Crystal Violet Binding Assay for Assessment of Biofilm Formation by Listeria monocytogenes and Listeria spp on Wood, Steel and Glass Surfaces

Abstract

Micro-organisms attach to surfaces and develop biofilms which are a concern in food and environmental safety. This study, quantified biofilms in 20 isolates viz: Listeria monocytogenes (14) and Listeria spp (6) from meat tables in Ibadan municipal abattoir, Nigeria on wood, glass and steel surfaces using crystal-violet binding assay. Listeria isolates formed biofilms on all three surfaces. At 24hours Listeria monocytogenes(SLM) formed more biofilms on wood(0.22±0.01) and glass(0.09±0.01) surfaces than Listeria spp(SLS) which was wood(0.17±0.02) and glass (0.06±0.01). But the reverse was the case on steel where Listeria spp (SLS) formed more biofilms (0.11±0.01) than Listeria monocytogenes (SLM) (0.10±0.00). A significant difference (P<0.01) exists between isolates for wood and glass at 24hours and (P<0.05) for wood at 72hours. Significant differences (P<0.05) occured in biofilms produced between 24hours and 72hours. It can be concluded that selection of suitable surfaces could reduce the ability of bacteria forming biofilms on meat contact-surfaces. This finding is very relevant in developing countries where wood is still in use as a major meat contact-surface in abattoirs.

Full text

Global Veterinaria 6 (1): 06-10, 2011
ISSN 1992-6197
© IDOSI Publications, 2011
Corresponding Author: Victoria O. Adetunji, Department of Veterinary, Public Health and Preventive Medicine,
University of Ibadan, Ibadan, Nigeria, E-mail: vadetunji@gmail.com & vo.adetunji@mail.ui.edu.ng.
6
Crystal Violet Binding Assay for Assessment of Biofilm Formation
by Listeria monocytogenes and Listeria spp on Wood, Steel and Glass Surfaces
Victoria O. Adetunji and Tajudeen O. Isola
Department of Veterinary, Public Health and Preventive Medicine, University of Ibadan, Ibadan, Nigeria
Abstract: Micro-organisms attach to surfaces and develop biofilms which are a concern in food and
environmental safety. This study, quantified biofilms in 20 isolates viz: Listeria monocytogenes (14) and
Listeria spp (6) from meat tables in Ibadan municipal abattoir, Nigeria on wood, glass and steel surfaces using
crystal-violet binding assay. Listeria isolates formed biofilms on all three surfaces. At 24hours Listeria
monocytogenes(SLM) formed more biofilms on wood(0.22±0.01) and glass(0.09±0.01) surfaces than Listeria
spp(SLS) which was wood(0.17±0.02) and glass (0.06±0.01). But the reverse was the case on steel where Listeria
spp (SLS) formed more biofilms (0.11±0.01) than Listeria monocytogenes (SLM) (0.10±0.00). A significant
difference (P<0.01) exists between isolates for wood and glass at 24hours and (P<0.05) for wood at 72hours.
Significant differences (P<0.05) occured in biofilms produced between 24hours and 72hours. It can be
concluded that selection of suitable surfaces could reduce the ability of bacteria forming biofilms on meat
contact-surfaces. This finding is very relevant in developing countries where wood is still in use as a major meat
contact-surface in abattoirs.
Key words: Biofilm % Listeria spp % Abattoir % Meat % Contact-surfaces
INTRODUCTION Various methods have been used for biofilm
Nearly 99% of micro-organisms living on the earthassay as described by Stepanovic and others [4, 22]. In
live in microbial communities known as biofilms [1].this assay, the dye bound to adherent cells is
Biofilms are formed by adhesion of bacterial cells toresolubilized and measured in optical density (OD).
surfaces through an exopolymeric matrix. This matrix isListeria spp are gram positive bacilli and are
important in formation and structure of the biofilm andtypified by Listeria monocytogenes, the causative
also on the protection of the bacterial cells as it preventsagent of listeriosis. Clinical infections are primarily by
antimicrobials and xenobiotics from gaining access toListeria monocytogenes although Listeria ivanovii is
the cells inside the biofilm [2]. Biofilm formation canalso pathogenic being particularly associated with
compromise the cleanliness of food contact surfaces andabortion in ruminants. The remaining species are
environmental surfaces by spreading detached organismsrecognized as non pathogenic. Listeria monocytogenes is
to food and other areas of processing plants [3]. Biofilmsquite hardy and resists dexterous effects of freezing,
formed in food processing environments are importantdrying and heat remarkably well for a bacterium that does
as they have the potential to act as a chronic source ofnot form spore [7].
