Minimum information guideline for 
spectrophotometric and fluorometric methods to 
assess biofilm formation in microplates
Jontana Allkja, Thomas Bjarnsholt, Tom Coenye, Paul Cos, 
Adyary Fallarero, Joe J. Harrison, Susana P. Lopes, Antonio 
Oliver, Maria Olivia Pereira, Gordon Ramage, Mark E. Shirtliff, 
Paul Stoodley, Jeremy S. Webb, Sebastian A.J. Zaat, Darla M. 
Goeres, Nuno Filipe Azevedo
Jontana Allkja, Thomas Bjarnsholt, Tom Coenye, Paul Cos, Adyary Fallarero, Joe J. Harrison, Susana P. 
Lopes, Antonio Oliver, Maria Olivia Pereira, Gordon Ramage, Mark E. Shirtliff, Paul Stoodley, Jeremy S. 
Webb, Sebastian A.J. Zaat, Darla M. Goeres, Nuno Filipe Azevedo (2020). Minimum information guideline for 
spectrophotometric and fluorometric methods to assess biofilm formation in microplates. Biofilm, 2, 100010. 
https://doi.org/10.1016/j.bioflm.2019.100010
This article is made available under the CC-BY 4.0 license
Made available through Montana State University’s ​ScholarWorks 
scholarworks.montana.edu 
Biofilm 2 (2020) 100010Contents lists available at ScienceDirect
Biofilm
journal homepage: www.elsevier.com/locate/bioflmMinimum information guideline for spectrophotometric and fluorometric
methods to assess biofilm formation in microplates
Jontana Allkja a,b, Thomas Bjarnsholt c,d,e, Tom Coenye e,f, Paul Cos g, Adyary Fallarero h,
Joe J. Harrison i, Susana P. Lopes j, Antonio Oliver k, Maria Olivia Pereira j, Gordon Ramage l,e,
Mark E. Shirtliff m,1, Paul Stoodley n,o,p, Jeremy S. Webb p, Sebastian A.J. Zaat q,
Darla M. Goeres b,*, Nuno Filipe Azevedo a
a LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465,
Porto, Portugal
b Montana State University, Center for Biofilm Engineering, 366 Barnard Hall, Bozeman, MT, 59717, USA
c Department of Clinical Microbiology, Rigshospitalet, 2100, Copenhagen, Denmark
d Department of Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health Sciences University of Copenhagen, 2200, Copenhagen, Denmark
e ESCMID Study Group for Biofilms, Basel, Switzerland
f Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium
g Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp,
Belgium
h Pharmaceutical Design and Discovery (PharmDD), Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, P.O. Box 56, FI-
00014, Helsinki, Finland
i Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada
j Centre of Biological Engineering (CEB), Laborat
orio de Investigaça~o Em Biofilmes Rosa
rio Oliveira (LIBRO), University of Minho, Braga, Portugal
k Servicio de Microbiología, Hospital Universitario Son Espases, Instituto de Investigaci
on Sanitaria Illes Balears (IdISBa), Palma de Mallorca, Spain
l Oral Sciences Research Group, University of Glasgow Dental School, School of Medicine, Dentistry and Nursing, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, United Kingdom
m Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD, 21201, USA
n Department of Microbial Infection and Immunity and Orthopedics, The Ohio State University, Columbus, OH, 43210, USA
o National Centre for Advanced Tribiology at Southampton (nCATS), Department of Mechanical Engineering, University of Southampton, Southampton, SO17 1BJ, UK
p National Biofilms Innovation Centre, School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK
q Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology, Amsterdam Infection and Immunity Institute, Meibergdreef 9, 1105AZ, Amsterdam, the
NetherlandsA R T I C L E I N F O
Keywords:
Biofilm
Reproducibility
Guidelines
Microplate
Spectrophotometry
Fluorometry* Corresponding author.
E-mail address: darla_g@montana.edu (D.M. Go
1 Deceased July 12, 2018.
https://doi.org/10.1016/j.bioflm.2019.100010
Received 31 July 2019; Received in revised form 8
Available online 19 November 2019
2590-2075/© 2019 The Author(s). Published by Els
nc-nd/4.0/).A B S T R A C T
The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the
field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is
the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the
necessary information that a manuscript should include for the experiments in a study to be clearly interpreted
and independently reproduced. As a result of several discussions between international groups working in the
area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm for-
mation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set
of recommendations. The intention of the minimum information guideline is to improve the quality of scientific
communication that will augment interlaboratory reproducibility in biofilm microplate assays.eres).
