Standardization and Classification ofIn vitroBiofilm Formation by Clinical Isolates of Staphylococcus aureus

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Abstract
Original Article
INTRODUCTION
Staphylococcus aureus is a leading cause of morbidity and
mortality in nosocomial and community-based infections.[1] It
is associated with a number of infections ranging from dental
caries, periodontitis, stye, carbuncle, impetigo, and pyoderma
to persistent tissue infections such as wound infection,
otitis media, osteomyelitis, rhinosinusitis, recurrent urinary
tract infection, and endocarditis.[2] It is also one of the most
important pathogens in implant-related infections.[3,4] Several
features of this bacterium render survival fi tness in a wide
variety of environments of which the biofi lm formation is one
of the special modes of persistent infections.[5-10]
Biofi lm formation is an adaptive protected mode of growth
enabling bacteria to survive in hostile environments as in
the human host. This mode also enables them to disperse
and colonize new niches as per their need which is mediated
by their chemical cross-talk called quorum sensing.[11,12] The
essential paradox of chronic infections is untreatability, and
in most cases, chronic infections are accompanied by the
formation of biofi lms. The National Institute of Health, USA,
claims the involvement of biofi lms in 80% of all bacterial
infections.[1] Neutrophil entrapment within biofi lms leads to
tissue injury by release of various infl ammatory mediators. It
has been observed that dead debris of neutrophils and/or other
immune cells also serve as a biological matrix to facilitate
biofi lm formation. Bacterial genomic DNA liberated from
Background: Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofi lm
exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However,
till date, consensus guidelines for in vitro biofi lm quantitation and categorization criterion for the bacterial isolates based on biofi lm-forming
capacity are lacking. Therefore, it was intended to standardize in vitro biofi lm formation by clinical isolates of S. aureus and then to classify
them on the basis of their biofi lm-forming capacity. Materials and Methods: A study was conducted for biofi lm quantitation by tissue
culture plate (TCP) assay employing 61 strains of S. aureus isolated from clinical samples during May 2015–December 2015 wherein several
factors infl uencing the biofi lm formation were optimized. Therefore, it was intended to propose a biofi lm classifi cation criteria based on the
standard deviation multiples of the control differentiating them into non, low, medium, and high biofi lm formers. Results: Brain-heart infusion
broth was found to be more effective in biofi lm formation compared to trypticase soy broth. Heat fi xation was more effective than chemical
fi xation. Although, individually, glucose, sucrose, and sodium chloride (NaCl) had no signifi cant effect on biofi lm formation, a statistically
signifi cant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and
1000 mM NaCl (P = 0.037). Conclusions: The present study puts forth a standardized in vitro TCP assay for biofi lm biomass quantitation
and categorization criteria for clinical isolates of S. aureus based on their biofi lm-forming capacity. The proposed in vitro technique may be
further evaluated for its usefulness in the management of persistent infections caused by the bacterium.
Keywords: Biofi lm, brain-heart infusion broth, tissue culture plate method, trypticase soy broth
Address for correspondence: Dr. Pradyot Prakash,
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu
University, Varanasi - 221 005, Uttar Pradesh, India.
E-mail: pradyotbhu@gmail.com
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How to cite this article: Singh AK, Prakash P, Achra A, Singh GP,
Das A, Singh RK. Standardization and classifi cation of In vitro biofi lm
formation by clinical isolates of Staphylococcus aureus. J Global Infect
Dis 2017;9:93-101.
Standardization and Classification of In vitro Biofilm Formation
by Clinical Isolates of Staphylococcus aureus
Ashish Kumar Singh, Pradyot Prakash, Arvind Achra, Gyan Prakash Singh1, Arghya Das, Rakesh Kumar Singh2
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 1Department of Community Medicine, Division of Biostatistics, Institute of
Medical Sciences, Banaras Hindu University, 2Department of Biochemistry, Institute of Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017
94
biofi lms is also an immunostimulant and is recognized by
toll-like receptor 9.[12] Therefore, biofi lms can be considered
as a special mode of persistent bacterial infection.[13]
Further, biofi lm formation is dependent on different parameters
including the characteristics of the nature of carbon source,
its concentration, pH, ionic strength, and temperature, etc.[14]
Although investigators have tried to optimize the conditions
required for biofi lm formation by staphylococcal isolates,
some of the parameters such as optimum concentration of
sugars, salt, and richness of medium have not been thoroughly
investigated.[15] Some investigators have used trypticase soy
broth (TSB) with glucose and/or brain-heart infusion (BHI)
broth with sucrose supplementations to assess the effect on
biofi lm phenotype.[16] However, some have comprehensively
elucidated sodium chloride (NaCl) dependence of biofi lms in
S. aureus.[17] However, their quantitative interpretation and
categorization based on biofi lm production criteria were not
clear and cannot be replicated in every laboratory settings.
Therefore, a simple and consensus guideline for in vitro biofi lm
synthesis by clinical isolates of S. aureus is direly needed.
To the best of our knowledge, the effect of growth medium,
fi xation and elution and then supplementation of different
sugars and salt levels to a larger range of concentrations on the
characteristics of S. aureus biofi lm has received comparatively
little attention as the majority of investigators have not screened
the sugar and salt concentration beyond 1%.[14,18] Further, there
is no method described till date by which the bacteria can be
differentiated on the basis of their biofi lm-forming ability.
Therefore, in the present study, we aimed for the standardization
of consensus protocol for achieving maximum in vitro biofi lm
formation by clinical isolates of S. aureus utilizing the
supplementation with the proper concentration of glucose,
sucrose, and NaCl. We also tried to put forth categorization
criteria for the bacterial isolates on the basis of their
biofi lm-forming capacity.
MATERIALS AND METHODS
Bacterial isolates
A study was conducted in which a total of 61 non-repetitive,
consecutive strains of S. aureus isolated from the clinical
samples received in the Microbiology laboratory over a
period of 7 months (May 2015–December 2015), from various
outpatients (outpatient departments [OPDs]) and inpatients
wards of University Hospital, Banaras Hindu University. Of
all the clinical isolates, majority were isolated from samples
received from the Dermatology and Venereology OPD
(n = 17), surgery OPD (n = 17), orthopedics ward (n = 10),
high dependency unit (n = 4), pediatrics ward (n = 3),
Intensive Care Unit (ICU) (n = 2), Neonatal ICU (n = 2), and
one each from obstetrics and gynecology, plastic surgery,
otorhinolaryngology, neurology, medicine, and urology wards
[Figure 1 and Supplementary Data 1].
The bacterial identifi cation was performed using conventional
bacteriological techniques, such as colony morphology,
Gram-staining, catalase test, coagulase test, mannitol
fermentation, bacitracin susceptibility test, and salt tolerance.
Staphylococcus epidermidis ATCC 35984 (high slime
producer), ATCC 35983 (moderate slime producer), and ATCC
12228 (non-slime producer) were used as reference strains
since similar biofi lm-producing reference strains of S. aureus
are not available till date.
Determination of antimicrobial resistance
Antibiotic susceptibility testing of the isolates was performed
by modifi ed Kirby–Bauer method in accordance with the
Clinical and Laboratory Standards Institute guidelines 2015
using 13 antibiotic discs including penicillin (10 Units),
cefoxitin (30 mcg), erythromycin (15 mcg), trimethoprim and
sulfamethoxazole (25 mcg), clindamycin (2 mcg), azithromycin
(15 mcg), linezolid (30 mcg), ciprofl oxacin (5 mcg), netilmicin
(30 mcg), moxifl oxacin (5 mcg), and amoxicillin/clavulanate
(30 mcg). Antimicrobial susceptibility to mupirocin and
fusidic acid was interpreted as described by Park et al.[19]
All the materials needed for the current study were procured
from HiMedia Laboratories, Mumbai, otherwise mentioned.
Tissue culture plates (TCPs) were procured from Tarsons,
Kolkata, India.
Standardization of in vitro synthesis of biofilm in tissue
culture grade microtiter plates
In the present study, the effect of various parameters on in vitro
biofi lm synthesis was at fi rst observed on S. epidermidis
American Type Culture Collection (ATCC) strains and
S. aureus clinical isolates using 96-well fl at bottom TCP.
Initial inoculum, media, and incubation
In the fi rst step, we evaluated the effect of growth conditions
for the preparation of initial inoculum (solid medium BHI
agar vs. liquid medium TSB), effect of nutritional media for
generation of biofi lm (TSB vs. BHI broth), and incubation
time (6, 12, 18, and 24 h) at 37°C.
In the fi rst method, briefl y fresh isolates were inoculated in
TSB and BHI broth in stationary condition overnight at 37°C
and diluted 1 in 100 with fresh medium for subsequent use.
Each well of TCP was fi lled with 200 μl aliquots of the diluted
Figure 1: Distribution pattern of isolates of Staphylococcus aureus from
different outpatient departments and wards

