understanding of the type and nature
of the soiling agent (sugar, fat, protein, mineral
salts etc.) and the microbial growth to be removed
from the surfaces (Czechowski & Banner 1990;
Troller 1993).
2. Cleaning and disinfection
Cleaning and disinfection is carried out in order to
produce safe products with acceptable shelf life
and quality. In the food industry there is a trend
towards longer production runs with short inter-
vals for sanitation. The cleaning programmes
should be performed as cost-eectively and safely
as possible, which means as infrequently as possi-
ble, in the shortest possible time, with low chemi-
cal, energy and labour costs, producing as little
waste as possible and with no damage to the
equipment (Lelieveld 1985; Holah 1992). The
mechanical and chemical power, temperature and
contact time in the cleaning regime should be
carefully chosen to achieve an adequate cleaning
eect (Czechowski & Banner 1990; Mosteller &
Bishop 1993).
The use of eective cleaning agents and disin-
fectants on surface-attached microbes minimises
contamination of the product, enhances shelf life
and reduces the risks of foodborne illness. A pro-
longed exposure of the surfaces to cleaning agents
and disinfectants enhances the removal (Troller
1993). Attention should also be paid to the quality
of the processing water, steam and other additives.
Using additives of poor quality easily spoils the
process. Furthermore, the tools and methods used
must also suit the process and the personnel must
be properly trained and responsible to maintain a
good level of plant hygiene (Lelieveld 1985; Mat-
tila-Sandholm & Wirtanen 1992).
An ecient cleaning and disinfection proce-
dure consists of a sequence of rinses and deter-
gent and disinfectant applications in various
combinations of temperature and concentration
(Frank & Ko 1990; Holah 1992; Troller 1993;
Gould 1994; Wirtanen 1995). In a wet open
process the gross soil should be removed by dry
methods, e.g. brushing, scraping or vacuuming
and visible soil rinsed o with low-pressure water.
Using water of sucient volume and temperature
increases the cleaning eect. However, a pure
water washing system is not practical due to
ineectiveness and cost limitation. Surfactants,
which suspend the adhered particles and microbes
from the surfaces in the water, are added to in-
crease the washing eect. After a production run
the equipment should be dismantled and cleaned.
The cleaned utensils should be stored on racks
and tables, not on the oor (Mattila-Sandholm &
Wirtanen 1992; Holah & Timperley 1999). The
cleaning of open process surfaces and surfaces in
the processing environment is carried out using
either foam or gel cleaning. The foam-units are
constructed to form foam of varying wetness and
durability depending on the cleaning to be per-
formed. The application of gels extends the con-
tact time with a soiled surface and can be used
with low-pressure system. The cleaning is mostly
carried out in combination with a nal disinfec-
tion, because there are likely to be viable mi-
crobes on the surfaces that could harm continued
production. Furthermore good ventilation in the
process facilities is needed to enable drying of the
process equipment and process lines (Holah 1992;
Wirtanen et al. 2000).
In the cleaning of closed processes, prerinsing
with cold water is carried out to remove loose soil.
The CIP treatment is normally performed using
hot cleaning solutions, but cold solutions can also
be used in the processing of fat-free products. The
warm alkaline cleaning solution, normally of 12%
sodium hydroxide, is heated to 7580 C and the
cleaning time is 1520 min. The equipment is
rinsed with cold water before the acid treatment is
performed at approximately 60 C for 5 min. The
cleaning solutions should not be reused in pro-
cesses aiming at total sterility because the reused
cleaning solution can contaminate the equipment.
The design of the tank should ensure that also
parts directly above the spray ball is also cleaned.
Drainage, minimisation of internal probes, cre-
vices and stagnant areas, arrangement of valves,
couplings and instrument ports and instrumenta-
tion should be planned carefully so that the
equipment is easily cleanable. Problems caused by
equipment constructions and materials cannot be
eliminated with CIP, because the CIP treatment
was not designed to eliminate biolms (Czechow-
ski & Banner 1990; Brackett 1992; Chisti & Moo-
Young 1994; Zottola & Sasahara 1994; Wirtanen
et al. 1997).
 295

3. Common disinfectants Pseudomonas sp. have also been found in concen-

Disinfectants have been developed to destroy mi-
crobes. Microbes have nevertheless, been found in
disinfectant solutions, which is due to their ability
to form resistant strains and build-up of protective
biolms (Gilbert & Allison 1999; McBain et al.
2000; Wirtanen et al. 2002). This means that
microbial contaminants can be spread on the sur-
face to be cleaned instead of being cleansed. As
early as 1967, it was reported that chlorhexidine
mixtures were contaminated with Pseudomonas sp.
trated iodine solutions (Marrie & Costerton 1981).
Serratia marcescens was found to be viable even
after 27 months in a disinfectant containing 2%
chlorhexidine. A concentration of 0.1% chlorhex-
idine is sucient to kill the cells of S. marcescens if
they are freely suspended in liquid (Marrie &
Costerton 1981; Costerton & Lashen 1983).
Microbial contamination of e.g. Alcaligenes
faecalis, Enterobacter cloacae, Eschericia coli,
Flavobacterium meningosepticum and Pantoea ag-
glomerans has also been found in solutions of