November 2019; Accepted 10 November 2019
evier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
J. Allkja et al. Biofilm 2 (2020) 100010
Fig. 1. Schematic diagram of the guideline and critical steps for spectro-
photometric and fluorometric methods of biofilm assessment. Schematic
diagram of the different sections of this guideline, highlighting the various
critical steps that can increase variability in biofilm experiments. Different ap-
proaches to washing were illustrated to showcase how variable these can be in
different protocols. (Illustration courtesy of Jill Story).Introduction
A major challenge facing science today is the lack of reproducibility
between published studies [1,2]. Many factors contribute to this phe-
nomenon, including the selective or insufficient reporting of experi-
mental details in the published literature, either in the methodology or
data processing, that are essential for conducting the experiment [3].
Furthermore, due to the rapid development of science, new terms are
often introduced, or existing terminology is repurposed, which can create
confusion when trying to understand a paper or reproduce a study [4].
Minimum information guidelines are an effective strategy for
addressing the reproducibility crisis [5]. These guidelines instruct au-
thors and reviewers on the minimum information required for the ex-
periments to be reproducible and the data to be comparable. They also
allow the scientific community to standardise terminology leading to the
development of ontology databases. However, they do not offer any in-
formation on whether a method is appropriate for a certain study nor
endorse any specific protocols. The Minimum Information for Biological
and Biomedical Investigations (MIBBI) Project is a web based platform
(www.mibbi.org) that gathers different minimum information guidelines
in the biological and biomedical field, as well as any databases or stan-
dard ontologies related to them [4].
Minimum information about a biofilm experiment (MIABiE) (www
.miabie.org) is one of the guidelines presented in MIBBI [6]. It offers a
broad view of the information necessary when conducting experiments
related to biofilms. Biofilms are defined as a community of microor-
ganisms embedded in an extracellular polymeric substance, often
attached to a biotic or abiotic surface, which are essential in certain
ecosystems but can also have detrimental effects in industry and
healthcare [7]. MIABiE includes several modules, each addressing spe-
cific parts of a biofilm study, and presents an initiative for a biofilm
ontology guide.
The present guideline will expand some of the MIABiE modules by
focusing on spectrophotometric and fluorometric methods of biofilm
assessment in microplate experiments. These are widely used biofilm
assessment methods due to their versatile applications in medical, in-
dustrial and environmental biofilm research [8]. They can serve as a
generic test, which does not require overly specialised or expensive
equipment or training, and can generate high-throughput data because
they are microplate compatible.
Although several options for photometric or fluorescence-based
methods in microplates exist, this guideline will focus on those
methods most frequently used. This includes spectrophotometric
methods used to quantify total biofilmmass based on the binding of dyes,
such as crystal violet and safranin to cells and negatively-charged mol-
ecules (such as polysaccharides) in the biofilm matrix [9–11]. Addi-
tionally, the guideline is applicable to fluorometric (or
fluorescence-based) methods used to quantify the metabolic activity of
cells within a biofilm, including those based on resazurin (also known as
alamarBlueTM), fluorescein diacetate (FDA) and various tetrazolium salts
like 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-
anilide (XTT), 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium
bromide (MTT) and 2,3,5-triphenyl-tetrazolium chloride (TTC) [8,
12–15]. Furthermore, methods that stain specific biofilm components
such as SYTO 9, which stains nucleic acids [8,16], and Wheat Germ
Agglutinin (WGA), which stains the extracellular polymeric substances
(EPS), are also compatible with the guideline [17].
The guideline
This guideline focuses on spectrophotometric and fluorometric mea-
surement of biofilm grown in microplates and is divided into 5 different
sections labelled 01–05 (Fig. 1). Although theremay beminor differences
between staining reagents and techniques, this outline is designed to
follow the chronological order in which the assays are typically per-
formed and described. Section 01 pertains to the experimental design.2
Here the investigators determine the research question and how they
may answer it. Once the experiment is mapped out, the next step is to
grow the biofilm (section 02). This step includes inoculum preparation as
well as biofilm growth in the microplate. Subsequently the biofilm is
typically quantified or assessed using a specific stain and this biofilm
assessment method is detailed in section 03. This process allows for
variations depending on the target and the stain; however, the main steps
are generally the same: washing, drying, staining, elution of stain and/or
measuring absorbance or fluorescence (Fig. 1). Once the reading is
concluded and the data are collected, the next step is to analyse them
(04). Moreover, in the interest of data sharing and communication we
propose that data should be submitted to biofilm databases in the future
(05).