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017 95
cultures and then investigated for biofi lm formation after 6,
12, 18, and 24 h at 37°C.
While in another method, the isolates were grown on BHI agar
overnight at 37°C. Then, colonies from overnight grown BHI
agar culture plates were suspended directly into physiological
saline (0.89% NaCl), and vortexed to achieve a suspension
of 0.5-McFarland turbidity (1.5 × 108 CFU/ml). Each well of
TCP was fi lled with 190 μl aliquots of BHI and then 10 μl of
bacterial suspension was added to it. Like above, the plates
were read after 6, 12, 18, and 24 h of incubation.
Fixation
After respective incubations, the plates were inverted and
gently tapped to remove residual broth. The wells were washed
thrice with 200 μl of phosphate buffer saline (PBS) (pH 7.2)
to remove planktonic bacteria before fi xation.
The two protocols as mentioned above were compared for
fi xation of cells in the plates by two different methods. In
the fi rst method, cells were fi xed with 200 μl of sodium
acetate (2% w/v) for 30 min, while in another, plates were
incubated for heat fi xation at 60°C for 20 min. After fi xation,
the plate with sodium acetate was washed with 200 μl PBS
thrice before staining.
Staining and elution
For staining, we used 175 μL of 0.5% crystal violet for 5 min.
The excess crystal violet was removed, and the plates were
washed with running tap water until runoff was clear. For
elution, we used 150 μl ethanol-acetone mixture (80:20)
and left at room temperature for 30 min. The elute was then
resuspended in wells of new TCP to take optical density (OD)
readings at λmax 550 nm in ELISA plate reader (Thermo
Scientifi c, USA).
Supplementation with sugars and salt
Glucose, sucrose, and NaCl in different molar concentrations,
namely, 55.6, 111.11, 166.7, and 222.2 mM for glucose; 29.2,
58.5, 116.9, and 175.4 mM for sucrose; and 500, 750, and
1000 mM for NaCl, respectively, were investigated to observe
for any possible effect on the biofi lm formation individually.
Based on the observations of maximum biofilm yielded
by supplementation of the individual ingredient, a solution
of optimum concentrations of glucose, sucrose, and
NaCl (supplement mix) was selected to supplement the above
method and the optimized method was then applied on all the
clinical isolates once again.
Categorization of isolates based on biofilm-forming
capacity
The following criteria were used for biofi lm gradation in
clinical isolates.
ODcut = O Davg of negative control + 3 × standard deviation (SD)
of ODs of negative control.
1. OD ≤ ODcut = Non-biofi lm-former (NBF)
2. ODcut < OD ≤ 2 × ODcut = Weak biofi lm-former (WBF)
3. 2 × ODcut < OD ≤ 4 × ODcut = Moderate biofi lm-former
(MBF)
4. OD ˃4 × ODcut = Strong biofi lm-former.
In this study, sterile broth and S. epidermidis ATCC 12228 served as
the negative control. However, S. epidermidis ATCC 35984 (high
slime producer) and ATCC 35983 (moderate slime producer) were
used as positive control. All experiments with clinical isolates were
done in quadruplet, i.e., each isolate were inoculated in four wells
simultaneously and repeated thrice (on different days), and then,
OD values were averaged and SD was calculated.
Statistical analysis
One-way ANOVA and one-tail t-test assuming equal variance
were used to compute and analyze the differences in OD values
obtained with different experimental variables of the in vitro
synthesis of biofi lm by TCP method. MS Excel data analysis
tool along with IBM SPSS version 21.0, Armonk, New York
was utilized for analysis. P ≤ 0.05 was considered statistically
signifi cant.
RESULTS
The following results were observed for different variables on
in vitro biofi lm synthesis by TCP assay in achieving conditions
required for maximum biofi lm biomass.
Effect of growth medium for harvesting bacterium for
inoculum preparation
Higher biofi lm formation was observed as inferred from increased
OD when initial bacterial inoculum was prepared from the growth
on BHI agar as compared to those grown in broths [Table 1].
Effect of growth medium
The absorbance was signifi cantly higher when BHI broth
was used as the nutritional medium as compared to TSB
(P = 0.00019, P < 0.05) [Figure 2 and Supplementary Data 2].
For instance, the average OD for S. epidermidis ATCC 35984
was 1.491 ± 0.017 (OD ± SD) in BHI broth, which was
34% higher when compared with average OD in TSB
(0.986 ± 0.019). Therefore, BHI broth was selected as the
medium for characterization of biofi lm formation of clinical
isolates of S. aureus in the present study.
Effect of incubation period
When ATCC control strains were assessed for the effect of
incubation period on biofi lm formation, maximum biofi lm
Table 1: Absorbance after in vitro biofilm assay using
tissue culture plates method using different initial
inoculums
Strains OD when
grown in broth
OD when grown
on BHI agar
ATCC 35984 1.452±0.019 1.961±0.017
ATCC 35983 0.471±0.013 0.577±0.016
ATCC 12228 0.106±0.016 0.197±0.014
BHI: Brain heart infusion, ATCC: American Type Culture Collection,
OD: Optical density