microbial contamination that eventually leads to foodListeria’s tolerance has attributed to it being
spoilage and transmission of diseases [1, 4]. Biofilms lead isolated from various meat samples, with chicken
to serious hygienic problems and hence, the importantbeing the best host for growth of the pathogen [8].
aspect of controlling biofilms represents one of theListeriosis is contracted through the consumption of
most persistent challenges within food and industrialcontaminated foods [9]. Listeria monocytogenes is
environments where microbial communities arefound in a variety of food products such as soft
problematic [5]. Biofilm forming bacteria persist andcheeses, dairy products raw foods, ready to eat
produce after sanitation thus making biofilms a potentialproducts and equipment surfaces [10, 11]. Listeria
threat to food safety [6]. monocytogenes is a food borne pathogen that
assessment; one of these is the crystal violet binding

Global Veterinaria, 6 (1): 06-10, 2011
7
account for less than 1% of food borne illness but isfrom the broth culture for biofilm quantification using the
responsible for 28% of deaths caused by food bornecrystal violet binding assay described by Stepanovic et
diseases [12]. al. [4]. Each set of chips was washed 3 times with 5ml of
Several studies have been conducted to determinesterile distilled water. The remaining adhered bacteria were
the ability of Listeria monocytogenes to adhere and formfixed with 2.5ml of methanol per chip. Each chip was
biofilms on food contact surfaces. All of whichstained with crystal violet (Fishers scientific, USA) for 15
determined that L. monocytogenes attach to food industry minutes and then the excess stain washed off under
surfaces such as plastics, glass, stainless steel and rubberrunning tap water. After the chip was air dried, the dye
surfaces [13-20]. However, it has been noted that there are bound to the adherent cells was re- solubilized with 2.5ml
differences in both the extent and the rate of adsorption.of 33% glacial acetic acid (Fishers scientific, USA) for
Luden et al [21] demonstrated that the most prevalenteach chip. The re-solubilized liquid for each chip was
strain of L. monocytogenes (strain 1/ c) found in foodpoured into a cuvette. The absorbance (optical density)
2
processing plants has good adhesion ability and requiredof each re-solubilized liquid was measured against the
only a short contact time for attachment. optical density of blank reading without inoculation
This work presents an assessment of biofilms formed (control) at wavelength of 620nm for the Listeria
by isolates of Listeria monocytogenes and Listeria sppstrains using a spectrophotometer (Springfield, UK). The
from a tropical abattoir on wood, glass and steel surfacesabsorbance of negative control was subtracted from the
using the crystal violet binding assay. absorbance values to determine the actual values [23].
MATERIALS AND METHODS Statistical Analysis: All results were presented in
Preparation of Listeria Isolates Culture for Attachment analysis of variance (ANOVA) was used to determine
and Biofilm: Listeria monocytogenes isolates (SLM 14)
and Listeria spp (SLS 6) were isolated from meat tables in
Ibadan municipal abattoir, Nigeria. Modified oxford agar
base (MOX) for Listeria isolation was supplemented with
antibiotic supplements (acriflavine, nalidixic acid and
cycloheximide) (Becton, Dickinson and company) was
used for Listeria isolation. Further identification of the
isolates was based on procedures described by Barrow
and Feltham [22]. One colony of each Listeria isolates
was cultured on nutrient agar slope (Fluka 7014, Germany)
and was transferred into each of the 20 sterile glass jars
with lids containing 150ml of nutrient broth (Fluka 7014,
Germany). One glass jar not inoculated with Listeria
isolate served as control in this study. The inoculated
broth culture was incubated at 37°C for 24hours. Two
hundred and seventy-six (276) chips (90 each (4cm x 2cm
x 1cm) for glass (Easy way medical, England), steel (type
304, #4, Ajaokuta steel co., Nigeria) and wood (softwood,
Oak) were used. The chips were washed with detergent
(Unilever, Lagos, Nigeria), rinsed with sterile distilled
water and air-dried before being placed into hot air oven
(Elektro-Helios, Sweden) at 75°C for 30mins. After 24hours
incubation of the inoculated broth culture, four of steel,
glass and wood chips were aseptically put into each of
the glass jars making a total of 12 chips per glass jar. All
the glass jars were incubated at ambient temperature of
(26-28°C for 24 hours and 72 hours. At the end of each
)
incubation period, a set of chips were aseptically removed
mean±standard error of mean (Mean±SEM). A one-way
significant differences between means for each surface
and strains. Student t- test was used to assess significant
differences between strains. All data were analyzed using
SPSS 15 [24], Chicago and IL., USA. Statistical
significance was evaluated at P<0.05 and P<0.01. Charts
were plotted using Microsoft Excel [25].