Table 1. Advantages and disadvantages of some disinfectants used in the food processes (according to Flemming 1991; Troller 1993
and Wirtanen 1995)

Disinfectant type Advantages Disadvantages

Alcohols Eective against vegetative cells, non-toxic, Microbistatic,

easy-to-use, colourless, harmless on skin, ineective against spores
soluble in water, volatile
Peracetic acid Eective in low concentration, broad microbial spectrum, Corrosive, unstable
kills spores, penetrates biolms, non-toxic ( acetic acid
and water)
Hydrogen peroxide Decomposes to water and oxygen, relatively non-toxic, High concentrations needed,
easy to use; weakens biolms and supports detachment corrosive

Chlorine Eective in low concentration, broad microbial spectrum, Toxic by-products, resistance
easy to use, supports microbial detachment, cheap development, residues, corrosive,
reacts with EPS, discolouration,
explosive gas

Hypochlorite Cheap, eective in a broad microbial spectrum, Unstable, toxic, oxidative, corrosive,
easy to use, supports detachment rapid regrowth, no prevention of
adhesion, discolouration of
products

Chlorine dioxide Eective in low concentration, can be produced on-site, Toxic by-products, explosive gas
low dependency in pH
Quaternary ammonium Eective, non-toxic, prevents regrowth, supports Inactivated in low pH and by salts
agents microbial detachment, non-irritating, non-corrosive, (Ca2+ and Mg2+), resistance
odourless, avourless development, ineective against
Gram-negative bacteria

Iodophor Non-corrosive, easy to use, non-irritating, broad Expensive, avour, odour,

activity spectrum forms purple compounds with
starch

Ozone Similar eect as chlorine, decomposes to oxygen, Corrosive, inactivated easily,

no residues, decomposes biolm reacts with organics
( epoxides)

Glutaraldehyde Eective in low concentrations, cheap, non-corrosive Low penetration in biolms,

degrades to formic acid, increased
DOC
296
quaternary ammonium compounds, aldehydes and
amfotensides (Heinzel 1988).
Disinfection is required in food plant opera-
tions where wet surfaces provide favourable con-
ditions for the growth of microbes. The aim of
disinfection is to reduce the surface population of
viable microbes after cleaning and to prevent
microbial growth on surfaces before restart of
production. Disinfective agents do not penetrate
the biolm matrix left on the surfaces after an
ineective cleaning procedure very well, and thus
do not destroy all the living cells in biolms
(Bloomeld 1988; Pontefract 1991; Brackett 1992;
Holah 1992; Carpentier & Cerf 1993). Disinfec-
tants are most eective in the absence of organic
material, e.g. fat-, sugar- and protein-based
materials. Interfering organic substances, pH,
temperature, concentration and contact time gen-
erally control the eciency of disinfectants
(Czechowski & Banner 1990; Mosteller & Bishop
1993; Gould 1994). The disinfectants must be
eective, safe and easy to use, and easily rinsed o
surfaces, leaving no toxic residues or residues that
aect the sensory values of the product. The use of
disinfectants in food plants depends on the mate-
rial used and the adhering microbes. Disinfectants
(Table 1) approved for use in the food industry are
alcohols, chlorine-based compounds, quaternary
ammonium compounds, oxidants (peracetic acid,
hydrogen peroxide and ozone), persulphates,
surfactants and iodophors. They should be chosen
based on the process (Sequeira et al. 1989; Larson &
Morton 1991; Troller 1993; Wirtanen 1995):
Is the agent eective in the pH range used?
Is the agent stable when diluted? Does it
vaporize?
Is the agent toxic, safe or irritating?
What is the spectrum of the agent?
How does temperature aect the activity of
the agent?
Is the agent corrosive on the surface?
Is the agent surface active?
Is the agent stable when reacting with organic
material?
Is the agent eective, and what are the costs?
There are several chlorine or chlorine-based
compounds, which are approved for use in food
plants, e.g. gaseous chlorine, chlorine dioxide, so-
dium and calcium hypochlorites. The antibacterial
active moiety is formed when the chlorine com-
pound is added to water. Hypochlorous acid is
formed and it dissociates further into protons and
hypochlorite anions. Stabilised hypochlorites are
used when a long duration is required. The range
of microbes killed or inhibited by chlorine-based
compounds is probably broader than by any other
approved sanitizer (Troller 1993; Stewart et al.
1994; Sanderson & Stewart 1997).
Hydrogen peroxide has been found to be
eective in removing biolms from equipment
used in hospitals. The eect of hydrogen peroxide
is based on the production of free radicals, which
aect the biolm matrix. The microbicidal eect of
peracetic acid on microbes in biolms was shown
to vary (Exner et al. 1987; Christensen 1989;
Kramer 1997). Aldehydes did not break the bio-
lm, but rather seemed to improve its stability.
The biolm must be disrupted in some way before
chemical agents such as peracetic acid and alde-
hydes can be used eectively (Exner et al. 1987).
The eect of ozone treatment has been found to
vary depending on the processing circumstances
and the microbes tested, e.g. ozonation has proved
very eective in treatment of cooling water systems
(Lin & Yeh 1993).
Also, iodophors are used in the food industry.
In the disinfection the iodine compound takes part
in the oxidation of essential parts of the microbial
cells. Like chlorine-containing products, iodo-
phors are active against Gram-positive and Gram-
negative bacteria, yeasts and molds (Holah et al.
1990; Holah 1992; Troller 1993). Bacterial spores,
however, are highly resistant to iodophors. Iodo-
phors cannot be used in food plants where starch-
containing products are produced because iodine
forms a purple complex with starch.
Quaternary ammonium compounds are used as
sanitizers in dairies and in the food industry, be-
cause they have good wetting properties and are
nonspecically described as cationic surface active
agents in which the cationic part is hydrophobic.
The greatest eect of quaternary ammonium
compounds is observed against Gram-positive
bacteria, whereas Gram-negative organisms, many
of them signicant in the contamination of food,
may not be aected (Troller 1993).
Fogging can be dened as chemical disinfection
using automatic spraying of disinfectant in a
closed room. The aim of disinfection testing on an
industrial scale using fogging is to study the e-
ciency of the disinfection on surfaces at dierent