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017
96
yield was found after 24 h with resultant average OD
0.991 ± 0.021 for ATCC 35984, 0.433 ± 0.012 for ATCC
35983, and 0.102 ± 0.017 for ATCC 12228. It was observed
that after 6 h of incubation, the majority of the S. aureus
isolates displayed insignifi cant absorbances with average
OD ranging from 0.147 ± 0.0301 to 0.236 ± 0.0410. After
18 h, all isolates were found to produce biofi lms as refl ected
by relative absorbances. The average OD for one of the
isolates of S. aureus (Isolate number 27) was 0.358 ± 0.04,
0.511 ± 0.02, and 0.726 ± 0.04 at 12, 18, and 24 h, respectively.
The similar pattern was also observed for other isolates.
Statistically signifi cant (P = 0.0015) results were observed
after 24 h of incubation compared to 18 h of incubation and
therefore was considered as the optimum incubation period
for the assessment of biofi lm-forming capacity of S. aureus
[Figure 3 and Supplementary Data 3].
Effect of fixation
When ATCC control strains were assessed for fi xation by heat,
it was found that there is a statistically signifi cant increase in the
absorbance as compared to sodium acetate fi xation (P = 0.004)
with average resultant OD 1.491 ± 0.017 for ATCC 35984,
0.478 ± 0.016 for ATCC 35983, and 0.129 ± 0.014 for ATCC
12228. However, with sodium acetate, average absorbance was
found to be 0.973 ± 0.016 for ATCC 35984, 0.311 ± 0.021 for
ATCC 35983, and 0.073 ± 0.017 for ATCC 12228.
Upon heat fi xation, signifi cantly enhanced absorbance (average
OD 0.653 ± 0.075) was observed compared to sodium
acetate fi xation with average OD ranging from 0.15 ± 0.01 to
0.38 ± 0.09 for most of the S. aureus isolates.
Effect of glucose
It was observed that most of the clinical isolates displayed a
perceivable biofi lm-positive phenotype when BHI broth was
supplemented with glucose [Supplementary Data 4]. Glucose
in almost all concentrations was positively added to the biofi lm
formation, but highest absorbance was observed at 222.2 mM
glucose. However, individual concentrations of glucose had
no signifi cant effect on absorbance (P = 0.135) [Figure 4].
Effect of sucrose
It was noted that less number of clinical isolates displayed a
biofi lm-positive phenotype when BHI broth was supplemented
with sucrose (P = 0.21). Sucrose also had no signifi cant effect
on absorbance. However, it has shown maximum absorbance at
concentration of 116.92 mM. Beyond 116.92 mM concentration
saturation was observed and in some cases, even the loss in
the biofi lm was observed as refl ected by ODs [Figure 5 and
Supplementary Data 4].
Effect of sodium chloride
S. epidermidis reference strains have shown enhanced
absorbance although observations were not statistically
signifi cant (P = 0.67). However, the response of S. aureus
was varying. It was observed that all the methicillin-sensitive
S. aureus (MSSA) isolates showed enhanced biofi lm phenotype
compared to methicillin-resistant S. aureus (MRSA) isolates
[Supplementary Data 5]. Although, upon supplementation
of NaCl, the enhancement was not statistically signifi cant
(P = 0.84) [Figure 6], highest absorbance was observed at
1000 mM NaCl.
Biofilm synthesis by clinical isolates of Staphylococcus
aureus employing proposed modified tissue culture plate
method
Based on the observations of different variables of in vitro
biofi lm synthesis including sugars and NaCl concentration
as described above, all the stains were subjected to biofi lm
formation on the selected combination of 222.2 mM glucose,
116.9 mM sucrose, and 1000 mM NaCl (supplement mix).
A signifi cant increase in the biofi lm formation (P = 0.031) was
observed after supplementation as compared to unsupplemented
BHI broth [Figures 7, 8 and Supplementary Data 6].
Categorization of Staphylococcus aureus isolates based
on biofilm-forming capacity
We tried to establish criteria for categorizing S. aureus isolates
based on their biofi lm-forming capacity. Based on the results
Figure 2: Enhancement in biofilm formation by clinical isolates of
Staphylococcus aureus using brain heart infusion and tr ypticase soy broth
Figure 3: Effect of incubation period on absorbance by clinical isolates
of Staphylococcus aureus

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017 97
obtained from TCP assay with supplement mix, a cut-off
OD (ODcut) was obtained by taking the average of all the ODs
of the negative control ATCC 12228 and thrice the value of
SD of the negative control was added to it.
In this study, the average OD of the negative control came to
be 0.147 ± 0.0305. Hence, the cutoff OD value in the current
study was set as 0.238. The isolates which have OD value lesser
than 0.238 were considered as NBFs [Table 2].
Upon employing differentiation criterion adopting ODcut, all
the 61 clinical isolates were observed to be biofi lm formers by
proposed method using supplement mix in this study. However,
15 (24.5%) isolates were observed to be non-former of biofi lm
by unsupplemented TCP method. Out of these 15 non-former
strains, 9 were MSSA and 6 were MRSA. Upon addition of
supplement mix, of total 9 NBF MSSA isolates, two (isolate
no. 28, 36) showed medium grade biofi lm and the rest seven
showed low-grade biofi lm formation, i.e., no isolate showed
the non-biofi lm producer phenotype. Similar to MSSA, upon
Figure 4: Effect of different concentrations of glucose supplementations
on absorbance
Figure 5: Effect of different concentrations of sucrose supplementations
on absorbance
Figure 6: Effect of different concentrations of sodium chloride
supplementations on absorbance Figure 7: Effect of optimized supplement mix on absorbance
Table 2: Categorization of biofilm made by strains of Staphylococcus aureus (n=61)
Average OD range Biofilm grade Number of strains older method Number of strains proposed method
<0.238 Non-former 15+1$0+1$
≥0.238 but ≤0.477 Low biofi lm former 20 13
≥0.477 but ≤0.954 Moderate biofi lm former 16+1@32+1@
≥0.954 High biofi lm former 10+1#16+1#
$ATCC 12228, @ATCC 34983, #ATCC 34984. ATCC: American Type Culture Collection, OD: Optical density

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017
98
supplementation of the supplement mix, all previously NBF
MRSA isolates showed enhanced biofi lm formation on the
addition of supplement mix. Of six NBF MRSA isolates, 5
shifted to low biofi lm-former grade while one (isolate no. 52)
showed medium-former grade phenotype (more enhance
biofi lm phenotype). All the low biofi lm-former (n = 4) showed
medium-biofilm forming phenotype except one (isolate
no. 41), which retained its low biofi lm-forming phenotype.
Without supplementation, only 11 MBF isolates were
observed. However, only 5 (45.45%) showed the shift into a
high biofi lm-former grade (isolate no. 3, 17, 27, 29, and 49) and
the remaining 6 isolates (isolate no. 4, 9, 12, 23, 24, and 26)
retained their biofi lm grade even after adding the supplement
mix [Table 3 and Supplementary Data 7].
Effect of opting Staphylococcus epidermidis ATCC 12228
as the negative control
It was observed that the ODs of moderate and high biofi lm
producing ATCC strains of S. epidermidis lied repeatedly in
the range of 2 × ODcut < OD ≤4 × ODcut and OD ˃4 × ODcut
respectively with respect to the non-former ATCC 12228
strain. Therefore, opting S. epidermidis ATCC 12228 as
the negative control was considered to be more useful in
deciding the precise cut-off criteria rather than the broth alone
[Figure 8 and Table 1].
The optimized protocol for the in vitro synthesis of biofi lm
by TCP assay for clinical isolates of S. aureus has been
summarized in Figure 9.
Antimicrobial sensitivity pattern
Out of 61 clinical isolates of S. aureus, 18 (29.51%) were MRSA.
The majority of S. aureus isolates were found to be resistant to more
than 9 antibiotics. All the clinical isolates were found to be sensitive
to linezolid and netilmicin. Only 3 isolates were penicillin sensitive.
Isolates have shown lesser susceptibility toward ciprofl oxacin as
the majority was either resistant or intermediate susceptible. The
majority of isolates (n = 37) showed intermediate resistance to the
erythromycin. However, compared to azithromycin, the incidence
of resistance was lesser with erythromycin. Most of the isolates
(n = 44) were resistant to co-trimoxazole. Four isolates were
resistant to fusidic acid while mupirocin resistance was detected
in only one strain [Supplementary Data 1]. Strong and moderate
biofi lm-producing isolates were found to be more resistant to
commonly used antibiotics compared to weak producing ones
[Table 4].
DISCUSSION
Biofi lm is a sessile microbial community wherein cells are
attached to a surface (biotic or abiotic) and are enmeshed within
a self-produced protective extracellular polymeric matrix. This
extracellular polymeric matrix in S. aureus/S. epidermidis is
poly-N-acetyl glucosamine (PNAG).[20] There are cases where
PNAG-independent proteinaceous biofi lms are also reported
in S. aureus.[21,22]
Schleifer and Kroppenstedt reported the surface association of
the infecting bacteria and speculated similarity of solid agar
Figure 8: Effect of supplementation - A phenotypic view. Lane 1: Row
A, B, C, and D show the unsupplemented brain-heart infusion while Row
E, F, G, and H show the supplemented brain-heart infusion for ATCC
1228. Lane 2: Row A, B, C, and D show the unsupplemented brain
heart infusion while Row E, F, G, and H show the effect of supplemented
brain-heart infusion for ATCC 35983. Lane 7: Row A, B, C, and D show
the unsupplemented brain-heart infusion while Row E, F, G and H show
the effect of supplemented brain-heart infusion for ATCC 35984. Lane
11: Row A, B, C, and D show the unsupplemented brain-heart infusion
while Row E, F, G, and H show the effect of supplemented brain-hear t
infusion for negative control
Table 3: Distribution of isolates in different classes in
toto and selective distribution of methicillin-susceptible
Staphylococcus aureus and methicillin-resistant
Staphylococcus aureus isolates in different classes
Total Before After MRSA Before After MSSA Before After
HBF 10 16 HBF 3 4 HBF 7 16
MBF 17 32 MBF 5 8 MBF 11 32
LBF 19 13 LBF 4 6 LBF 16 13
NBF 15 0 NBF 6 0 NBF 9 0
MRSA: Methicillin-resistant Staphylococcus aureus,
MSSA: Methicillin-susceptible Staphylococcus aureus, NBF: Nonbiofi lm
former, MBF: Moderate biofi lm forming, LBF: Low biofi lm formers,
HBF: High biofi lm former
Fixation
Inoculum,
incubation
& Media
Staining
and
elution
• Grow isolates overnight on BHI agar at 37oC
• Then prepare 0.5 McFarland bacterial suspension using physiological saline
• Fill TCP wells with 190 µl BHI broth supplemented with 222.2 mM glucose,
116.9 mM sucrose and 1000 mM NaCl and then add 10 µl of bacterial
suspension to it. Then incubate for next 24 h at 37oC
• S. apidermidis ATCC 35984, 35983, 12228 were included in each run.
• Remove residual BHI broth and wash thrice with PBS
• Heat fix at 60oC for 20 min in incubator.
• Stain with 175 µl of 0.5% crystal violet dye for 5 min
• Wash off the exess stain under tap water. Air dry the plate
• Then, elute the dye with 150 µl of alcohol:acetone (80:20)
• Resuspend the dye in other plate and read the absorbance at 550 nm
wavelength
• Result will be considered valid only when OD of the medium and high biofilm
formers are within the OD range of cutoff defined for these strains.
Figure 9: A simplified flowchart of the proposed method

