Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa

and Staphylococcus epidermidis
Kalimuthu Kalishwaralal, Selvaraj BarathManiKanth, Sureshbabu Ram Kumar Pandian,
Venkataraman Deepak, Sangiliyandi Gurunathan ∗
Department of Biotechnology, Division of Molecular and Cellular Biology, Kalasalingam University, Anand Nagar, Krishnankoil 626190, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix.
Received 7 January 2010 Formation of these sessile communities and their inherent resistance to anti-microbial agents are the
Received in revised form 14 April 2010 source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major
Accepted 15 April 2010
role in development of ocular related infectious diseases in human namely microbial keratitis. Different
Available online 22 April 2010
approaches have been used for preventing biofilm related infections in health care settings. Many of these
methods have their own demerits that include chemical based complications; emergent antibiotic resis-
Keywords:
tant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the
Anti-biofilm activity
Silver nanoparticles
present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm
Keratitis activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epider-
Pseudomonas aeruginosa midis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than
Staphylococcus epidermidis 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As
a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial
keratitis based on its potential anti-biofilm activity.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction charide that is involved in adherence and biofilm formation. These

Contact lenses (CL) often get infected with bacteria, and pro-
longed usage of such lenses leads to microbial keratitis in eye.
Bacteria frequently adhere to the surface of the lens through a
biofilm matrix, a three-dimensional, gel-like, highly hydrated and
locally charged environment, and using such lens can cause infec-
tions in eye. Adhesion of these bacteria to CL may contribute
to the pathogenesis of infection and may be influenced by lens
surface properties [1–3]. The first step in biofilm formation is
the adhesion of microbial cell to the surface by the exopolysac-
charides synthesized by the bacteria [4]. Pseudomonas aeruginosa
and Staphylococcus epidermidis have been well-known as the
major causative agents of infectious keratitis. The Gram-negative
opportunistic aerobic rod P. aeruginosa is ubiquitous and well
adapted for growth in an aquatic environment, also synthesizes an
adhesive alginate extracellular matrix for biofilm formation. The
Gram-positive aerobic coccus S. epidermidis is present as normal
saprophytic flora on the skin, and it produces a surface polysac-
∗ Corresponding author at: Department of Biotechnology & Chemical Engineering,
Division of Molecular and Cellular Biology, Kalasalingam University (Kalasalingam
Academy of Research and Education), Anand Nagar, Krishnankoil 626 190, Tamil-
nadu, India. Tel.: +91 4563 289042; fax: +91 4563 289322.
E-mail address: lvsangs@yahoo.com (S. Gurunathan).
two organisms are capable of adhesion and biofilm formation on CL
and even on the inner walls of lens storage cases [5–10]. The cells
in the biofilm are distinct from their planktonic counterparts, since
the biofilm augments resistance to drug therapy, disinfectants, and
the immune response of the host [7–9]. The biofilm on CL prolongs
the bacteria’s contact with the surface of the eye, thus increasing
its pathogenicity [10].
Earlier, in the 19th century, microbial infections were treated
with 0.5% AgNO3 like Ophthalmia neonatorum (by German obste-
trician Carl Crede), and for the prevention of infection in burns.
When the era of the antibiotics began with the discovery of peni-
cillin, the use of silver slowly diminished [11]. But in the present
scenario due to the emergence of biocide-resistant strains, once
again the use of silver for treating infections has gained impor-
tance. However, the use of ionic silver has one major drawback;
they are easily inactivated by complexation and precipitation thus
limiting the uses [12]. Here zerovalent silver nanoparticles can be
a valuable alternative for ionic silver [13].
Nanosilver is one of non-toxic and safe antibacterial agents to
the human body. Besides, silver nanoparticles are also reported
to possess anti-fungal activity [14], anti-inflammatory effect [15],
anti-viral activity [16] and anti-angiogenic activity [17,18]. But, sil-
ver nanoparticles can be well applied in therapy safely when the
effective concentrations of silver nanoparticles on various types of
organisms are determined.