RESULTS
The mean absorbance values at 24hours showed
that Listeria monocytogenes (SLM) (Figure 1) formed
more biofilms on wood (0.22±0.01) and glass (0.09±0.01)
surfaces than Listeria spp (SLS) which was wood
(0.17±0.02) and glass (0.06±0.01). But the reverse
was the case on steel where Listeria spp (SLS) formed
more biofilms (0.11±0.01) than Listeria monocytogenes
(SLM) (0.10±0.00) respectively (Table 1). The mean
absorbance value at 72hours showed that Listeria
monocytogenes (SLM) formed more biofilms on wood
(0.35±0.01) and glass (0.12±0.01) surfaces than Listeria
spp (SLS) which was wood (0.28±0.01) and glass
(0.11±0.01) respectively (Table 1). The mean absorbance
values (nm) of biofilm adherence to surfaces were
observed at 24 and 72hours on wood more significantly
(P<0.05) than on glass and steel surfaces for the isolates
except Listeria spp at 24hours where there were
significant differences (P<0.05) in biofilm formation on the
three surfaces (Table 1).

Global Veterinaria, 6 (1): 06-10, 2011
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Fig. 1: Showing differences in biofilms produced byharveyi. Cunliffe et al. [33]; Sinde and Carballo [19];
SLM-Listeria monocytogenes and SLS-ListeriaDonlan [34] also reported that Listeria monocytogenes
spp on the 3 surfaces at 24 and 72hours adhere in higher numbers to more hydrophobic materials.
Table 1: Comparison of Listeria biofilms on wood, steel and glass surfaces
at 24 and 72 hours incubation period
SURFACES
---------------------------------------------------------------
Strains Wood (mean Steel(mean Glass (mean
(Codes) Periods absorbance (nm) absorbance (nm) absorbance (nm)
SLM 24hrs 0.22±0.01 0.10±0.00 0.09±0.01
a b b
SLS 24hrs 0.17±0.02 0.11±0.01 0.06±0.01
a b c
SLM 72hrs 0.35±0.02 0.13±0.01 0.12±0.01
a b b
SLS 72hrs 0.28±0.03 0.13±0.01 0.11±0.00
ab b
* *Means with the same superscript are not significantly different at
0.05 levels
DISCUSSION
In this study, biofilm formation by Listeria
monocytogenes and Listeria spp revealed that these
bacteria possess a high capacity for biofilm formation on
the 3 surfaces used (wood, glass and steel) but with
differences in the extent of adhesion. Previous studies
have confirmed the biofilm formation by strains of
Listeria monocytogenes and Listeria spp on these
surfaces [13, 14, 19, 20, 26]. The higher level of biofilm
production by isolates of Listeria monocytogenes when
compared to Listeria spp on wood and glass at 24hours
has been explained in other studies done by Hood and
Zottola [13]; Gulsun et al. [27] where they reported that
the level of biofilm production of isolates is a virulence
characteristic of such isolates. In the current work, the
mean absorbance values (nm) of biofilms produced by
Listeria showed that wood retained the greatest biofilms
followed by steel and glass. This result is in agreement
with the report of Sinde and Carballo, [19]; Donlan [28]
who reported that glass and stainless steel are hydrophilic
materials while wood and plastic are hydrophobic
materials. Hydrophobic materials are reported as surfaces
that provide a greater bacterial adherence [29]. Fletcher
and Loeb [30] noted that large numbers of bacteria
attached to hydrophobic surfaces with little or no surface
charge and moderate numbers attached to hydrophobic
metals with positive charge or neutral charge and very few
attached to hydrophilic negatively charged substrates.
The importance of hydrophobicity in bacteria attachment
has also been highlighted by other investigators [31].
Variation in biofilm density depending on surface was
also reported by Karunasagar et al. [32] with Vibrio
Donlan [34] also reported that adhesion is the first step in
the complex process of biofilm formation.
The finding of significant differences (P < 0.05) in
biofilm formation between 24 and 72 hours incubation
periods for all the Listeria strains (Figure 1) in this study
has also been demonstrated by Moltz and Martin; [35];
Adetunji [36]; Adetunji and Adegoke [37] on stainless
steel chips and quantitative recovery of purified biofilm
and cellulose from culture respectively. In these studies
increase in biofilms formation with extension of incubation
period was reported.