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017 99
grown bacteria to natural infection settings and then to the
pathogens grown in liquid media.[23] When initial inoculum
was prepared from the bacteria grown on BHI agar, we noticed
their comparatively higher effi ciency in biofi lm production as
compared to those grown in broths. This could be probably
a result of the higher expression of surface proteins required
for adherence when bacteria are grown on solid media. The
expression of these proteins is also reported as a prerequisite
for infectivity in various studies.[24]
The richness of nutrients is another important factor which
infl uences the ability of bacteria to produce biofi lm.[15] Some
investigators have utilized TSB for biofi lm quantitation.[25,26] In
the current study, BHI broth was found to be signifi cantly more
effective in biofi lm formation [Supplementary Data 2]. Proteins
especially rich in leucine, proline, serine, and aspartate are
abundant in BHI broth since these amino acids may be essential
for the production of adhesins such as fi bronectin-binding
protein and clumping factors which are necessary for adherence.
The presence of lipids such as choline and sphingosine in BHI
may have added advantage in biofi lm formation and provide
resistance from desiccation. Further, it is a source of sugars
such as inositol/myoinositol which cannot be fermented by
S. aureus leading to resistance in pH fall, which, in turn,
may be needed for robust biofi lm architecture. These results
indicate a strong dependence of biofi lm formation in S. aureus
and the environmental conditions required for growth, which
seems to be even more pronounced in S. aureus than in
S. epidermidis.[22,27-29] Similarly, while observing the effect of
incubation period on in vitro biofi lm formation, it was noticed
that after 6 h of incubation, the majority of the S. aureus isolates
remained NBF and for some of the isolates biofi lms were even
non-detectable. Adhesion of bacterial cells to microtiter plate
appeared to be a function of time and increased linearity was
observed with time progression. Although biofi lm formation
was observed in all isolates after 18 h of incubation, the
maximum biofi lm yield as refl ected in ODs was observed after
24 h of incubation as also noticed by other investigators.[15,16]
The fi xation of attached cells by heating at 60°C for 20 min was
found to be statistically more signifi cant than fi xation by sodium
acetate in our study. Therefore, we opted for heat fi xation. Heat
disrupts hydrogen bonds and non-polar hydrophobic interactions
of bacterial cell surface proteins leading to coagulation and in
some cases its denaturation. Further, it dehydrates the sugar
content leading to the crude biomass estimation. While sodium
acetate has a protective effect against denaturation.[30] These results
are in consonance with the observations of Baldassarri et al.[31]
During elution step, only 150 μL of eluent (ethanol:acetone
[80:20]) was added per well, to evade interference with
the stained matter at the liquid–air interface, which is not
considered to be indicative of biofi lm formation.
We examined the biofi lm formation in both MRSA and MSSA
isolates in media supplemented with different concentration of
glucose, sucrose, and NaCl. Although the addition of sugars
and salts individually has increased the biofi lm phenotype
as manifested by an increase in OD, it was not statistically
signifi cant (P ˃ 0.05). On the other hand, when the supplement
mix was added to the broth in a defi ned ratio, the signifi cant
increase in OD was observed (P = 0.037, P < 0.05). Therefore,
it is strongly recommended to use the proposed method for
in vitro biofi lm quantitation.
Among MSSA isolates, isolate-to-isolate variation was
observed with respect to biofi lm-forming ability with nature
of supplementation used. Glucose in almost all concentrations
was positively added to the biofi lm formation while sucrose at
concentration beyond 116.92 mM showed almost saturation and
in some cases even the loss in the biofi lm. NaCl at 1000 mM
concentration showed the maximum increase in absorbance.
This observation was found consistent with Lim et al. who
found enhanced expression of rbf gene involved in the signal
transduction pathway for biofi lm production when the NaCl
concentration is above 1.6% but not when it is below 1.6%.[17]
While observing biofi lm synthesis by MRSA isolates, the
strong correlation existed between the biofilm phenotype
Table 4: Biofilm-forming ability of strains of different resistance pattern
Name of antibiotic Isolate resistance
including intermediate
percentage
Biofilm grade
Penicillin (10 units) 95.08 (58/61) 2 sensitive strains are weak biofi lm formers while one is strong former
Cefoxitin (30 mcg) 29.51 (18/61) 5 resistant strains were high former, 7 were medium formers, and 6 were
weak formers
Erythromycin (15 mcg) 83.6 (51/61) 5 resistant strains were high formers, 2 were medium formers while 7 were
weak formers
Trimethoprim and sulfamethoxazole (25 mcg) 83.6 (51/61) 22 were weak formers, 11 were strong formers, and 11 were medium formers
Clindamycin (2 mcg) 8.19 (5/61) 3 were weak formers and 2 were medium formers
Azithromycin (15 mcg) 40.98 (25/61) 8 were strong formers, 4 were medium formers, and 7 were weak formers
Ciprofl oxacin (5 mcg) 93.44 (57/61) 9 were strong formers, 11 were medium formers, and 12 were weak formers
Moxifl oxacin (5 mcg) 72.13 (44/61) 11 were strong formers, 10 were medium formers, and 13 were weak formers
Amoxicillin and clavulanate (30 mcg) 45.90 (28/61) 7 were strong formers, 11 were medium formers, and 9 were weak formers
Mupirocin (200 mcg) 1.63 (1/61) 1 is medium former
Fusidic acid (10 mcg) 6.55 (4/61) 1 is strong former, 2 are medium formers, and 1 is weak former