places in the room. Controlled experiments have
been carried out at two cheese-producing dairies.
Neither of the fogging trials showed clear reduc-
tion of the microbial load. Critical control points
in fogging are the amount of fog used, the disin-
fectant concentration used for the fog, thorough
rinsing of equipment and drying of facilities
(Wirtanen et al. 1997, 2002).
4. Ecacy and residue testing methods
Ecacy of disinfectants and antimicrobial agents
are usually determined in free cell suspensions,
which do not mimic the growth conditions on
surfaces where the agents are required to inactivate
the microbes (Frank & Ko 1990; Wirtanen
1995). The agent must reduce the microbial pop-
ulations by 5 log units in suspensions in order to be
considered eective. The goal for reduction of
surface-attached bacteria with disinfectants is 3 log
units (Mosteller & Bishop 1993). The standard
suspension tests have proved suciently reliable
because the variations of results are within
acceptable limits when replication is adequate.
There can, however, be problems with the repeat-
ability and reproducibility of suspension tests
performed with an organic load. It is obvious that
the surface tests are even more dicult to perform
than suspension tests because of the carrier mate-
rial used and the viability of dried cells on the
surfaces (Bloomeld et al. 1994). In developing a
proposal for the testing of disinfectants on surfaces
to an analytical standard, it is important to iden-
tify the major sources of variation in the proce-
dure. Microbes growing or dried on surfaces are
not susceptible to disinfectants from all sides as
they are in suspensions. Due to the requirement
for penetration, disinfectants are thus used in
higher concentrations on surfaces than in suspen-
sions (Mattila-Sandholm & Wirtanen 1992).
4.1. Suspension tests
Determination of disinfectant eciency is often
performed in suspension tests with ready-to-use
dilutions. The European Committee for Stan-
dardization (CEN) has launched many standards.
The microbes used are standard test organisms as
well as spoilage bacteria, pathogens and spores of
297
concern in hygiene. All disinfectants passing the
ecacy test should reduce the number of vegeta-
tive cells by 5 log units and the number of bac-
terial spores by 1 log unit (Wirtanen 1995). The
suspension tests in European Standards are:
in EN 1040:1997 the basic bactericidal activ-
ity of a disinfectant is tested against both
Gram-negative (Pseudomonas aeruginosa)
and Gram-positive bacteria (Staphylococcus
aureus),
in EN 1275:1997 the basic fungicidal activity
of a disinfectant is tested against both yeast
(Candida albicans) and mould (Aspergillus
niger),
in the quantitative suspension test EN
1276:1997 the bactericidal activity of a disin-
fectant for use in food, industrial, domestic
and institutional areas is tested against both
Gram-negative (P. aeruginosa, and Esche-
richia coli) and Gram-positive (S. aureus and
Enterococcus hirae) bacteria (additionally the
following bacteria Salmonella typhimurium,
Lactobacillus brevis and Enterobacter cloacae
can be used if needed) in hard water,
in the quantitative suspension test EN
1650:1997 the fungicidal activity of a disin-
fectant for use in food, industrial, domestic
and institutional areas is tested against both
yeast (C. albicans and if needed Saccharo-
myces cerevisiae can additionally be used) and
mould (A. niger) in hard water,
in the quantitative suspension test EN
1656:2000 the bactericidal activity of a disin-
fectant for use in veterinary eld is tested
against both Gram-negative (P. aeruginosa,
and Proteus vulgaris) and Gram-positive (S.
aureus and Enterococcus hirae) bacteria in
hard water with an organic load of bovine
albumin or a mixture of bovine albumin and
yeast extract or skimmed milk,
in the quantitative suspension test EN
1657:2000 the fungicidal activity of a disin-
fectant for use in veterinary eld is tested
against both yeast (C. albicans) and mould
(A. niger) in hard water with organic load of
bovine albumin or a mixture of bovine albu-
min and yeast extract or skimmed milk,
in the quantitative suspension test prEN
13704:1999 the sporicidal activity of a disin-
fectant for use in food, industrial, domestic
and institutional areas is tested against
298
bacterial spores of Bacillus subtilis (if needed
spores of B. cereus and Clostridium sporogenes
can additionally be used) in hard water,
in EN 1499:1997 the basic activity of hygienic
handwash products is tested against E. coli on
test persons hands,
in EN 1500:1997 the basic activity of hygienic
handrub products is tested against E. coli on
test persons hands.
The activity of disinfectants is at VTT Biotech-
nology tested using a Dutch 555-suspension test
protocol. The 555-suspension test is performed to
nd out the bactericidal, fungicidal and sporicidal
activity of the disinfectant. The activity is mea-
sured after a challenging time of 5 min. The
product has both bactericidal and fungicidal
activity if the microbial reduction is at least 5 log-
units for vegetative cells and sporicidal activity if
the reduction of spores is at least 1 log-unit. The
microbes used are Salmonella Choleraesuis,
P. aeruginosa, S. aureus, B. cereus (spores) and
S. cerevisiae and the test is carried out using
bovine albumin as the organic load. In a modied
555-suspension test the disinfectant is tested
against a chosen panel of process contaminants
(consisting of bacteria, yeasts and/or moulds) in
bovine albumin solution (Wirtanen 1995;
Wirtanen & Juvonen 2002).
4.2. Tests using biolms
Various surface tests have shown that surface-at-
tached cells are more resistant to disinfectant
treatment than are cells in suspension (Wirtanen
1995; Wirtanen et al. 1997). Results obtained using
only one assessment method in testing can be
inaccurate. For example, cultivation and CTC-
DAPI staining in a comparison based on biolms
showed underestimation of viable bacteria in the
cultivation (Wirtanen et al. 1997). Microscopy
techniques have often been used as a reference
method for cultivation based on swabbing. It has
been reported that counting cells by direct
microscopy consistently give a result at least one
log unit higher than the cultivation method (Holah
et al. 1988). This may be because the bacteria do
not grow under the conditions provided in the plate
count method or that large numbers of bacteria
remain on the surfaces after swabbing (Holah
1992). In our experiments, epiuorescence micros-
Figure 1. Diagram of the biolm-based disinfectant test. Bio-
lm is grown on test coupons using an inoculated lter paper
placed on top of a suitable nutrient agar (Charaf et al. 1999).
copy clearly revealed that even vigorous swabbing
detached only a small portion of the actual biolm
and the cells within it (Wirtanen 1995). Therefore
methods used in assessment and in detachment
should both be chosen carefully. One testing pro-
cedure, based on cultivation, image analysis,
impedance and metabolic indicators e.g. CTC-
DAPI staining, seems to give a good estimation of
both removal of biolm from surfaces and killing
of bacteria on surfaces (Wirtanen et al. 1997).
Microbial cells dried on test surfaces have also been
used in disinfectant carrier tests e.g. Draft prEN
13697. The model biolms have many of the
characteristics of wild biolms. The microbial
cells are adhered to test surfaces, they produce
slime and they show increased resistance to disin-
fectants. In response to the need for a relatively
realistic, simple and reliable test for disinfectant
ecacy, Charaf et al. (1999) have developed a
method for culturing laboratory model biolms.
The method involves growing biolm on test cou-
pons using inoculated lter papers placed on top of
a suitable nutrient agar (Figure 1). After the re-
moval of the lter paper, the coupons are subjected
to disinfectant testing as per suspension tests. The
above mentioned methods for disinfectant testing,
however, require further validation.
4.3. Tests with biolm constructs
The poloxamer hydrogels demonstrate thermo-
reversible gelation properties, being liquid and fully
miscible with water at temperatures <15 C, but
rm gels at temperatures >15 C. This means high
cell densities can be cultured within the gels at 30 C
and subsequently exposed to a disinfectant. After
treatment, a full recovery of the individual cells can
be achieved simply by moving the hydrogels into
neutraliser solutions/diluents at <15 C (Wirtanen