0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.04.014
 K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
There are many methods available for the synthesis of sil-
ver nanoparticles. Most chemical methods use a reducing agent
(e.g, sodium borohydride) to reduce Ag+ to Ag0 and a stabilizer
(e.g., polyvinylpyrrolidone) to control particle growth and prevent
aggregation. However, these preparations often have problems
with particle stability and are difficult to scale up. In addition, there
is a demand for more environment-friendly production methods.
Alternatively, silver nanoparticles can also be synthesized biologi-
cally using bacteria [13].
The present study divulges the anti-microbial and anti-biofilm
ability of biologically synthesized silver nanoparticles against P.
aeruginosa and S. epidermidis, the important causative agents of
keratitis. To our knowledge, this is the first report on the antibi-
otic effect of silver nanoparticles on P. aeruginosa and S. epidermidis
and its effect on the biofilm formation.
2. Materials and methods
2.1. Strains used
Wild-type of Bacillus licheniformis, P. aeruginosa and S. epider-
midis were maintained in nutrient agar as well as sub cultured from
time to time in the microbiology laboratory during the study period.
2.2. Synthesis of silver nanoparticles
In a typical experiment, 2 g of wet B. licheniformis biomass was
taken in an Erlenmeyer’s flask. 1 mM AgNO3 solution was prepared
using deionized water and 100 mL of the solution mixture was
added to the biomass. Then the conical flask was kept in a shaker
at 37 ◦ C (200 rpm) for 24 h for the synthesis of nanoparticles [19].
2.3. Purification of silver nanoparticles
The cells from each Erlenmeyer flask were washed twice with
50 mM phosphate buffer (pH 7.0) and re-suspended in 5 mL of the
same buffer. Ultrasonic disruption of cells was carried out with an
ultrasonic processor (Sonics Vibra Cell VC-505/220, Newtown, USA)
over three 15 s periods, and with an interval of 45 s between peri-
ods. The resulting solution was centrifuged (16,000 rpm, 30 min)
and filtered through a 0.22 ␮m filter (Millipore,) to remove cell-
debris. Characterization of synthesized and purified particles was
carried out according to the method described previously [19].
Samples for transmission electron microscopy (TEM) analysis were
prepared on carbon-coated copper TEM grids. TEM measurements
were performed on a JEOL model 1200EX instrument operated at
an accelerating voltage of 120 kV.
2.4. Determination of concentration of the silver nanoparticles
The concentration of silver nanoparticles was determined by the
method which has been previously reported for Liu et al. [20] for
gold nanoparticles. The calculation is as follows [20].
• To determine the average number of atoms per nanoparticle (Liu
et al. [20])
D3
N= NA
6M
where N is the number of atoms per nanoparticles,  = 3.14,  is
the density of face centered, cubic (fcc) silver (=10.5 g/cm3 ), D is
the average diameter of nanoparticles (=50 nm = 50 × 10−7 cm),
M is the atomic mass of silver (=107.868 g), NA is the number of
atoms per mole (Avogadro’s number) (=6.023 × 1023 ).
341
Therefore assuming 100% conversion of all silver ions to silver
nanoparticles,
3
3.14 × 10.5 × (50.0 × 10−7 ) × 6.023 × 1023
N=
6 × 107.868
i.e. N = 3837233.003
• Determine the molar concentration of the nanoparticle solution
using the following formula: (Liu et al. [20])
NT
C=
NVNA
where C is the molar concentration of nanoparticle solution, NT
is the total number of silver atoms added as AgNO3 = 1 M, N is the
number of atoms per nanoparticle (from calculation 1), V is the
volume of the reaction solution in L, NA is the Avogadro’s number
(=6.023 × 1023 ).
1 × 6.023 × 1023
C=
3837233.003 × 1 × 6.023 × 1023
C = 2.606 × 10−7 M/L = 260 nM
Further, the required concentration are made out from the
obtained values.
2.5. Determination of anti-microbial activity by well-diffusion
method
The AgNPs synthesized from B. licheniformis were tested for anti-
microbial activity by conventional well-diffusion method against P.
aeruginosa and S. epidermidis [21]. The pure cultures of organisms
were sub cultured on nutrient broth at 37 ◦ C on a rotary shaker at
200 rpm. Wells of 6-mm diameter were made on Muller–Hinton
agar plates using gel puncture. Each strain was spread uniformly
onto the individual plates using sterile cotton swabs. Using a
micropipette, 100 nM of the sample of nanoparticles solution was
filled onto each well on all plates. After incubation at 37 ◦ C for 28 h,
the different levels of zone of inhibition were measured.
2.6. Determination of biofilm formation by Congo red agar
method (CRA)
The determination of biofilm formation was carried out by the
method described by Freeman et al. [22]. This method utilizes a
specially prepared solid medium – brain heart infusion broth (BHI)
supplemented with 5% sucrose and Congo red for screening the for-
mation of biofilm by P. aeruginosa and S. epidermidis. The medium
composes of BHI (37 g/L), sucrose (50 g/L), agar No.1 (10 g/L) and
Congo red stain (0.8 g/L). Congo red was prepared in the form of
concentrated aqueous solution and it was autoclaved at 121 ◦ C
for 15 min, separately from other medium constituents. Following
autoclave, the concentrated solution was added to agar which was
previously cooled to 55 ◦ C. Plates were inoculated and incubated
aerobically for 24–48 h at 37 ◦ C.
2.7. Tissue culture plate method (TCP) – in vitro biofilm
formation assay
To determine the efficacy of silver nanoparticles in elimination
of formed biofilm, TCP method was carried out with suitable mod-
ifications [23]. Individual wells of sterile, polystyrene, 96-well-flat
bottom tissue culture plates were filled with 180 ␮L of BHI broth
and inoculated with 10 ␮L of overnight culture. To the mixture
342 K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344