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017
100
and the concentration of the sugar supplemented. Even some
isolates showed exceptional behavior to this generalized
rule [Supplementary Data 3-5]. Although this sort of
heterogeneity in biofi lm-forming capacity of MRSA has been
addressed earlier, isolate-wise exceptional behavior has never
been highlighted. Each isolate responded differently from one
another regarding response to the sugar and, in turn, in biofi lm
phenotype. Pozzi et al. (2012) proposed that acquisition of
methicillin resistance appears to repress polysaccharide-type
biofi lm production and promote the formation of proteinaceous
biofi lms as evidenced by biofi lm phenotype observations made
in the present study.[32,33] However, there are certain MRSA
isolates which showed the exception to this generalized rule. The
universality to this generalized rule is just an enigma. Biofi lm
development in MRSA isolates is primarily glucose induced
but not solely, and apparently, involves a protein adhesin.[21,34]
Till date, there is no consensus view regarding categorization
of S. aureus isolates based on their biofi lm-forming capacity.
Therefore, the definition of a strong, medium, weak, and
non-biofi lm producer varies greatly among the studies.[15,16,35,36]
Mathur et al. have recently proposed the criteria for grading the
isolates based on their ability to form a biofi lm which considered
non-former isolates when the OD was <0.120, while OD range for
medium-former was ˃0.120–≤0.240 and for those of high former
was >0.240.[16] Similarly, Stepanovic et al. have also proposed
the criteria for biofi lm classifi cation and used the same old gold
standard of Christensen et al. using the same ATCC 35984, 35983,
and 12228 reference isolates.[15] Christensen et al. have used only
an approximation of distance plotted in a graph, by dividing
the graph into three portions: nonadherent (OD in both media,
<0.120), weakly adherent (OD in either medium, >0.120 but
0.240), and strongly adherent (OD in either medium, >0.240).[37]
In the present study, a need of new cut-off criteria was felt
because of the aforesaid reason and signifi cant increase of the
OD expanding the limit of OD in previously described non,
moderate, and high biofi lm-former category. A plethora of
literature is available where only broth was taken as the negative
control. In this study, S. epidermidis ATCC 12228 as the negative
control was found to be more accurate in deciding the precise
cutoff criteria rather than the broth alone. Broth can be used to
ensure the sterility during the execution of the experiment. As
negative and positive controls are a must in any experimental
setup, we propose the OD cutoff criteria based on the OD of
the negative control and the addition of some factor to its SD
value. And then, various multiples (even) of ODcut can be used
to distinguish clinical isolates based on their biofi lm-forming
capacities. By adopting the proposed method and criteria, it
was observed that reference strains ATCC 35984, 35983, and
12228 remained in their respective classes as high, medium,
and non-formers. However, it was interesting to observe that
when the new criterion was applied on all the clinical isolates of
S. aureus, all the previously declared nonformer isolates were
either shifted to low former or to the medium-former category.
Therefore, instead of using uninoculated broth, ATCC 12228
may be used as negative control for error free and concordant
results. This method can, therefore, be unequivocally used for
all clinical staphylococcal isolates to adapt the low/WBFs as
reported by other investigators also.[15,16,28,38]
It was observed that ODs of a number of clinical isolates
of S. aureus lied between the non and the moderate biofi lm
range. Therefore, a new category of WBFs is needed to be
introduced in the study of biofi lm quantitation and also for
the sake of uniformity. To further strengthen the validity of
results on biofi lm quantitation, one may need a higher number
of reference strains of both S. epidermidis as well as S. aureus
of all the four grades of biofi lm producers.
In the present study, strong and MBF isolates were found to
be more resistant to commonly used antibiotics compared to
WBFs. Strong biofi lm producers are more adapted pathogenic
strains and have acquired resistance over the period due to
continuous exposure to the antibiotics or by acquiring genes
through horizontal gene transfer or by both. This may be the
consequence of biofi lm providing an appropriate environment
for the transfer of drug resistance determinants.[39]
Further, investigators claimed that as much as thousand times
increased MIC of biofi lm-dwelling cells than the planktonic
cells.[1] This may be due to interruptions posed by the biofi lm slimy
matrices in the form of electrostatic repulsion and/or sequestration
of antibacterial substances apart from being diffusion barrier.[40,41]
There are attempts which were made to design a number of
anti-biofi lm compounds mainly short peptides, which seems to be
promising strategy against staphylococcal biofi lm.[42] However, in
the future, for these and several other candidate drugs, there will
be a need for a standardized method for in vitro biofi lm synthesis
by S. aureus along with classifi cation criterion for conclusive
authentication of drugs as potential antibiofi lm agents.
However, the limitation of the current study is that the method
of biofilm formation proposed here may not be useful for
Gram-negative isolates. This is because, among Gram-negative
bacteria, altogether, different operon arrays are responsible for
controlling biofi lm biogenesis. In Gram-negative bacteria, some of
the polysaccharides are neutral or polyanionic due to the presence
of uronic acids or ketal-linked pyruvates.[40] However, classifi cation
criteria can be used with properly established negative control.
CONCLUSIONS
The results indicate that the different variables including
supplement mix containing glucose, sucrose, and NaCl in a
defi ned ratio enhances the biofi lm-forming ability of S. aureus
significantly in the proposed method of in vitro biofilm
formation assay employing TCP. The present study puts forth
a standardized in vitro TCP assay for biofi lm synthesis by
S. aureus and its categorization indicating their differential
ability to produce biofi lm. The proposed in vitro technique
may be further evaluated for its usefulness in the management
of persistent infections caused by the bacteria.
Financial support and sponsorship
The present work is accomplished by the grant sanctioned