While developing the guideline, it became clear that methodological
details that may be essential to achieve reproducibility of a biofilm
experiment are often lacking critical information or omitted entirely.
Therefore, in Table 1 we describe the most common omissions in
reporting microplate methods and reflect on the potential impact of these
omissions on the outcome of the experiment. At the end of each section of
the guideline, we provide an example of a hypothetical simple experi-
ment related to biofilm formation using crystal violet. An example of
similar guidance for a more complex analysis, involving the effect of
antibiotic exposure on biofilms, can be found as supplementary infor-
mation (Table S1). Additionally, it was of vital importance to gather a
group of international researchers actively working in the area of bio-
films in order to provide a balanced view on what can realistically be
requested of most groups reporting these methods. All researchers
involved in the process are listed as authors in this article.
01. Experimental design
1. Describe the main question to be addressed in the study. This includes
proposed main (and possibly secondary) hypothesis(es).
2. Explain the experimental design for the study, in other words, what
type of experiment is being conducted to test the hypothesis (es)? For
example, a comparison between different treatments or factors;
different microbial bacterial strains (i.e. reference “type” strains,
mutant constructs or clinical isolates) or different concentrations and
exposure times?
3. State the number of biological replicates, meaning the independent
repeats of the same experiment. Ideally, these should be day-to-day
replicates to account for changes in humidity and room
J. Allkja et al. Biofilm 2 (2020) 100010
Table 1
Common omissions in reporting spectrophotometric and fluorometric methods of
biofilm assessment.
Omission Impact
References Often papers cited as containing the protocol followed in
the study do not describe the full protocol and redirect
you to another paper. This can create confusion when
trying to understand the protocol that was followed.
Replicates The number of replicates within one experiment is not
reported in the published paper. Furthermore, there are
inconsistencies in the terminology used when describing
replicates. For example, biological and technical
replicates vs day-to-day and within experiment
replicates.
Controls While controls are mostly mentioned in the published
articles, their values and variation are usually not
reported. This makes it difficult to understand the
variability associated with the method and how the raw
data was processed.
Inoculum preparation Different culturing methods can affect the behaviour of
microorganisms, their ability to attach to a surface,
formation of aggregates, and response to different
stimuli, chemicals, or other microorganisms [32].
Environmental factors In dry conditions, the microplate wells easily dry out,
which affects biofilm formation. Hence, investigators
take precautions to avoid the problem which are usually
not reported in the methodology section.
Position of samples in The layout of plates is often not reported, but the
the wells position of samples in the microplate can affect the
results. For example, the “edge effect” is a suspected
phenomenon which might be due to differences in
evaporation between the outer and inner wells, as well
as thermal changes in the plate.
Orbital shaker settings Most papers only refer to the rpm settings on their
orbital shaker and omit other details such as the orbital
diameter which can affect the shear stress exerted in the
wells [33].
Washing Description of this step is often omitted or vague terms
such as, “gently rinse” or “slowly tip over plate” are
used, which leave it up to the reader to determine how
to perform the step [11,34].
Drying This step is very often omitted altogether from the
method description or contains very little detail on how
it was performed.
Raw data Most articles do not provide their raw data and omit
information on how this was analysed [35].
Outliers Outliers are very often not included in the paper or, if
reported, their exclusion is simply mentioned with little
argumentation for it and how the final data analysis was
affected by their removal.
Data presentation The most common way of presenting microplate
experiment data is through bar charts. However, often
they do not provide all the relevant information from a
dataset (distribution, outliers, paired data relations).
Hence the way data is presented can limit its
interpretation. Changing to a scatter plot or a box plot
can provide more details for the same dataset [19].temperatures, for example. Include the number of technical replicates
within the experiment, meaning the number of replicates for each
sample group in the experiment. If applicable state whether the
technical replicates are within one plate or in separate ones.