Figure 2. Test protocol in ecacy testing of disinfectants using biolm constructs (Wirtanen et al. 1998, 2001, 2003).
et al. 1998, 2001, 2003). Poloxamer F127 is a di-
block co-polymer of polyoxyethylene and poly-
oxypropylene. It has been investigated earlier for its
potential as an agar substitute in microbiology.
Solutions are unaected by autoclaving and appear
to be non-toxic to all the bacterial species so far
tested (Gilbert et al. 1998; Wirtanen et al. 1998,
2001). The poloxamer matrices we have studied not
only reproduce the reaction-diusion resistance
properties of the biolms but also simulate other
aspects of the biolm mode of growth (Figure 2).
The use of articial biolms i.e. biolm-constructs
inoculated with process contaminants in disinfec-
tant testing is suitable for screening the activity of
various disinfectants.
4.4. Microbial based residue test
As mentioned above, chemical cleanliness is also
essential in food plants. Chemically clean surfaces
are surfaces from which undesirable chemical res-
idues have been removed. The total cleanliness of
Figure 3. Test protocol for testing of chemical residues
(Lappalainen 1991; Lappalainen et al. 2003).
299
the process facilities prior to initiating production
is mainly based on measuring the microbial load
using culturing techniques. It is possible that these
results do not indicate the total cleanliness, they
only show that there are no viable microbes on the
surface. It is possible that there are chemical resi-
dues of the cleaning agents and disinfectants left
on the surface. This is not allowed but usually no
tests are run to avoid this. The luminescent bac-
teria light inhibition method can be used to mea-
sure low amounts of residues both in liquids and
on surfaces. The luminescence inhibition method
with the photobacterium Vibrio scheri is a useful
tool to estimate the toxicity of dierent samples,
which is based on the reduction in light output due
to interactions between bacteria and toxic com-
pounds (Lappalainen 2001; Lappalainen et al.
2003). Specied volumes of the test sample or
diluted sample is mixed with the suspension of
luminescent bacteria in a cuvette (Figure 3). The
decrease in light output is measured in a lumi-
nometer after a contact time of 5 min. The values
measured are compared to a control sample and
the changes in intensity are taken into account by
using a correction factor when calculating the re-
sults (Lappalainen et al. 2003).
5. Results of disinfectant ecacy and residues
5.1. Suspensions
Tests carried out at VTT Biotechnology have
shown that microbes in suspensions are easily
300

