Vol. 13  No. 1/2019
© Inštitut za sanitarno inženirstvo, 2019
International Journal of Sanitary Engineering Research4
Biofilm formation capacity of 
Bacillus cereus on silicone, 
polyethylene terephthalate, 
Teflon, and aluminium food 
contact materials
1 Department of Sanitary engineering, 
University of Ljubljana Faculty of Health 
Sciences, Zdravstvena pot 5,  
1000 Ljubljana, Slovenia
*	Corresponding author
Assist. Prof. Dr. Rok Fink 
University of Ljubljana 
Faculty of Health Sciences 
Zdravstvena pot 5 
1000 Ljubljana 
E-mail: rok.fink@zf.uni-lj.si
Martina ODER1, Rok FINK1*
ABSTRACT
Biofilms on food contact materials represent public health issues because they 
are resistant to cleaning and disinfection. This study aims to assess the Bacillus 
cereus biofilm formation capacity on silicone, polyethylene terephthalate, 
Teflon, and aluminium food contact materials. The biofilm biomass was 
analysed with the crystal violet assay method. We used the standard strain B. 
cereus CCM 2010, wild strain B. cereus 100 and spores of those two strains. 
The results show that both the vegetative form the bacteria and it spores form 
large amounts of biofilm on silicone, followed by polyethylene terephthalate, 
Teflon, and aluminium. More detailed analysis has shown that spores form 
more biomass on all materials in comparison to the vegetative form and that 
the standard strains form low levels of biofilm in contrast to the wild strains. 
Selecting proper material with the lowest biofilm formation potential can 
prevent or reduce food contamination and consequently increase food safety. 
Key words: biofilm; Bacillus cereus; food contact materials
POVZETEK
Biofilmi na kontaktnih površinah predstavljajo pomemben javno zdravstveni 
izziv, saj so bolj odporni na čiščenje in dezinfekcijo kot planktonske celice. 
Namen raziskave je bil ovrednotiti količino biofilma na materialih za stik z živili, 
kot so silikon, polietilen tereftalat, Teflon in aluminij. Količina biomase biofilma 
na površini je bila ocenjena z metodo kristal vijolično. V raziskavi smo uporabili 
standardni sev B. cereus CCM 2010, divji sev B. cereus 100 in spore obeh 
omenjenih sevov. Rezultati kažejo, da tako vegetativna oblika, kot spore obeh 
sevov tvorijo velike količine biofilma na silikonu, sledi mu polietilen tereftalat, 
teflon in aluminij. Bolj natančna analiza kaže, da spore tvorijo več biomase na 
vseh materialih v primerjavi z vegetativno obliko ter da standardni sev B. cereus 
tvori manj biofilma v primerjavi z divjim sevom. Izbira primernega materiala z 
najmanjšim možnim potencialom za nastanek biofilmov lahko zmanjša ali 
prepreči kontaminacijo živil in posledično izboljša varnost.
Ključne besede: biofilm; Bacillus cereus; materiali za stik z živili 
 Original scientific article
Received: 26. 06. 2019 
Accepted: 30. 12. 2019
International Journal of Sanitary Engineering Research Vol. 13  No. 1/2019 5
 
INTRODUCTION
Most household food contact materials are in permanent contact with 
foodstuff; therefore, the probability of acquiring surface contaminants 
from contact materials into the food is high [1]. The contamination of 
food contact surfaces during food handling due to bacteria present in 
foodstuff is one of the main causes of alimentary intoxication [2]. 