Singh, et al.: Standardization and classifi cation of Staphylococcus aureus biofi lms
Journal of Global Infectious Diseases ¦ Volume 9 ¦ Issue 3 ¦ July-September 2017 101
as contingency grant offered to AKS as JRF by University
Grants Commission, New Delhi, and DST-PURSE GRANT
sanctioned to Department of Microbiology, Institute of Medical
Sciences, Banaras Hindu University.
Conflict of interest
There are no confl icts of interest.
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Supplementary Data 1: Epidemiological profile and resistance pattern of Staphylococcal strains
Strain number Clinical strain number Resistant to antimicrobials Ward/OPD
1 1114/2015 1, 4, 8, 11 Skin/pus
2 1115/2015 1, 4, 10, 11 Skin/pus
3 774/2015 10 ICU/ET aspirate
4 1269/2015 1, 8, 12, 10, 11,12 ICU/ET aspirate
5 792/2015 1, 2, 13, 8,10, 11 Orthopedics/pus
6 1229/2015 1, 4, 8, 10, 11 Skin/pus
7 1975/2015 1, 4, 8, 11 Skin/pus
8 1968/2015 1, 4, 8, 10 Skin/pus
9 1360/2015 1, 3, 4, 6, 8, 10, 11 Pediatrics emergency/pleural fl uid
10 1458/2015 1, 4, 10 Orthopedics/pus
11 1659/2015 1, 4, 8, 10 Skin/pus
12 1573/2015 1, 8, 10, 11 Skin/pus
13 2037/2015 1, 4, 11 Orthopedics/pus
14 2018/2015 1, 3, 4, 6, 8,10 Skin/pus
15 2034/2015 1, 3, 4, 6, 8, 10 Skin/pus
16 771/2015 1, 2, 4, 8, 10 SOPD/pus
17 775/2015 1, 6, 8 NSW/pus
18 876/2015 1, 3, 4, 6, 8, 10 SOPD/pus
19 2028/2015 1, 2, 3, 6, 8, 10, 11 NSW/tracheal aspirate
20 1104/2015 1, 4, 10, 11 Skin/pus
21 699/2015 1, 2, 3, 4, 6, 10, 11 HDU/IJV tip
22 1115/2015 1, 4, 10, 11 Skin/pus
23 1114/2015 1, 4, 6, 8 Skin/pus
24 749/2015 1, 4 SOPD/pus
25 1371/2015 1, 4, 11 ENT/pus
26 1378/2015 1, 4, 8 Pediatrics emergency/pus
27 753/2015 1, 4, 11 SOPD/pus
28 704/2015 4, 8, 11 SOPD/pus
29 3862/2015 1, 4, 6 SOPD/pus
30 4042/2015 1, 2, 3, 4, 6, 8, 10, 11 Skin/pus
31 492/2015 1, 11 Orthopedics/pus
32 962/2015 1, 2, 3, 4, 6, 8, 10 Urology/pus
33 2179/2015 1, 2, 4, 11 Skin/wound
34 619/2015 1, 4 Gynecology/HVS
35 961/2015 1, 4, 8 Orthopedics/pus
36 742/2015 1, 3, 5, 6 Orthopedics/pus
37 619/2015 1, 2, 4, 8, 10, 11 HDU/pus
38 625/2015 1, 5, 6 Neurology/pus
39 758/2015 1, 2, 10 HDU/IJV tip
40 756/2015 1, 2, 4, 8, 10, 11 SOPD/pus
41 699/2015 1, 2, 3, 4, 5, 8, 10, 11 HDU/IJV tip
42 994/2015 1, 4, 11, 12 SOPD/pus
43 935/2015 4, 8, 10 ENT/pus (ear swab)
44 869/2015 1, 4, 11 SOPD/pus
45 394/2015 1, 2, 3, 4, 5, 6 SOPD/pus
46 775/2015 1, 8, 10 NSW/pus
47 676/2015 1, 4, 8, 10, 11 Orthopedics/pus
48 617/2015 1, 2, 3, 4, 6 Skin/pus
49 517/2015 1, 3, 6, 12 SOPD/pus
50 999/2015 1, 4, 8, 10 SOPD/pus
51 740/2015 1, 11 Medicine/pus
52 408/2015 1, 2, 3, 6, 13 NICU/pus
53 719/2015 1, 2, 8, 10 Orthopedic/pus
54 328/2015 1, 4 SOPD/pus
Contd...

Supplementary Data 1: Contd...
Strain number Clinical strain number Resistant to antimicrobials Ward/OPD
55 907B/2015 1, 4, 10 Orthopedic/pus
56 643/2015 1, 4, 10 Orthopedic/pus
57 545/2015 1, 6, 8, 10, 12 Skin/pus
58 1823/2015 1, 4, 8, 10 SOPD/pus
59 771B/2015 1, 2, 4, 8, 10, 11 SOPD/pus
60 2972/2015 1, 2, 4, 11 Skin/pus
61 394/2015 1, 2, 3, 6, 10 SOPD/pus
Penicillin (1), Cefoxitin (2), Erythromycin (3), Trimethoprim and Sulfamethoxazole (4) , Clindamycin (5), Azithromycin (6), Linezolid (7), Ciprofl oxacin (8),
Netilmicin (9), Moxifl oxacin (10), Amoxicillin and clavulanate (11), Fusidic Acid (12), Mupirocin (13). OPD: Outpatient department, ICU: Intensive Care
Unit, SOPD: Surgical outpatient department, HDU: High dependency unit, ENT: Ear, nose, and throat, NICU: Neonatal Intensive Care Unit, ET: Endotracheal
aspirate, HVS: High vaginal swab, IJV: Internal jugular venous catheter tip
Supplementary Data 2: Comparison of OD’s when media
used was trypticase soy broth and brain-heart infusion
over biofilm formation
Strain number With TSB With BHI
1 0.197 0.253
2 0.211 0.298
3 0.438 0.827
4 0.329 0.594
5 0.327 0.595
6 0.981 2.006
7 0.183 0.269
8 0.199 0.376
9 0.349 0.606
10 0.207 0.36
11 0.276 0.445
12 0.342 0.579
13 0.873 1.715
14 0.826 1.667
15 0.823 1.404
16 0.867 1.631
17 0.329 0.697
18 0.798 1.13
19 0.812 1.351
20 0.849 1.606
21 0.753 1.024
22 0.831 1.462
23 0.506 0.827
24 0.379 0.614
25 0.237 0.428
26 0.411 0.666
27 0.527 0.726
28 0.103 0.247
29 0.627 0.901
30 0.103 0.22
31 0.173 0.325
32 0.091 0.194
33 0.397 0.663
34 0.206 0.318
35 0.197 0.306
36 0.124 0.236
37 0.631 0.927
Contd...

Supplementary Data 2: Contd...
Strain number With TSB With BHI
38 0.122 0.233
39 0.193 0.349
40 0.147 0.28
41 0.185 0.301
42 0.186 0.329
43 0.185 0.328
44 0.069 0.153
45 0.073 0.174
46 0.213 0.428
47 0.207 0.362
48 0.239 0.472
49 0.403 0.768
50 0.117 0.303
51 0.079 0.174
52 0.109 0.211
53 0.053 0.157
54 0.069 0.172
55 0.097 0.144
56 0.062 0.141
57 0.091 0.187
58 0.257 0.409
59 0.328 0.563
60 0.103 0.199
61 0.421 0.627
TSB: Trypticase soy broth, BHI: Brain-heart infusion, OD: Optical density
Supplementary Data 3: Contd...
Strain number After 6 h After 12 h After 18 h After 24 h
15 0.181 0.387 0.828 1.404
16 0.192 0.401 0.913 1.631
17 0.163 0.307 0.511 0.697
18 0.422 0.681 0.904 1.13
19 0.487 0.624 0.926 1.351
20 0.513 0.731 0.964 1.606
21 0.399 0.573 0.869 1.024
22 0.483 0.631 0.915 1.462
23 0.161 0.391 0.623 0.827
24 0.114 0.309 0.481 0.614
25 0.093 0.119 0.207 0.428
26 0.142 0.286 0.434 0.666
27 0.185 0.358 0.511 0.726
28 - 0.087 0.111 0.247
29 0.245 0.417 0.689 0.901
30 - 0.083 0.108 0.22
31 - 0.093 0.194 0.325
32 - - 0.096 0.194
33 0.153 0.321 0.469 0.663
34 - 0.089 0.194 0.318
35 - 0.097 0.204 0.306
36 - 0.084 0.121 0.236
37 0.369 0.583 0.716 0.927
38 - 0.076 0.109 0.233
39 0.094 0.123 0.211 0.349
40 0.076 0.114 0.186 0.28
41 0.086 0.128 0.195 0.301
42 0.098 0.154 0.216 0.329
43 0.098 0.155 0.213 0.328
44 - - 0.083 0.153
45 - - 0.094 0.174
46 0.091 0.157 0.239 0.428
47 - 0.134 0.209 0.362
48 0.103 0.211 0.342 0.472
49 0.218 0.467 0.532 0.768
50 - 0.131 0.214 0.303
51 - - 0.086 0.174
52 - - 0.153 0.211
53 - - 0.096 0.157
54 - - 0.102 0.172
55 - - 0.088 0.144
56 - - 0.087 0.141
57 - - 0.107 0.187
58 0.104 0.211 0.297 0.409
59 0.159 0.267 0.385 0.563
60 - - 0.088 0.199
61 0.166 0.310 0.423 0.627
OD: Optical density
Supplementary Data 3: Comparative OD’s when subjected
to four different incubation periods
Strain number After 6 h After 12 h After 18 h After 24 h
1 - 0.061 0.120 0.253
2 - 0.068 0.137 0.298
3 0.151 0.342 0.528 0.827
4 0.097 0.143 0.337 0.594
5 0.089 0.152 0.362 0.595
6 0.316 0.581 1.211 2.006
7 - 0.062 0.171 0.269
8 - 0.087 0.229 0.376
9 0.105 0.294 0.459 0.606
10 - 0.083 0.213 0.36
11 0.061 0.192 0.306 0.445
12 0.084 0.216 0.389 0.579
13 0.187 0.396 0.838 1.715
14 0.187 0.394 0.832 1.667
Contd...