4. Include the number of replicates for the controls used in the experi-
ment. Additionally, describe what these controls were and report
their data to improve overall understanding of results. Depending on
the test hypothesis they could be very straightforward, such as a
growth check as a positive control and a sterility check as a negative
control. On the other hand, they could bemore complex. For example,
if an antimicrobial agent is added and then rinsed off, an appropriate
control is to use a mock carrier (e.g. saline) which accounts for the
removal of microbes resulting from the exchange of fluids. Other
appropriate controls for antimicrobial testing include solvent controls
(e.g. DMSO) to verify that decrease in biofilm is due to the compound
and not the solvent, and pre-treatment controls to verify that the3
effects observed are due to bactericidal not bacteriostatic activity.
Furthermore, when biocide tests are performed it is recommended to
perform a neutralizer verification, as well as checking for interactions
between the microplate material, biocide (e.g. bleach) and dye. It
should be noted that controls are highly dependent on the experi-
mental design, therefore it is important to report all the relevant
controls.
5. When applicable, reference published protocols followed, ideally to
the original articles containing all the necessary information. Addi-
tionally, if any changes were made to these published protocols they
need to be described in detail.
6. Provide a link to any supplementary information or data not reported
in the main body of the article, such as more detailed method de-
scriptions, a metadata sheet containing raw data and layouts of
microplate designs, etc.
“This study investigated the effect of growth media concentration on
Staphylococcus aureus biofilm formation in a microplate. Total biofilm
mass formation after 24 hours for four different concentrations of Tryptic
Soy Broth (TSB) was compared. Each experiment consisted of one plate
that used 6 sample wells per TSB concentration, and 6 negative control
wells containing only TSB for all four concentrations tested. Each experi-
ment was repeated in three independent weeks. A more detailed description
of the methodology together with a schematic illustration of the sample and
negative control positions within the plate can be found in our supple-
mentary data section [Link].”02. Biofilm formation
1. Describe the microorganisms selected for the experiment. List the
species and strain number, and if available the strain numbers
assigned in international culture collections, e.g. ATCC, BCCM/LMG
bacteria Collection, or DSMZ, or provide a reference in which the
relevant details of the strains are reported. Alternatively, if clinical or
environmental isolates are used, provide all available and relevant
background and ethical information. Describe the stock preservation
conditions, and any modifications made to the microorganism
(plasmid insertions, gene knockouts, etc) using established genetic
nomenclature.
2. Describe the inoculum preparation protocol. Include information on
incubation conditions such as concentration, growth phase, temper-
ature, time, shaking (rpm and orbital diameter or static conditions)
and growth media (ingredients, concentration, origin). Depending on
the microorganism, include other applicable incubation conditions
such as light, CO2 concentration, humidity, etc. Additionally, if any
washing steps were performed include detailed information on
centrifugation conditions (g force, time, equipment) and the washing
agent used (water, PBS, etc). Other important factors might be
whether a culture was grown up then diluted to a specific concen-
tration, and how this was measured, i.e. optical density is commonly
used.
3. Describe the compounds or conditions being tested. In case of anti-
microbials, describe their concentration (molarity, g/L or any other
appropriate SI units), origin (manufacturers if purchased and cata-
logue numbers if allowed by the journal of choice), and time point in
the experiment when they were added, and whether an agent was
used to neutralize the active ingredient. If applicable, describe pH,
any solvents used, activity corrections and whether agents were
filtered prior to use.
4. Provide information on microplates used. This includes type of plate
(clear, white or black), number of wells (6, 9, 24, 96 or 384), shape of
the wells (flat, rounded, U-shaped or V-shaped), the material and the
manufacturing company, including catalogue numbers if allowed by
the journal of choice. Report any modifications made to the
J. Allkja et al. Biofilm 2 (2020) 100010manufactured microplate such as pre-coating of the wells or addition
of coupons.
5. Describe how the microplate was prepared. Provide information on
the inoculum conditions at harvest such as growth phase, optical
density (wavelength, zero solution, equipment) and concentration of
microorganism (CFU/mL for bacteria or cells/mL for yeast) and
growth media (if different from point 2). If a biofilm prevention
experiment is being conducted provide information on the anti-
biofilm agent used (concentration in relevant SI units, preparation
and origin).
If possible, provide a short description of the layout of the microplate
showing the position of controls and samples. Additionally, if appli-
cable mention any extra steps taken such as adding water to the outer
wells to avoid “edge effects”.
6. Provide a description of incubation conditions for the microplate.
Include information on temperature, time and shaking (rpm and
orbital diameter or stationary). Similar to point 2 include a descrip-
tion of any other relevant conditions such as light, CO2 concentration
or humidity. Additionally, if applicable mention any extra steps such
as sealing the plate with parafilm or other films or incubating within a
humidified container.