Use of wood as a food contact surface has reduced
in the developed countries because it is a porous and
absorbent material where organic matter along with
bacteria can become entrapped; cross-contamination is a
main concern [38] but wood is still in use in the
developing countries for food processing because it's
readily available and cheap. The preferred surface for food
processing is glass. Glass is sometimes used for food
contact surfaces because of its smooth and corrosion-
resistant surface [39]. On the other hand, stainless steel
resists impact damage better than glass but is vulnerable
to corrosion, while rubber surfaces are prone to
deterioration and may develop surface cracks where
bacteria can accumulate [40]. Equally important in meat
contact surface is the cleanability of the surface once
bacteria forming biofilm have attached. The use of wood
tables for meat display should be discouraged in the
developing countries based on the findings in this study.
CONCLUSION
It is concluded that Listeria monocytogenes and
Listeria spp isolates from meat tables in the abattoir
under study are capable of forming biofilms on wood,

Global Veterinaria, 6 (1): 06-10, 2011
9
steel and glass surfaces but with some variations.12. Pan, Y.F., J.R. Breidt and S. Kathariono, 2006.
The food processing units in the developing countries
should therefore consider the application of findings in
this study with a view to preventing biofilms in food
processing environment. Further studies will be necessary
to understand whether the ability to form biofilms may
influence the survival of strains in meat contact surfaces.
REFERENCES
1. Costerton, J.W., O.S. Stewart and E.P. Greenberg,
1999, Bacterial biofilms: A common cause of
persistent infections. Sci., 284(5418): 1318-1322.
2. Davey, E.P. and A.G. O9toole, 2002, Microbial biofilm
from ecology to molecular genetics. Microbial Mol.
biol., 64: 847-86.
3. Trachoo, N., 2004. Biofilm removal technique using
sands as a research tool for accessing microbial
attachment to surface: Songklangkaria J. Sci.
Technol., 26(1): 109-115.
4. Stepanovic, C.S., M.L. Cirkoric, L. Ranin and
A.L. Svabicviahocic, 2004. Biofilm formation by
Salmonella spp and Listeria monocytogenes on
plastic surface. Appl. Microbiol., 28: 326-432.
5. Kumar, C.G. and S.K. Anand, 1998. Significance of
microbial biofilms in food industry. A review: Int. J.
Food Mirobiol., 42(1): 9-27.
6. Hyde, F.W., M. Alberg and K. Smith, 1997.
Camparison of fluorinated polymers against stainless
steel, glass and poly polyprophylene in microbial
biofilm adherence and removal. J. Ind. Microbiol. and
Biotechnol., 19: 142-147.
7. USFDA-CFSAN. United States Food and Drug
Administration 2006.
8. Borucki, M.K., J.D. Peppin, D. White, F. Lodge and
D.R. Galt, 2003. Variation in biofilm formation among
strains of Listeria monocytogenes. Appl. Environ.
Microbiol., 69: 7336-7342.
9. Taormina, P.J. and L.R. Beauchat, 2002. Survival of
Listeria monocytogenes in commercial food
processing equipment cleaning solutions and
subsequent sensitivity to sanitizers and heat. J. Appl.
Microbial., 92: 71-80.
10. Jeyasekan, G. and J. Karunasagar, 2000. Effect of
Sanitizers on Listeria biofilm on contact surfaces.
Asian Fisheries Sci., 13: 209-213.
11. Mafu, A.A., D. Roy, J. Goulet, L. Savioe and R. Roy,
2006. Efficacy of sanitizing agents for destroying
Listeria monoccytogenes on contaminated surfaces.
J. Dairy Sci., 13: 3428-3432.
Resistance of Listeria monocytogenes biofilms
to sanitizing agents in a stimulated food
processing environment. Appl. Environ. Microbiol.,
21: 123-128.
13. Hood, S.K. and E.A. Zottola, 1997. Isolation &
identification of adherent Gram negative micro-
organisms from 4 meat processing facilities of food.
J. Food Prot., 60(9): 1135-1138.
14. Wong, A.C.L., 1998. Biofilms in food processing
environments. J. Dairy Sci., 81: 2765-2770.
15. Sommer, P., C. Martin-Rouas and E. Mettler, 1999.
Influence of the adherence to population level on
biofilm population, structure and resistance to
chlorination. Food Microbiol., 16: 503-515.
16. Chae, M.S. and H. Scraft, 2001. Cell viability of
Listeria monocytogenes biofilms. Food Microbiol.,
18: 103-112.
17. Chae, M.S. and H. Scraft, 2000. Comparative
evaluation of adhesion and biofilm formation of
different Listeria monocytogenes strains. Int. J. Food
Microbiol., 62: 103-111.
18. Leriche, V. and B. Carpentier, 2000. Limitation of
adhesion and growth of Listeria monocytogenes on
stainless steel surfaces by Staphylococcus sciuri
biofilms. J. Appl. Microbial., 88: 594-605.