Biofilm formation is a biological phenomenon as bacteria tend to live on 
surfaces rather than in a planktonic state. When embedded in a biofilm, 
cells are protected against harsh environmental conditions, such as 
chemicals, physical stresses, and antimicrobial agents, because their 
exopolysaccharide matrices act as protective barriers that limit 
penetration into the biofilm [3]. Recent foodborne outbreaks have 
focused on biofilms on food contact materials, examining the sources of 
food contamination [4]. The most commonly used materials in 
household environments are wood, ceramics, glass, different types of 
metals, silicones, Teflon, and polyethylene terephthalate [5]. Those 
materials are used for kitchenware, such as bottles, jars, tubs, models 
for baking and freezing, pastry brushes, lids, pots, pans, containers, 
wrappings, baking sheet, milk jugs and others. B. cereus is a gram-
positive microorganism which can form spores under harsh 
environmental conditions. They are pathogenic, facultative anaerobic 
bacteria that produce toxins. Some vegetative strains are harmful to 
humans and cause foodborne illness, including nausea, vomiting, and 
diarrhoea [6]. B. cereus is a pathogenic bacterium that is frequently 
found in various types of raw and cooked foods, and its ability to 
survive high cooking temperatures requires that cooked foods be served 
hot or cooled rapidly to prevent the growth of this bacterium [7]. 
Because of its ability to form highly resistant spores and its natural 
spread in the wild, B. cereus is a major food safety concern. The spores 
are common in soil and spread easily to cows’ udders and from there to 
the raw milk. In addition to the ability to survive pasteurization, they 
also attach very well to most household materials [8] from which they 
can spread throughout the kitchen environment. It is well known that B. 
cereus in vegetative cells or spores tends to adhere to rough surfaces 
[9, 10]. One reason for this can be the presence of appendages, 
proteins, polysaccharides, and lipids that allow attaching and 
consequently forming the biofilm [11]. Moreover, some authors have 
reported that the surface energy of B. cereus, which is highly 
hydrophobic, is able to adhere firmly to various materials such as those 
found during food processing in household environments [12]. A more 
specific study by Ekman et al. [13] demonstrated a transfer of B. cereus 
from paper surfaces to foods. Similarly, Le Gentil et al. [14] analysed 
the attachment and detachment of B. cereus in cleaning processes and 
found that re-attachment can be a reason for surface contamination. 
Furthermore, Fink et al. [6] reported that the removal of B. cereus from 
polyurethane conveyor belts with industrial cleaning agent is difficult if 
not impossible. The persistence of microbial biofilms represents a 
significant challenge to the establishment and maintenance of hygienic 
conditions in different environments. The possibility of bacterial 
The contamination of food 
contact surfaces during food 
handling due to bacteria 
present in foodstuff is one of 
the main causes of 
alimentary intoxication.
B. cereus is a pathogenic 
bacterium that is frequently 
found in various types of raw 
and cooked foods, and its 
ability to survive high cooking 
temperatures requires that 
cooked foods be served hot 
or cooled rapidly to prevent 
the growth of this bacterium.
 M. Oder, R. FinkBiofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
© Inštitut za sanitarno inženirstvo, 20196

multiplication in foods after storage and/or handling must be taken into 
account when defining safe levels for human consumption [15]. The 
objective of this study was to analyse the capacities of B. cereus biofilm 
formation on silicone, polyethylene terephthalate, Teflon, and aluminium 
food contact materials and to provide consumer information on material 
hygiene. 
METHODS
Bacteria, growth and sporulation media
In the experiment, wild strain B. cereus 100 (isolated from milk and 
kept at the University of Ljubljana, Faculty of Health Sciences), standard 
strain B. Cereus CCM 2010 (Czech Collection of Microorganisms, Brno, 
Czech Republic), and spores of these two strains were used. 
Methods
In this study, four different food contact materials that are often used in 
the home kitchen environment were tested for B. cereus biofilm 
formation: aluminium, silicon, Teflon, and poliethylenetherephalate 
(PET). The materials were cut into the coupons of 10 × 10 mm, which 
were washed with 98% ethanol (Sigma-Aldrich, Misuri, ZDA) and 
destilled water and dried before being autoclaved. An Olympus CX40 
optical microscope with an off-the-bench illuminator and CCD CMOS 
camera (Camera Digital microscope Electronic Eyepiece for Image) was 
used to visualize the structures of the materials (Figure 1). The surface 
roughness of the selected material was determined by mechanical 
profilometer Form Talysurf Series 2 from Taylor-Hobson Ltd., Leicester, 
Great Britain. 