10 ␮L of silver nanoparticles were added from the stock so that

final concentration was made between 10 nM and 100 nM. The
tissue culture plates were incubated for 24 h at 37 ◦ C. After incu-
bation, content of each well was gently removed. The wells were
washed four times with 0.2 mL of phosphate buffer saline (PBS pH
7.2) to remove free-floating ‘planktonic’ bacteria. Biofilms formed
by adherent ‘sessile’ organisms in plate were fixed with sodium
acetate (2%) and stained with crystal violet (0.1%, w/v). Excess stain
was rinsed off by thorough washing with deionized water and
plates were kept for drying. After drying, 95% ethanol was added to
the wells and the optical densities (OD) of stained adherent bacteria
were determined with a microplate reader (model 680, Bio-rad) at
595 nm (OD595 nm). These OD values were considered as an index
of bacteria adhering to surface and forming biofilms. Experiment
was performed in triplicate, the data was then averaged and the
standard deviation was calculated.
Fig. 1. TEM image obtained from purified AgNPs synthesized using B. licheniformis.
3. Results Several fields were photographed and were used to determine the diameter of
nanoparticles. The range of observed diameter was 50 ± 5 nm.

3.1. Biological synthesis of AgNPs

Table 1
Silver nanoparticles were synthesized using B. licheniformis and Diameter of the zone of inhibition by silver nanoparticles against microbes respon-
sible for the disease pathogenesis. Results are mean ± SD (n = 4).
purified. The synthesized nanoparticles were primarily character-
ized by UV–vis spectroscopy, which has proved to be a very useful Microorganism Diameter of inhibition zones (in mm) (mean of
technique for the analysis of nanoparticles [19]. Further charac- four replicates)
terization was carried out using transmission electron microscopy. Staphylococcus epidermidis 12 ± 1.2 mm
Electron microscopic images show that purified nanoparticles are Pseudomonas aeruginosa 9.5 ± 0.9 mm
spherical with a mean diameter of 50 nm (Fig. 1).