Table 2. The microbicidal eect of four commercial disinfectants (alcohol-based, hydrogen peroxide-based, hypochlorite-based and
persulphate-based products) against Listeria innocua, L. monocytogenes, E. coli, Salmonella Infantis, S. Choleraesuis and Bacillus
cereus grown in suspension (reduction unit is log cfu/ml)

Disinfectant/Concentration Alcohol- Hydrogen Hypochlorite-based Persulphate Control

based peroxide-based based

100% 75% 1% 0.50% 0.25% 1.40% 0.70% 0.35% 1/1.251

Escherichia coli >6.7 >6.7 >6.7 >6.7 >6.7 >6.7 >6.7 >6.7 >6.7 7.46
Listeria innocua >6.4 >6.4 >6.4 >6.4 >6.4 >6.4 >6.4 >6.4 >6.4 7.11
Listeria monocytogenes >6.3 >6.3 >6.3 >6.3 >6.3 >6.3 4.9 >6.3 >6.3 7.04
Salmonella Choleraesuis >6.5 >6.5 >6.5 >6.5 >6.5 >6.5 >6.5 >6.5 >6.5 7.29
Salmonella Infantis >6.8 >6.8 >6.8 >6.8 >6.8 >6.8 >6.8 >6.8 >6.8 7.67
Bacillus cereus (spores) ND 0.14 0.20 0.26 ND ND ND ND ND 6.04

ND = no microbicidal eect observed.

destroyed, which is in agreement with several
studies from other laboratories (Table 2). LeCh-
evallier et al. (1988) showed that unattached
Gram-negative bacteria in drinking water were
susceptible to chlorine disinfectants whereas at-
tached cells were not. Eagar et al. (1988) reported
that planktonic cells of Pseudomonas uorescens
were more sensitive towards glutaraldehyde than
sessile cells. Best et al. (1990) stressed that the
selection of appropriate disinfectants for use on
contaminated surfaces should be carried out
carefully. They tested the eects of disinfectants on
Listeria monocytogenes in carrier and suspension
tests and found that sodium dichloroisocyanurate
was the only eective solution in the presence of
milk containing 2% fat.
Available reports about the susceptibility of
foodborne yeasts to various chemicals used in the
food industry are sporadic and covers only a few
disinfectants against some spoilage yeasts (Juvo-
nen et al. 2001). McGrath et al. (1991) showed
that ready-to-use concentrations of a hypochlo-
rite disinfectant killed most of the S. cerevisiae
tested in suspension by 1520 min. The asco-
spore-containing cultures of Pichia and Saccha-
romyces species are more resistant to
disinfectants than the vegetative populations
(McGrath et al. 1991). Hypochlorite, peracetic
and phosphoric acid as well as anionic com-
pound in ready-to-use concentrations were eec-
tive against suspended yeasts isolated from
orange juice (Winniczuk & Parish, 1997). Chlo-
rine dioxide appeared to be eective against yeast
and mould contaminants (Han et al. 1999). The
ecacy of various types of disinfectants against
food-spoilage was thoroughly studied using a
modied 5-5-5 suspension tests against yeast
contaminants isolated from various food pro-
cessing environments. The results of the suspen-
sion tests (Table 3) showed that the alcohol,
peroxide and tenside based disinfectants were
ecient. The disinfectants containing chlorine
and persulphate did not destroy suspended yeast
cells (Wirtanen & Juvonen 2002).
5.2. Biolms
The microbicidal eect of the four commercial
disinfectants (hydrogen peroxide-based, alcohol-
based, persulphate-based and hypochlorite-based
products) were also tested using biolms of six
dierent bacteria (L. innocua, L. monocytogenes,
E. coli, S. Infantis, S. Choleraesuis and B. cereus).
The bacteria were allowed to form biolms on the
surface of steel coupons (Figure 1) according to
the method described by Charaf et al. (1999).
When the results of the suspension test and the
biolm test were compared, the protection of the
biolm against the disinfectants was shown clear-
ly. In the suspension tests all disinfectants had a
sucient microbicidal eect towards all vegetative
bacterial types, but in the biolm tests the micro-
bicidal eects of the same disinfectants was lower.
Only the alcohol-based and the hydrogen perox-
ide-based agents were ecient enough giving a log-
reduction greater than three for most vegetative
bacteria tested. Spore forming bacteria were also
used in the biolm test. The test showed (Table 4)
that the biolm protects also spores against the
disinfectants.
Table 3. The in-use concentrations of alcohol-based (A1-A3), hydrogen peroxide-based (H1-H3), chlorine-based (C1-C2), tenside-based (T1-T2) and persulphate-based (S1)
disinfectants needed to kill various yeast isolates obtained from dierent food processes. The disinfectant ecacy was tested using a modied 5-5-5 suspension test using
Saccharomyces servazzii, S. cerevisiae, Zygosaccharomyces rouxii, Candida krusei, C. lambica, C. lipolytica, C. boidinii, C. intermedia, C. parapsilosis, Cryptococcus albidus,
Debaromyces hansenii, Dekkera anomala, Rhodotorula glutinis, R. rubescens and R. mucilaginosa (Wirtanen & Juvonen 2002)