Supplementary Data 4: Effect of glucose and sucrose supplementation in different concentration over biofilm formation by
proposed method
Serial
number
OD at glucose
(55.55 mM)
OD at glucose
(111.11 mM)
OD at glucose
(222.22 mM)
OD at glucose
(333.33 mM)
OD at sucrose
(29.23 mM)
OD at sucrose
(58.47 mM)
OD at sucrose
(116.92 mM)
OD at sucrose
(175.38 mM)
OD without
any sugar
1 0.359 0.362 0.397 0.411 0.352 0.359 0.361 0.363 0.253
2 0.371 0.379 0.403 0.415 0.37 0.378 0.382 0.382 0.298
3 0.83 0.851 0.897 0.898 0.829 0.828 0.837 0.836 0.827
4 0.594 0.613 0.679 0.679 0.591 0.626 0.631 0.629 0.594
5 0.598 0.613 0.627 0.629 0.597 0.609 0.613 0.613 0.595
6 2.013 2.097 2.169 2.182 2.009 2.017 2.157 2.161 2.006
7 0.431 0.472 0.498 0.498 0.441 0.463 0.469 0.469 0.269
8 0.383 0.397 0.418 0.423 0.381 0.398 0.419 0.417 0.376
9 0.612 0.619 0.647 0.649 0.619 0.638 0.641 0.639 0.606
10 0.367 0.371 0.383 0.385 0.368 0.378 0.378 0.373 0.36
11 0.447 0.451 0.468 0.467 0.445 0.458 0.456 0.46 0.445
12 0.581 0.589 0.596 0.598 0.581 0.587 0.591 0.589 0.579
13 1.781 1.792 1.851 1.857 1.737 1.793 1.797 1.787 1.715
14 1.673 1.681 1.837 1.838 1.669 1.783 1.769 1.719 1.667
15 1.405 1.521 1.581 1.589 1.459 1.539 1.517 1.514 1.404
16 1.636 1.641 1.724 1.729 1.633 1.685 1.639 1.646 1.631
17 0.713 0.727 0.893 0.854 0.722 0.849 0.798 0.824 0.697
18 1.139 1.142 1.267 1.249 1.131 1.187 1.191 1.159 1.13
19 1.362 1.367 1.457 1.413 1.356 1.394 1.369 1.373 1.351
20 1.616 1.621 1.763 1.753 1.61 1.735 1.714 1.719 1.606
21 1.049 1.057 1.126 1.129 1.024 1.098 1.107 1.108 1.024
22 1.476 1.491 1.587 1.551 1.462 1.498 1.463 1.469 1.462
23 0.831 0.837 0.869 0.865 0.829 0.854 0.856 0.856 0.827
24 0.618 0.621 0.659 0.657 0.616 0.643 0.643 0.643 0.614
25 0.613 0.628 0.643 0.648 0.599 0.629 0.634 0.631 0.428
26 0.669 0.673 0.741 0.743 0.668 0.724 0.729 0.726 0.666
27 0.729 0.732 0.791 0.793 0.726 0.751 0.757 0.759 0.726
28 0.249 0.257 0.353 0.361 0.247 0.339 0.327 0.318 0.247
29 0.912 0.933 0.967 0.971 0.917 0.958 0.961 0.964 0.901
30 0.224 0.239 0.297 0.291 0.225 0.231 0.263 0.261 0.22
31 0.331 0.348 0.409 0.417 0.329 0.389 0.396 0.394 0.325
32 0.206 0.218 0.237 0.263 0.201 0.214 0.269 0.268 0.194
33 0.670 0.677 0.729 0.732 0.668 0.687 0.693 0.691 0.663
34 0.321 0.329 0.441 0.452 0.318 0.396 0.379 0.379 0.318
35 0.311 0.319 0.458 0.461 0.313 0.321 0.436 0.438 0.306
36 0.241 0.249 0.354 0.355 0.236 0.327 0.33 0.331 0.236
37 0.934 0.941 1.027 1.033 0.934 0.987 0.997 0.989 0.927
38 0.239 0.243 0.328 0.331 0.235 0.306 0.314 0.31 0.233
39 0.352 0.357 0.373 0.362 0.352 0.351 0.362 0.362 0.349
40 0.289 0.297 0.309 0.316 0.282 0.287 0.304 0.306 0.28
41 0.311 0.324 0.331 0.334 0.307 0.319 0.327 0.329 0.301
42 0.337 0.342 0.425 0.429 0.336 0.391 0.397 0.395 0.329
43 0.334 0.357 0.478 0.481 0.331 0.437 0.442 0.442 0.328
44 0.159 0.172 0.269 0.273 0.159 0.247 0.253 0.251 0.153
45 0.181 0.187 0.194 0.196 0.176 0.182 0.189 0.193 0.174
46 0.434 0.497 0.583 0.59 0.434 0.517 0.523 0.524 0.428
47 0.369 0.376 0.458 0.462 0.367 0.427 0.439 0.443 0.362
48 0.478 0.487 0.512 0.523 0.475 0.489 0.516 0.516 0.472
49 0.775 0.813 0.871 0.883 0.771 0.828 0.841 0.84 0.768
50 0.379 0.385 0.436 0.447 0.374 0.414 0.415 0.415 0.303
51 0.196 0.265 0.305 0.313 0.183 0.287 0.298 0.301 0.174
52 0.219 0.227 0.231 0.239 0.211 0.221 0.229 0.226 0.211
Contd...