3.2. Anti-microbial activity of AgNPs against P. aeruginosa and
S. epidermidis
AgNPs are one of the successfully employed anti-microbial
agents commercially. Here, the anti-microbial activity of AgNPs was
tested against P. aeruginosa and S. epidermidis, using well-diffusion
method (Fig. 2). The zone of inhibition is found to be slightly higher
for S. epidermidis in comparison to its counterpart. The zone of
inhibition is given as a mean of four replicates of the diameter of
inhibition zones (in mm) around each well with AgNPs solution
(Table 1).
3.3. Detection of biofilm formation
Biofilm formation is detected in many organisms synthesizing
exopolysachharides. The biofilm is made up of microorganisms
adhering to the surface coated with slime – the exopolysaccha-
ride matrix which protects the microbes from the unfavorable
environmental factors [23]. Biofilm formation by P. aeruginosa and
S. epidermidis were tested by growing the organism in Brain heart
infusion agar supplemented with Congo red (BHIC) with and with-
out silver nanoparticles. When the colonies were grown without
AgNPs in the medium, the organisms appeared as dry crystalline
black colonies, indicating the production of exopolysachharides,
which is the prerequisite for the formation of biofilm (Fig. 3).
Whereas when the organisms were grown on BHIC with AgNPs,
the organisms did not survive. During the treatment with reduced
concentrations of AgNPs (10 nM), the organisms continued to grow,
but AgNPs treatment has inhibited the synthesis of exopolysach-
harides, indicated by the absence of dry crystalline black colonies
(Fig. 3). The presence of nanoparticles at a certain level inhibited
bacterial growth by more than 90%. When the exopolysachharide
synthesis is arrested, the organism cannot form biofilm. There-
fore, 50 nM of silver nanoparticles significantly arrested biofilm
formation without affecting viability, whereas 100 nM inhibited the
growth of the organism itself.

Fig. 2. Anti-microbial activity of the purified silver nanoparticles against P. aeruginosa (A) and S. epidermidis (B) at 100 nM concentration.
 K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344 343

Fig. 3. Ability of the organisms was checked for biofilm formation in brain heart infusion agar supplemented with Congo red. The appearance of black crystalline colonies
indicate the exopolysachharide production by P. aeruginosa (A) and S. epidermidis (C), whereas the addition of 50 nM silver nanoparticles blocked the exopolysachharide
synthesis by P. aeruginosa (B) and S. epidermidis (D).

3.4. Quantification of biofilm detachment

As biofilm formation is a prerequisite for Microbial keratitis,

the ability of P. aeruginosa and S. epidermidis, the major particip-
itants, in the microbial biofilm formation was investigated in vitro
by monitoring the binding of the dye crystal violet to adherent
cells which directly revealed their effective ability in formation of
biofilm [24]. Crystal violet staining, a spectrophotometrical method
was adopted to quantify biofilm density, a technique used for
quantification of biofilm [23]. A calibration curve was previously
plotted using various concentration of crystal violet to arrive at the
results.
P. aeruginosa and S. epidermidis were grown to form biofilm
for 24 h in microtiter plate wells and then treated with varying
concentrations of silver nanoparticles (Fig. 4). Treatment for 2 h
with concentration of 100 nM of silver nanoparticles resulted in a
decrease of 95% and 98% of the biofilm formed and 50 nM resulted
in a 50% reduction in biofilm. These data demonstrate that silver
nanoparticles induced detachment of P. aeruginosa and S. epider-
midis biofilms was rapid, efficient and also occurred at clinically
achievable concentrations of silver nanoparticles.