Yeast Control Disinfectant treatmentsa

A1b A2 A3 H1 H2 H3 C1 C2 Tl T2 S1

S. servazzii C-00362 4.75.1 100% 75% 75% 0.5% 1.0% 0.3% 0.3% 1.0% 0.2% 2%
S. cerevisiae C-00370 5.86.4 100% 75% 75% 0.5% 0.25% 1.3% 1.0% ME 4.3 2% 2%
S. cerevisiae C-96203 5.05.6 100% 75% 75% 0.25% 0.25% 0.3% 0.7% 1.0% 0.2% 2%
Z. rouxii C-00363 5.96.1 100% 75% 75% 0.25% 0.25% 0.3% ME 3.7 0.1 % 0.2% 2%
Z. rouxii C-00367 4.64.7; 6.16.3 100% 75% 75% 0.25% 0.25% 0.3% 0.3% 1.0% 0.2% 2%
Z. rouxii C-95218 4.65.1 100% 75% 75% 0.25% 0.25% 0.3% 0.3% 1.0% 0.2% 4%
C. lambica C-00365 5.66.0 ME 2.0 75% 75% ME 1.4 0.25% 0.3% 0.7% ME 4.0 2% 2%
C. lipolytica C-00365 5.05.7; 6.26.5 100% 75% 75% ME 1.5 ME 3.6 ME 2.2 ME 2.1 ME 2.8 0.2% 2%
C. lipolytica C-00380 4.95.1; 6.7 100% 75% 75% 1.0% 0.25% 1.3% 1.0% ME 3.1 0.2% 2% ME 1.1
C. boidinii C-00366 5.76.1; 6.56.6 100% 75% 75% 0.25% 0.25% 0.3% 0.7% ME 4.3 0.2% 2%
C. intermedia C-00372 6.36.5 100% 75% 75% 1.0% 0.25% ME 2.8 1.0% 1.0% 2% 2%
C. parapsilosis C-00373 6.06.7 100% 75% 75% 1.0% 0.25% 1.3% ME 1.3 ME 3.5 2% 2%
C. parapsilosis C-00381 5.96.4 100% 75% 75% 1.0% 0.25% ME 3.3 1.0% ME 1.7 2% 2% ME 0.9
C. krusei C-00371 5.96.1 100% 75% 75% 0.5% 0.25% 0.3% ME 3.7 ME 3.0 ME 4.4 ME 3.7
C. albidus C-00392 4.95.4 100% 75% 75% 0.25% 0.25% 0.3% 0.7% ME 2.3 0.2% 2%
C. albidus C-00397 4.65.2 100% 75% 75% 0.25% 0.25% 0.3% 0.3% ME 2.7 0.2% 2%
D. hansenii C-00382 5.36.1 100% 75% 75% 0.25% 0.25% 0.3% 0.3% ME 3.9 0.2% 2% 1 tab/1.25 l
R. glutinis C-00391 5.3-6.0 100% 75% 75% 0.25% 0.25% 0.3% 0.3% 1.0% 0.2% 2%
R. rubescens C-00393 5.76.0 100% 75% 75% 0.25% 0.25 % 0.3% ME 3.0 ME 3.0 2% 2%
R. mucilaginosa C-00396 5.86.1 ME 2.7 75% 75% 0.25% 0.25% 1.3% ME 2.5 0.2% 2%
D. anomala C-91183 6.37.0 100% 75% 75% 0.25% 0.25% 0.3% 0.3% 1.0% 0.2% 2% ME 3.5
R. mucilaginosa 5.66.1 100% 75% 75% 0.5% 0.25% 1.3% ME 4.4 0.2% 2%
a
The percentage given is the lowest in-use concentration of the agent tested,which kills the yeast tested; if a microbial eect (ME) value is given in a shadowed cell the highest
concentration of the agent tested is given (ME unit is log cfu/ml).
b
The agent A1 was tested only as undiluted (recommended concentration).
301
302