Determining the biofilm biomass formation capacity
To determine the biofilm’s biomass formation, a modified method by 
Bohinc et al. [1] and Kubota et al. [16] was used. Staining biofilm 
biomass remains a useful baseline technique to provide a practical, 
inexpensive, and reliable method for the detection of biofilms [17]. 
Bacteria from the collection were transferred on the nutrient agar and 
incubated at 37 °C 24h. After that, a single colony of strain was 
transferred from the nutrient agar to the nutrient broth (Biolife, Italy) 
and incubated under the same conditions. Next, the bacterial culture 
was diluted in a 1:300 ratio, with fresh nutrient broth. Sterile coupons 
were transferred in a sterile petri dish and exposed to the bacterial 
suspension; 4 mL of the nutrient broth with bacterial cultures in a ratio 
of 1:300 was added. The bacterial suspension and coupons were 
incubated for 24 hours at the temperature of 37 °C. After the incubation 
time, the bacterial suspension was removed and the coupons were 
rinsed three times with phosphate buffered saline (PBS) (80 g of NaCl, 
2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4 in 1 L) to remove unattached 
or loosely attached cells. The coupons with adhered bacterial cells were 
exposed to 3 mL 0.1% (w/v) crystal violet suspension (Merck, Germany) 
for 5 min. Then the coupons were rinsed three times with the PBS 
Four different food contact 
materials that are often used 
in the home kitchen 
environment were tested for 
B. cereus biofilm formation: 
aluminium, silicon, Teflon, 
and poliethylenetherephalate 
(PET).
Staining biofilm biomass 
remains a useful baseline 
technique to provide a 
practical, inexpensive, and 
reliable method for the 
detection of biofilms.
M. Oder, R. Fink Biofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
International Journal of Sanitary Engineering Research Vol. 13  No. 1/2019 7
 
buffer to remove excess dye. In the next step, the dye was extracted 
from the cells with 200 µL 96% ethanol. The optical density (OD) of 
the ethanol/dye solution was measured with an Infinite 200® PRO 
microplate reader (Tecan, Austria, GmbH) at the wavelength of 620 nm 
(Figure 1). 
Figure 1. 
B. cereus biofilm capacity assessment 
process flowchart.All the experiments were performed with five parallels and three 
repetitions. For assay of the spores biofilm, the sporulation Casein-
Casein-Yeast (CCY) medium (Sigma-Aldrich, USA) was used. The 
method of spore production was introduced by Abbas et al. [18] and 
modified as follows. To obtain spores form vegetative cells, both 
bacterial strains were incubated in a CCY medium for 24 hours. In the 
next step, bacterial culture was centrifuged with 4000 × g for 10 
minutes to separate the cells from the liquid medium. The cells were re-
suspended with a PBS buffer. The process was repeated three times to 
remove the entire liquid medium. At the final step of the culture process, 
the suspension was exposed to 80 °C for 10 min to destroy the 
 M. Oder, R. FinkBiofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
© Inštitut za sanitarno inženirstvo, 20198

remaining vegetative cells. To determine the quantity of B. Cereus 
spores biofilm, the same procedure as for the vegetative form of B. 
cereus described above was used.
Statistical analysis was provided using R software version 3.1.3 and a 
Student’s t-test comparing the OD of crystal violet dye released from 
the biofilm regarding the form and material. The statistical significance 
was set to p < 0.05.
RESULTS AND DISCUSSION 
Food contact materials are the main source of alimentary intoxication in 
the domestic environment. Several studies indicate that the materials of 
kitchen accessories (e.g. cutlery, knives, and chopping boards) 
represent a high risk for bacterial cross-contamination [19]. The results 
of material characterization show that PET has the highest roughness of 
1.2 µm, followed by silicone with 0.9 µm, Teflon 0.4 µm and aluminium 
with 0.2 µm. The results show that B. Cereus standard and wild 
strains, the vegetative form, and spores grow on all analysed food 
contact materials. The results show the least biofilm biomass on 
aluminium surfaces and the highest amounts on silicone (Figure 2). 