Supplementary Data 5: Effect of sodium chloride supplementation in different concentration over biofilm formation by
proposed method
Serial
number
OD at 500 mM NaCl
concentration
OD at 750 mM NaCl
concentration
OD at 1000 mM NaCl
concentration
OD without
supplementation
1 0.363 0.387 0.419 0.253
2 0.384 0.397 0.421 0.298
3 0.852 0.871 0.892 0.827
4 0.621 0.653 0.676 0.594
5 0.597 0.602 0.614 0.595
6 2.107 2.162 2.193 2.006
7 0.443 0.467 0.498 0.269
8 0.391 0.422 0.443 0.376
9 0.615 0.639 0.653 0.606
10 0.373 0.382 0.406 0.36
11 0.457 0.492 0.509 0.445
12 0.588 0.595 0.593 0.579
13 1.767 1.793 1.816 1.715
14 1.69 1.715 1.742 1.667
15 1.419 1.543 1.578 1.404
16 1.635 1.657 1.691 1.631
17 0.734 0.829 0.883 0.697
18 1.171 1.235 1.327 1.13
19 1.361 1.37 1.377 1.351
20 1.621 1.644 1.72 1.606
21 1.068 1.099 1.114 1.024
22 1.647 1.704 1.769 1.462
23 0.846 0.872 0.891 0.827
24 0.627 0.649 0.67 0.614
25 0.644 0.671 0.676 0.428
26 0.729 0.757 0.783 0.666
27 0.742 0.787 0.799 0.726
28 0.358 0.358 0.381 0.247
29 0.951 0.963 0.977 0.901
30 0.229 0.237 0.242 0.223
31 0.373 0.399 0.413 0.325
32 0.208 0.237 0.244 0.194
33 0.674 0.687 0.706 0.663
34 0.352 0.397 0.449 0.318
Supplementary Data 4: Contd...
Serial
number
OD at glucose
(55.55 mM)
OD at glucose
(111.11 mM)
OD at glucose
(222.22 mM)
OD at glucose
(333.33 mM)
OD at sucrose
(29.23 mM)
OD at sucrose
(58.47 mM)
OD at sucrose
(116.92 mM)
OD at sucrose
(175.38 mM)
OD without
any sugar
53 0.168 0.172 0.187 0.187 0.163 0.168 0.183 0.184 0.157
54 0.181 0.251 0.293 0.309 0.179 0.264 0.281 0.281 0.172
55 0.157 0.237 0.27 0.276 0.153 0.249 0.261 0.268 0.144
56 0.151 0.231 0.272 0.272 0.149 0.243 0.251 0.251 0.141
57 0.188 0.241 0.279 0.287 0.19 0.252 0.26 0.264 0.187
58 0.419 0.496 0.571 0.58 0.412 0.511 0.52 0.523 0.409
59 0.569 0.581 0.617 0.619 0.566 0.593 0.606 0.609 0.563
60 0.217 0.226 0.239 0.241 0.222 0.222 0.236 0.241 0.199
61 0.638 0.649 0.686 0.689 0.621 0.642 0.661 0.659 0.627
62 1.993 2.021 2.124 2.117 1.972 2.094 2.121 2.112 1.961
63 0.589 0.597 0.609 0.615 0.582 0.592 0.604 0.601 0.577
64 0.202 0.211 0.218 0.216 0.2 0.209 0.21 0.214 0.197
OD: Optical density
Contd...

Supplementary Data 5: Contd...
Serial
number
OD at 500 mM NaCl
concentration
OD at 750 mM NaCl
concentration
OD at 1000 mM NaCl
concentration
OD without
supplementation
35 0.319 0.333 0.459 0.306
36 0.299 0.347 0.361 0.236
37 0.932 0.939 0.94 0.927
38 0.267 0.308 0.334 0.233
39 0.356 0.36 0.367 0.349
40 0.287 0.293 0.309 0.281
41 0.309 0.311 0.316 0.301
42 0.364 0.383 0.427 0.329
43 0.369 0.389 0.467 0.328
44 0.251 0.263 0.274 0.153
45 0.179 0.186 0.191 0.174
46 0.497 0.517 0.579 0.428
47 0.429 0.455 0.491 0.362
48 0.479 0.483 0.487 0.472
49 0.791 0.838 0.874 0.768
50 0.389 0.424 0.447 0.303
51 0.289 0.312 0.327 0.174
52 0.222 0.231 0.243 0.211
53 0.163 0.169 0.172 0.157
54 0.296 0.323 0.338 0.172
55 0.197 0.267 0.294 0.144
56 0.251 0.268 0.275 0.141
57 0.246 0.271 0.283 0.187
58 0.526 0.558 0.581 0.409
59 0.568 0.571 0.577 0.563
60 0.219 0.227 0.239 0.199
61 0.632 0.638 0.641 0.627
62 1.993 2.013 2.117 1.961
63 0.582 0.596 0.611 0.577
64 0.198 0.208 0.215 0.197
OD: Optical density NaCl: Sodium chloride
Supplementary Data 6: Comparative OD’s obtained with
and without supplementation in proposed method
Serial
No
OD without
supplementation
OD at final
supplementation
1 0.253 0.479
2 0.298 0.477
3 0.827 0.962
4 0.594 0.688
5 0.595 0.693
6 2.006 2.224
7 0.269 0.482
8 0.376 0.521
9 0.606 0.654
10 0.36 0.487
11 0.445 0.536
12 0.579 0.603
13 1.715 1.857
14 1.667 1.893
15 1.404 1.588
16 1.631 1.771
Contd...

Supplementary Data 6: Contd...
Serial
No
OD without
supplementation
OD at final
supplementation
17 0.697 0.959
18 1.13 1.329
19 1.351 1.537
20 1.606 1.796
21 1.024 1.324
22 1.462 1.793
23 0.827 0.886
24 0.614 0.667
25 0.428 0.678
26 0.666 0.781
27 0.726 0.993
28 0.247 0.491
29 0.901 0.98
30 0.22 0.397
31 0.325 0.478
32 0.194 0.298
33 0.663 0.733
34 0.318 0.483
35 0.306 0.494
36 0.236 0.487
37 0.927 1.103
38 0.233 0.391
39 0.349 0.492
40 0.28 0.511
41 0.301 0.427
42 0.329 0.483
43 0.328 0.481
44 0.153 0.297
45 0.174 0.286
46 0.428 0.597
47 0.362 0.509
48 0.472 0.619
49 0.768 0.981
50 0.303 0.492
51 0.174 0.318
52 0.211 0.481
53 0.157 0.283
54 0.172 0.341
55 0.144 0.297
56 0.141 0.28
57 0.187 0.302
58 0.409 0.587
59 0.563 0.648
60 0.199 0.353
61 0.627 0.739
62 1.961 2.125
63 0.577 0.669
64 0.197 0.216
OD: Optical density
Supplementary Data 7: Comparative class obtained with
and without supplementation in proposed method
Grading of biofilm without
supplementation
Grading after Isolate number
LBF MBF 1
LBF MBF 2
MBF HBF 3
MBF MBF 4
MBF MBF 5
HBF HBF 6
LBF MBF 7
LBF MBF 8
MBF MBF 9
LBF MBF 10
LBF MBF 11
MBF MBF 12
HBF HBF 13
HBF HBF 14
HBF HBF 15
HBF HBF 16
MBF HBF 17
HBF HBF 18
HBF HBF 19
HBF HBF 20
HBF HBF 21
HBF HBF 22
MBF MBF 23
MBF MBF 24
LBF MBF 25
MBF MBF 26
MBF HBF 27
NBF MBF 28
MBF HBF 29
NBF LBF 30
LBF MBF 31
NBF LBF 32
MBF MBF 33
LBF MBF 34
LBF MBF 35
NBF MBF 36
MBF HBF 37
NBF LBF 38
LBF MBF 39
LBF MBF 40
LBF LBF 41
LBF MBF 42
LBF MBF 43
NBF LBF 44
NBF LBF 45
LBF MBF 46
Contd...

Supplementary Data 7: Contd...
Grading of biofilm without
supplementation
Grading after Isolate number
LBF MBF 47
LBF MBF 48
MBF HBF 49
LBF MBF 50
NBF LBF 51
NBF MBF 52
NBF LBF 53
NBF LBF 54
NBF LBF 55
NBF LBF 56
NBF LBF 57
LBF MBF 58
MBF MBF 59
NBF LBF 60
MBF MBF 61
HBF HBF 62
MBF MBF 63
NBF NBF 64
NBF: Non biofi lm former, MBF: Moderate biofi lm former, LBF: Low
biofi lm formers