<-20 100.0
3% -20 - 0
10 %
50.0
50 - 100
38 %
0.0

Inh/ %
0 - 20
21 %
0 100 200 300 400 500
-50.0

-100.0

20 - 50 -150.0
28 %

Figure 4. Residue test results of 501 dairy samples. The results are divided in ve groups according to the inhibition result. The
distribution of all results shows clearly that there are residues left on the surfaces in about 40% of the samples, in which the inhibition
was greater than 50% (Lappalainen et al. 2003).

Abundant growth of unwanted yeast during
production can lead to defects in the nal products
and, furthermore, problems in process hygiene
(McGrath et al. 1991; Winniczuk & Parish 1997;
Han et al. 1999; Juvonen et al. 2001). Low pH,
high sugar or salt content favours the growth of
yeast. Spoiling yeast takes part in the deterioration
of syrups, pralines, jams, berries and fruits, fruit
juices, pickled vegetables and dairy products. In
many cases the risk caused by growth of spoilage
yeast in products has been underestimated because
many of these yeast are not known to be oppor-
tunistic pathogens (Wirtanen & Juvonen 2002).
The yeast also readily form biolms on process
surfaces at both low and elevated temperatures.
Therefore, the spoilage yeast can be a hygiene risk,
when failures occur in the cleaning and disinfec-
tion procedure. The aim of our study was to assess
the ecacy of various types of disinfectants
against yeast isolated from various food processes
growing on surfaces (Charaf et al. 1999). The re-
sults of the surface test showed that the alcohol-
based agent was the most ecient disinfectant
against the yeasts tested (Table 5). After 5 min

Table 4. The microbicidal eect of four commercial disinfectants (alcohol-based, hydrogen peroxide-based, hypochlorite-based and
persulphate-based products) against biolms of Listeria innocua, L. monocytogenes, E. coli, Salmonella Infantis, S. Choleraesuis and
Bacillus cereus (reduction unit is log cfu/cm2)

Disinfec- Alcohol-based Hydrogen peroxide-based Hypochlorite- Persul- Control

tant/Con- based phate
centration based
100% 75% 1% 0.50% 0.25% 1.40% 0.70% 0.35% 1/1.251

Escheri- >5.07 >5.07 429 [0.80] 2.81 1.44 2.11 0.44 0.29 1.54 5.39
chia coli [0.00] [0.00] [2.10] [3.18] [0.84] [0.77] [0.65] [0.29] [0.51]
Listeria >2.99 >2.99 >2.99 >2.99 >2.99 >299 1.76 2.68 2.35 3.32
innocua [0.00] [0.00] [0.00] [0.00] [0.00] [0.00] [0.63] [0.55] [1.11] [0.37]
Listeria >5.30 >5.30 >5.30 4.72 4.20 0.47 0.91 1.19 0.00 5.62
monocyto- [0.00] [0.00] [0.00] [1.01] [1.91] [0.99] [0.24] [0.08] [0.00] [0.00]
genes
Salmonella >3.14 >3.14 1.81 2.47 1.80 1.98 1.81 2.22 0.58 3.47
Cholerae- [0.00] [0.00] [1.20] [1.17] [1.31] [2.02] [1.26] [0.67] [1.15] [0.32]
suis
Salmonella >6.06 >6.06 5.09 4.30 5.48 1.55 0.34 0.43 1.61 6.38
Infantis [0.00] [0.00] [1.68] [1.95] [1.01] [0.52] [0.15] [0.06] [0.13] [0.25]
Bacillus ND ND ND ND ND ND ND ND 0.36 5.21
cereus [0.23] [0.59]
(spores)

ND = no microbicidal eect observed.