4. Discussion

Contact lenses associated MK is a severe eye infection arising

from the presence of adhered microorganisms on the lens sur-
face. MK is common among patients suffering from ocular injury
[25], however other factors like continuous and overnight wear Fig. 4. The anti-biofilm ability checked by tissue culture plate method showing that
[25,26] contaminated lens care solutions and lens cases [27] are addition of increasing concentrations of silver nanoparticles reduced the ability of
also involved in the development of MK. P. aeruginosa and Staphylo- the organisms to form biofilm and attach to the surface of the wells. 100 nM silver
nanoparticles completely inhibited the exopolysachharide synthesis by both the
cocci sp. are the common causative pathogens of the MK. Therefore
orgamisms (silver nanoparticle treatment to the adhered (A) P. aeruginosa and (B)
the ability of the nanoparticles to inhibit the formation of the S. epidermidis on the wells).
biofilms and formed biofilms were checked against the aforesaid
organisms. The exopolysaccharides synthesized by the bacteria
344 K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
not only protects the bacteria from the host defense mechanism
but also mediates the adhesion of the organism between the
lens and the corneal epithelium [28,29]. Moreover the ability of
the organisms to form biofilm was confirmed by the formation
of dry crystalline colonies on BHIC. Here we checked the abil-
ity of the AgNPs to inhibit the growth of the organisms under
consideration, by well-diffusion method of antibiotic assay. The
organisms’ growth was effectively impeded by the silver nanopar-
ticles at the concentration of 100 nM. The concentration of silver
nanoparticles was lesser than the previous reports for the toxic
concentration of the AgNPs in vitro against the mammalian retinal
cells [18].
The recurrence of biofilms in spite of treatment with various
anti-microbial agents was attributed to the impedance created by
the biofilm matrix [30]. Although water channels are present in
the biofilms, the deep lying organisms escape the treatment as the
matrix hinders the diffusion of the drug. Therefore, inhibition of
biofilm formation is very much essential in the case of prevention
of MK and various other disorders, as the exopolysachharide slime
formed reduces the susceptibility of the organism to the adminis-
tered drug. Antibiotic treatments effectively kill the bacteria which
remain individual, but the efficiency is very much reduced when
the organism forms slime. This makes the organisms to revert the
disease when the antibiotic treatment is finished because of the
healing of symptoms. This is the reason behind the biofilm infec-
tions showing recurring symptoms even after cycles of antibiotic
therapy until the mass is surgically removed. Therefore in this
report we investigated whether AgNPs has anti-microbial activity
and also anti-biofilm function by using BHIC. Here, AgNPs not only
inhibited the growth but also the ability of the organism to syn-
thesize the exopolysachharide. This is evident from Fig. 3 where
the BHI agar containing Congo red is supplemented with reduced
the concentration of AgNPs (50 nM). The concentration was cho-
sen based on the non-inhibitory concentration values which only
impeded the synthesis of the exopolysachharides but not the via-
bility of the organism (data not shown). The organism continued
to grow, but its ability to synthesize the exopolysachharide is
blocked. This shows that silver nanoparticles have the ability to
block the exopolysachharide synthesis of the organism otherwise
the biofilm. Another method which was found to be useful in deter-
mining the ability of the moiety to disrupt the biofilm formation
is the crystal violet method on TCP. The TCP assay described by
Christensen et al. [23] is most widely used and was considered
as standard test for detection of biofilm formation. The ability of
the organism to form biofilm and the effect of various concen-
trations of silver nanoparticles in inhibiting the formation was
checked on a 96-well plate, where an increase of silver nanopar-
ticles concentration negatively regulated the biofilm formation.
This is evident from Fig. 4 where 100 nM of silver nanoparticles
treatment almost completely inhibited the formation of biofilm
(>95%).
Therefore the results obtained directly reveals that biologically
synthesized silver nanoparticles at a concentration of 100 nM not
only effectively inhibited the growth of the bacteria, but also wiped
out the biofilm formed by it. This inhibitory effect of AgNPs on
the existing biofilm may due to be presence of water channels
though out the biofilm. Since in all biofilms water channels (pores)
are present for nutrient transportation, AgNPs may directly diffuse
through the exopolysaccharide layer through the pores and may
impart anti-microbial function. The experiment with 96-well plate
shows that AgNPs eliminate the biofilm formed previously besides
inhibiting the formation of biofilm in existing bacteria.
5. Conclusion
The present study characterizes the anti-biofilm activity of sil-
ver nanoparticles against two common bacterial pathogens that
are been proven for their efficient biofilm formation. It is notewor-
thy that for prevention of Microbial keratitis, silver nanoparticles
composing gel may be used as a safe biocide for destroying differ-
ent bacterial biofilms. Observations made through microtiter plate
assay (0.1% crystal violet staining) discloses the potential of sil-
ver nanoparticles in effective inhibition of biofilm formation which
affirms the futuristic applications of AgNP based contact lens care
solutions, for biofilm based human ocular problems, thereby serv-
ing mankind through an economic therapeutic alternative.
Acknowledgements
The authors gratefully acknowledge Dr. Pushpa Viswanathan,
Professor, Cancer Institute (WIA), Chennai, India, for her immense
support in analyzing samples under Transmission Electron Micro-
scope. The author Kalishwaralal Kalimuthu is grateful to CSIR for
providing senior research fellowship (Ack. No. 142070/2K9/1).
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