Furthermore, the biofilm formation capacity for standard strain B. 
cereus CCM 2010 initially inoculated from vegetative cells shows, on 
average, the highest biofilm capacity for silicone, followed by PET, 
Teflon, and aluminium (Figure 2a). Similar results can be obtained for 
standard strain B. cereus CCM 2010 inoculated from spores, for which 
abundant biofilm formation was found on silicone, but the fewest spores 
on aluminium (Figure 2b). The wild strain of B. cereus 100 vegetative 
cells formed high biofilm biomass on silicone, PET, aluminium but 
much less biomass was found on Teflon (Figure 2c). Complementary to 
that, B. cereus 100 wild strain biofilm inoculated from spores show the 
highest biofilm formation on silicone, followed by PET and aluminium. 
The lowest amount of biofilm inoculated from spores was found on 
Teflon (Figure 2d). This demonstrates that, generally (apart from PET), 
total amounts of biofilm biomass correspond to material roughness. It is 
generally accepted that the smoother the surface is, the lower the 
number of adhered cells is present [1, 20]. More importantly, this study 
indicates that a significant difference in total biofilm biomass exists 
when comparing material, bacterial strain, and form (vegetative form or 
spores).
Shaheen et al. [21] studied adhesion potential of different strains of B. 
cereus and found that spores adhere to the surface more firmly than 
vegetative cells do. Similar results were presented by Kolari et al. [22], 
who reported that hydrophobic spores of B. cereus are the most 
adhesive, one reason for which can be that strong adhesion makes 
favourable conditions for the spread of spores with rinse water from one 
location to another. Exosporium plays a significant role in spore 
interaction with materials, probably by providing a larger contact 
surface with materials. Kumariand Sarkar [23] reported that the strong 
adhesion potential of B. cereus spores has been attributed to the 
Several studies indicate that 
the materials of kitchen 
accessories (e.g. cutlery, 
knives, and chopping boards) 
represent a high risk for 
bacterial cross-contamination.
B. cereus 100 wild strain 
biofilm inoculated from 
spores show the highest 
biofilm formation on silicone, 
followed by PET and 
aluminium. The lowest 
amount of biofilm inoculated 
from spores was found on 
Teflon.
M. Oder, R. Fink Biofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
International Journal of Sanitary Engineering Research Vol. 13  No. 1/2019 9
 
hydrophobic character of exosporium, which varies between strains. We 
also found that wild strain B. cereus biofilm causes more biomass 
growth on all material in comparison to the standard strain. Comparable 
to our study, Hayrapetyan et al. [3] analysed standard and the 
undomesticated food isolate strain B. cereus and found significant 
differences in OD after 24 hours of incubation on stainless steel 
surfaces. Similar to that, other researchers [24, 25] reported that the 
amounts of biofilm biomass can vary between the strains of the same 
species.
Comparison of optical densities of released crystal violet dye from 
biofilm biomass reveals statistically significant higher optical densities 
for biofilm inoculated from spores on all materials and both strains 
(p < 0.05). The most abundant differences between biofilm inoculated 
from vegetative form and spores can be observed for silicone, in the 
case of both strains (ΔOD B. cereus CCM 2010 = 0.1317; ΔOD B. 
cereus 100 = 0.1220). In contrast, the smallest difference between 
biofilm inoculated from vegetative form and spores was found for Teflon 
when comparing the standard strain B. cereus CCM 2010 (ΔOD = 
0.521) and the wild strain B. cereus 100 (ΔOD = 0.068) (Table 1).
Figure 2. 
Optical densities (mean, quartiles, min 
and max) of released crystal violet dye 
from B. cereus biofilm inoculated from 
vegetative form (a, b) and spores (c, d) 
on silicone, PET, Teflon, and 
aluminium.
Wild strain B. cereus biofilm 
causes more biomass growth 
on all material in comparison 
to the standard strain.
 M. Oder, R. FinkBiofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
© Inštitut za sanitarno inženirstvo, 201910

Table 1. Comparison of optical densities of crystal violet dye released from B. cereus biofilm inoculated from vegetative 
form and spores on silicone, PET, teflon, and aluminium. 