 303

treatment the hydrogen peroxide-based disinfec-
tant was also eective in some cases and especially
when the duration of the disinfectant treatment
was extended to 15 min the peroxide-based and
quaternary ammonium containing disinfectants
were also ecient (Table 5). The disinfectants
containing chlorine and persulphate were ineec-
tive in destroying yeast biolms.
5.3. Biolm constructs
The biolm construct test is a severe measure of
disinfection eciency giving reproducible results.
Whilst the results do not necessarily reect the
likely eects of a formulation against microbial
contamination in situ, they enable discrimination
between the disinfectant formulations at normal
use levels and the choice of the most eective one.
The survival levels obtained made the test able to
distinguish the performances of many dierent
disinfectant formulations against a variety of test
strains where this had been impossible with con-
ventional testing methods (Wirtanen et al. 1998,
2001, 2003). Conventional suspension tests fail to
discriminate between the agents in terms of their
ecacy and are therefore, not of assistance in the
nal selection of agents (Gilbert et al. 1998; Wir-
tanen et al. 1998). The results showed that Gram-
negative bacteria are more resistant to disinfectant

Table 5. Ecacy of disinfectant treatments on yeast biolms grown on stainless steel after 5 min (upper table) and 15 min (lower
table): The yeast isolates used were Saccharomyces cerevisiae, Candida lipolytica, C. intermedia, C. parapsilosis, C. krusei, Cryptococcus
albidus, Debaromyces hansenii, Rhodotorula glutinis, R. rubescens; R. Mucilaginosa, Dekkera anomala and Trichosporon asahii

Yeasts Disinfectant treatments

Alcohol-based Hydrogen Hypochlor- Quaternary Persul-

peroxide-based ite-based ammonium phate-
based

Concentration 100% 75% 50% 1.0% 0.5% 0.25% 1.4% 0.7% 4% 2% 0.2% 1 tab/
1.25 l

S. cerevisiae C-00370 ND ND ND ND ND ND ND ND ND
S. cerevisiae C-96203 ND ND ND ND ND ND
C. lipolytica C-00380 ND ND ND ND
C. intermedia C-00372 ND ND ND ND ND ND ND ND ND
C. parapsilosis C-00373 ND ND ND ND ND ND ND ND
C. parapsilosis C-00381 ND ND ND ND ND ND ND ND
C. krusei C-00371 ND ND ND ND ND
C. albidus C-00392 ND ND ND ND ND ND ND ND ND
D. hansenii C-00382 ND ND ND ND ND ND
R. glutinis C-00391 ND ND ND ND ND
R. rubescens C-00393
R. mucilaginosa C-00396 ND ND ND ND
D. anomala C-91183 ND ND ND
T. asahii G-10 ND ND ND ND ND ND ND

S. cerevisiae C-00370 ND ND ND
C. lipolytica C-00380 ND ND

C. intermedia C-00372 ND ND ND ND ND ND
C. parapsilosis C-00373 ND ND ND ND ND
C. parapsilosis C-00381 ND ND ND ND
C. albidus C-00392 ND ND ND ND ND ND ND
R. mucilaginosa C-00396 NO ND ND ND ND ND

Reduction greater than 3 log-units; h Reduction below 3 log-units but exceeding 1.5 log-units; ND reduction lower than 1.5
log-units; Not tested.
304
treatments than Gram-positive bacteria. This is in
agreement with the general observations (Nikaido
& Vaara 1985; McKane & Kandel 1996) as well as
studies using surfaces with dried bacterial cells
(Gronholm et al. 1999) and biolms (Wirtanen
1995; Wirtanen et al. 1997, 2002).
5.4. Microbial based residue test
The method based on photobacteria is rapid to
perform, because the incubation time is only
5 min. The results show that residues do exist on
production surfaces prior to production start-up
(Figure 4). Most of the samples taken in dairies
were swab samples because of ease of sampling.
There were in total 501 samples from dierent
countries. The samples from factories can be di-
vided into three groups: samples with very clear
inhibition (inhibition percentage >50), samples
with moderate inhibition (inhibition percentage
between 20 and 50%) and samples that induced
light production (inhibition value negative).
About 40% of the samples showed a clear inhi-
bition. In other words, this means that there are
residues on surfaces before starting the produc-
tion if no preventive rinsing is performed. This
method oers a useful alternative for testing
chemical residue left on surfaces after cleaning
and disinfection.
6. Conclusions
Disinfection is required in food plant operations
where wet surfaces provide favourable conditions
for the growth of microbes. The use of eective
disinfectants minimizes contamination of the
product, enhances shelf life, and reduces the risks
of foodborne illness. A prolonged exposure of the
surfaces to disinfectants enhances the microbicidal
eect. Disinfectants approved for use in the food
industry are alcohols, oxidants, iodophor- and
chlorine-based compounds, persulphates, surfac-
tants and quaternary ammonium compounds.
The ecacy testing based on suspensions gives
an answer to whether the disinfectant is eective
against the microbe tested. According to the
standard tests available a reduction of 5 log-units
is needed for the agent to be eective against
vegetative microbial cells and 1 log-unit for bac-
terial spores. Various laboratory studies have
shown that surface-attached cells are more resis-
tant to disinfectant treatment than suspended cells.
This has lead to development of various types of
carrier tests, e.g. tests using microbial cells dried
on surfaces, biolm-constructs, and biolms
grown on test coupons. The above mentioned
methods require further validation and improved
repeatability.
Research has shown that there can be chemical
residues on the surfaces prior to production. The
results from using a photobacterial method can be
categorized into the following levels: very clear,
moderate and no inhibition. This method oers a
useful alternative for testing chemical residue on
surfaces and is worthy of further study.
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