19. Sinde, E. and J. Carballo, 2000. Attachment of
Salmonella spp and Listeria monocytogenes to
stainless steel, rubber and polytetrafluro ethylene:
the influence of free energy and the effect of
commercial sanitizers. Food Microbiol., 17: 439-447.
20. Joseph, B. S.K. Otta and J. Karunasagar, 2001. Biofilm
formation by Salmonella spp on food contact
surfaces and there sensitivity to sanitizer. Int. J. Food
Microbiol., 64: 367-372.
21. Luden, J.M., M.K. Mlettinen, T.J. Autio and
H.J. Korkeala, 2002. Persistent Listeria
monocytogenes strains show enhanced adherence to
food contact surface after her short contact time. J.
Food Prot., 53(6): 7426.
22. Barrow, G.I. and R.K.A. Feltham, 1993. Cowan and
steel’s manual for the identification of medical
bacteria (3 Ed.) Cambridge University Press, pp: 331.
rd
23. Pawar, D., M. Rossman and J. Chen, 2005. Role of
curli fimbriae in mediating the cell of
enterohemorrhagic Escherichia coli to attach to
abiotic surfaces. J. Appl. Microbiol., 99: 418-425.
24. SPSS-15 Statistical Package for Social Sci., 2006.
25. Microsoft Excel, 2009.

Global Veterinaria, 6 (1): 06-10, 2011
10
26. Stepanovic, C.S., M.L. Cirkoric and M. Svabic-34. Donlan, R.M., 2002. Biofilms: microbial life surfaces.
Vlahovic, 2003. Influence of the incubationEmerg. Infect. Dis., 8: 881-890.
temperature, atmosphere and dynamic conditions on35. Moltz, A.G. and S.E. Martin, 2005. Formation of
biofilm formation by Salmonella spp. Foodbiofilms by Listeria monocytogenes under various
Microbiol., 20: 329-343. growth conditions. J. Food Prot., 68(1): 92-97.
27. Gulsun, S., N. Ogzolu, A. Inan and N. Leran, 2005.36. Adetunji, V.O., 2007. Public health Aspect of
The virulence factors and antibiotic sensitivity of E.processing and storage of wara in Oyo and Kwara
coli isolated from recurrent urinary tract infections.state, Nigeria. A thesis submitted to the Department
Saudi med. J., 26(11): 1755-1758. of Veterinary Public Health, the Preventive Medicine,
28. Donlan, R.M., 2000. Role of biofilms in AntimicrobialUniversity of Ibadan.
Resistance. Asaio J., 46: 547- 552. 37. Adetunji, V.O. and G.O. Adegoke, 2008. Formation of
29. Djordjevic, D., M. Wiedmann andbiofilms by strains of Listeria monocytogenes
L.A. Mclandsborough, 2002. Microtiter plateisolated from soft cheese ‘wara’ and its processing
assay for assessment of Listeria monocytogenesenvironment. Afr. J. Biotech., 7(16): 2893-2897.
biofilm formation. Appl. Environ. Microbiol.,38. Lauzon, H.L., 1998. The suitability of materials used
68(6): 2950-2958. in the food industry, involving direct or indirect
30. Fletcher, M. and G.I. Loeb, 1979. Influence ofcontact with food products. Project P 98076 "Wood
substratum characteristics in the attachment of ain the Food Industry Nordic industrial fund. Center
marine psuedomonad to solid surfaces. Appl.for innovation and commercial development and the
Environm Microbiol., 37: 67-72. industry partners in Denmark, Iceland, Norway and
31. Characklis, W.G., 1990. Process analysis. In:Sweden, pp: 5.
Characklis W.G. Marshall, K.C., Edition 1990.39. Dunsmore, D.G., A. Twomey, W.G. Whittlestone and
Biofilms, Wiley-Interscience, New York, pp: 17-54. H.W. Morgan, 1981. Design and performance of
32. Karunasagar, I. S.K. Otta and I. Karunasagar, 1996.systems for cleaning product-contact surfaces of
Bioflm formation by Vibrio harveyi on surfaces.food equipment: a review. J. Food Prot., 44: 220-240.
Aquaculture, 140: 241-245. 40. LeClercq-Perlat, M. and M. Lalande, 1994.
33. Cunliffe, D., C.A. Smart, C. Alexander andCleanability in relation to surface chemical
E.N. Vulfson, 1999. Bacterial adhesion atcomposition and surface finishing of some materials
synthetic surfaces. Appl. and Environ. Microbiol., commonly used in food industries. J. Food Eng.,
65: 4995-5002. 23: 501-517.