Material B. cereus
OD620 vegetative 
form
OD620 spores Δ OD (/) t-value p-value
Silicone
Standard 
strain
CCM 2010
0.0810 0.2127 0.1317 11.925 0.000008**
PET 0.0712 0.1438 0.0726 10.367 0.000006**
Teflon 0.0188 0.0709 0.0521 23.969 <0.000000**
Aluminium 0.0163 0.0805 0.0642 7.396 0.000049**
Silicone
Wilde strain
100
0.0778 0.1998 0.1220 9.278 0.000003**
PET 0.0921 0.1625 0.0704 8.137 0.000005**
Teflon 0.0280 0.0968 0.0688 34.32 <0.000000**
Aluminium 0.0613 0.1343 0.0730 4.085 0.001805*
Legend: * p<0.05; **p<0.000
CONCLUSIONS 
The selection of proper material with the lowest adhesion potential, 
along with cleaning procedures and good hygiene behaviour, represents 
the primary strategy for decreasing the risks of food poisoning in 
household environments. The results of our study demonstrated that 
aluminium and Teflon have much lower biofilm capacity in comparison 
to others. Moreover, the results of our study indicate that biofilm 
biomass formation depends not only on material properties but also on 
bacterial strain and form. By understanding the relationship between 
material surface properties and bacterial adhesion, strategies can be 
developed that would greatly inhibit, if not prevent, biofilm growth in 
domestic environments. 
REFERENCES
 [1] Bohinc K, Jevšnik M, Fink R, Dražič G. Raspor P. Surface characteristics 
dictate microbial adhesion ability. V: Prokopovich P. ed. Biological and 
pharmaceutical applications of nanomaterials. Forida: CRC Press; 2015. 
p. 193-214. 
 [2] Fink R. Higienically Relevant Biofilms. New York: Nova Science Publishers; 
2015.
 [3] Hayrapetyan H, Muller L, Tempelaars M, Abee T, Nierop Groot M. 
Comparative analysis of biofilm formation by Bacillus cereus reference 
strains and undomesticated food isolates and the effect of free iron. Int J 
Food Microbiol. 2015;200:72-9.
 [4] Wang H, Ding S, Wang G, Xu X, Zhou G. In situ characterization and 
analysis of Salmonella biofilm formation under meat processing 
environments using a combined microscopic and spectroscopic 
approach. Int J Food Microbiol. 2013;167(3):293-302. 
 [5] Al Meslmani BM, Mahmoud GF, Leichtweiß T, Strehlow B, Sommer FO, 
Lohoff MD, Bakowsky U. Covalent immobilization of lysozyme onto 
woven and knitted crimped polyethylene terephthalate grafts to minimize 
the adhesion of broad spectrum pathogens. Mat Sci Eng. 2016;58:78-
87. 
 [6] Fink R, Oder M, Stražar E, Filip S. Efficacy of cleaning methods for the 
removal of Bacillus cereus biofilm from polyurethane conveyor belts in 
bakeries. Food Control. 2017;80:267-72. 
The selection of proper 
material with the lowest 
adhesion potential, along 
with cleaning procedures and 
good hygiene behaviour, 
represents the primary 
strategy for decreasing the 
risks of food poisoning in 
household environments.
Biofilm biomass formation 
depends not only on material 
properties but also on 
bacterial strain and form.
M. Oder, R. Fink Biofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...
International Journal of Sanitary Engineering Research Vol. 13  No. 1/2019 11
 
 [7] El-Arabi TF, Griffiths MW. Bacillus cereus. V: Glenn Morris J Jr., Potter 
ME, eds. Foodborne infections and intoxications. San Diego: Academic 
Press; 2013. p. 401-07. 
 [8] Tewari A, Abdullah S. Bacillus cereus food poisoning: international and 
Indian perspective. J Food Sci Technol. 2014;52(5):2500-11. 
 [9] Elhariry HM. Attachment strength and biofilm forming ability of Bacillus 
cereus on green-leafy vegetables: Cabbage and lettuce. Food Microbiol. 
2011;28(7):1266-74. 
 [10] Lemos M, Gomes I, Mergulhao F, Melo L,Simoes M. The effects of 
surface type on the removal of Bacillus cereus and Pseudomonas 
fluorescens single and dual species biofilms. Food Bioprod Process. 
2015;93:234-41.
 [11] Tauveron G, Slomianny C, Henry C, Faille C. Variability among Bacillus 
cereus strains in spore surface properties and influence on their ability 
to contaminate food surface equipment. Int J Food Microbiol. 
2006;110(3):254-62. 
 [12] Peng JS, Tsai WC, Chou CC. Surface characteristics of Bacillus cereus 
and its adhesion to stainless steel. Int J Food Microbiol. 2001;65(1–2): 
105-11. 
 [13] Ekman J, Tsitko I, Weber A, Nielsen-LeRoux C, Lereclus D, Salkinoja-
Salonen M. Transfer of Bacillus cereus spores from packaging paper into 
food. J Food Protect. 2009;72(11):2236-42. 
 [14] Le Gentil C, Sylla Y, Faille C. Bacterial re-contamination of surfaces of 
food processing lines during cleaning in place procedures. J Food Eng. 
2010;96(1):37-42. 
 [15] European Food Safety Authority (EFSA). Panel on Biological Hazards. 
Risks for public health related to the presence of Bacillus cereus and 
other Bacillus spp. including Bacillus thuringiensis in foodstuffs. EFSA 
Journal; 2016.
 [16] Kubota H, Senda S, Nomura N, Tokuda H, Uchiyama H. Biofilm 
formation by lactic acid bacteria and resistance to environmental stress. 
J BiosciBioeng. 2008;106(4):381-6. 
 [17] Azeredo J, Azevedo NF, Briandet R et al. Critical review on biofilm 
methods. Crit Rev Microbiol. 2017;43(3):313-51. 
 [18] Abbas AA, Planchon S, Jobin M, Schmitt P. A new chemically defined 
medium for the growth and sporulation of Bacillus cereus strains in 
anaerobiosis. J Microbiol Meth. 2014;105:54-8. 
 [19] Giaouris EE, Simoes MV. Pathogenic biofilm formation in the food 
industry and alternative control strategies. V: Holban AM, Grumezescu 
AM, eds. Foodborne Diseases. Cambridge: Academic Press; 2018. p. 
309-77.
 [20] Nieto Pozo I, Olmos D, Orgaz B, Božanić DK, González-Benito J. Titania 
nanoparticles prevent development of Pseudomonas fluorescens biofilms 
on polystyrene surfaces. Mater Lett. 2014;127(0):1-3. 
 [21] Shaheen R, Svensson B, Andersson MA, Christiansson A, Salkinoja-
Salonen M. Persistence strategies of Bacillus cereus spores isolated 
from dairy silo tanks. Food Microbiol. 2010;27(3):347-55. 
 [22] Kolari M, Nuutinen J, Salkinoja-Salonen M. Mechanisms of biofilm 
formation in paper machine by Bacillus species: the role of 
Deinococcusgeothermalis. J IndMicrobiolBiot. 2001;27(6):343-51. 
 [23] Kumari S, Sarkar PK. Bacillus cereus hazard and control in industrial 
dairy processing environment. Food Control. 2016;69:20-9. 
 [24] Kurinčič M, Jeršek B, Klančnik A, Možina SS, Fink R, Dražić G, Raspor 
P, Bohinc K. Effects of natural antimicrobials on bacterial cell hydro-
phobicity, adhesion, and zeta potential. ArhHig Rada Toksikol. 2016; 
67(1):39-45.
 [25] Luo K, Kim SY, Wang J, Oh DH. A combined hurdle approach of slightly 
acidic electrolyzed water simultaneous with ultrasound to inactivate 
Bacillus cereus on potato. LWT Food Sci Technol. 2016;73: 615.
 M. Oder, R. FinkBiofilm formation capacity of Bacillus cereus on silicone, polyethylene terephthalate...