“Staphylococcus aureus strain ATCC 25923TM was used. To prepare the
inoculum, -80 
C glycerol stocks were streaked out on Tryptic Soy Agar
[Manufacturer] plates. One colony from the plate was transferred into 15
ml TSB and incubated at 37 
C, 125 rpm in a shaker incubator [Model
number] with an orbital diameter of 1.9 cm. After 18 hours a 1:100
dilution of the inoculum was incubated at 37 
C, 125 rpm until it reached
the exponential growth phase (OD¼0.300 [595nm; Model number]). Four
2 ml aliquots of the suspension were made and washed by centrifugation
(2000 g for 15 minutes [Model number]) and resuspending the pellets in
PBS [pH 7.4; Manufacturer] twice. Subsequently, the pellets were resus-
pended in 4 different TSB broths (30 g/ml, 3 g/ml, 0.3 g/ml and 0.03 g/
ml) and 200 μl per well of each of these suspensions was added to a flat
bottom polystyrene 96 well plate [Manufacturer] according to the layout in
the supplementary data. The plate was incubated at 37 
C under static
conditions in a non-humidified incubator for 24 hours. To prevent excess
drying the outer wells were filled with 200 μl/well of sterile water.”03. Biofilm assessment method
1. Describe the method followed to discard the planktonic suspension,
e.g. pipetting, suction manifold.
2. Describe all washing steps in detail. Provide information on the
washing agent such as sterility, origin, concentration and pH, if
applicable. Additionally, describe the number of washes and
method(s) used to add and remove the washing agent (immersion,
rinsing or pipetting). When possible, avoid the sole use of vague terms
such as “gently” which are subject to interpretation and include more
detailed descriptions. For example, describing the angle and depth at
which a pipette tip was inserted into the well or stating the number of
times the plate was shaken to remove excess liquid when inverted. If
automatic liquid handling devices are used provide information of
equipment and settings.
3. Describe the staining process. This includes information on the stain:
origin (manufacturer), stock and working solution concentrations,
solvents used as well as information on the staining: time and incu-
bation conditions (light, temperature, volumes, shaking etc.). If
applicable, provide information on any standard curves performed
with the experiment.
4. In cases where extra steps such as fixation, drying and elution are
required, describe how these were performed and any solvents or
chemicals (origin, concentration) used.
5. Describe how the spectrophotometric or fluorometric signal was
measured. Provide information on the equipment (model number,4
company, software) used as well as its settings (excitation, emission
and detection wavelengths, end-point or continuous read, shaking).
When using fluorometric reading, provide information on the type of
readout (top or bottom reading). If bottom reading is performed,
provide information on the number and distribution of the points
measured across each well.
Furthermore, if imaging functions of the microplate reader were used,
describe the settings (time, shaking, imaging mode, filters, camera).
“The planktonic suspension was carefully removed using a multichannel
pipette [Model, Manufacturer] fitted with a 300 μL tip inserted slowly at a
45
 angle while making sure to avoid touching the sides and bottom of the
wells. The plate was washed twice with 250 μl/well of PBS using a
multichannel pipette fitted with a 300 μL tip and left to air-dry for 15 min
in under laminar flow at room temperature (RT, 20  5 
C). The biofilm
was fixed for 15 min with 200 μl/well of 99% v/v ethanol [Manufacturer]
and then allowed to air-dry until fully dry, between 5 and 10 minutes. The
plate was stained with 200 μL of 0.1% v/v Crystal violet [Manufacturer]
for 15 min at RT, under static conditions. After staining the plates were
washed twice with 250 μl/well of MilliQ water using a multichannel pipette
and left to air-dry for 15 min in laminar flow. The stain was eluted with
200 μl/well of 99% v/v ethanol for 30 min at RT, no shaking. The eluted
stain was mixed by pipetting up and down 4 times and 100 μl/well of it
were transferred to an empty 96-well plate using a multichannel pipette.
The absorbance was measured at 595 nm using a [Company; Model
number] plate reader.”04. Statistical assessment and data presentation
1. Describe how the raw data were processed and/or transformed. If
possible, include raw data in the supplementary data section.
2. Present all outliers. Argumentation should be given if they were
removed from the analysis in the results and ideally how their
removal affected the data.
3. Test the data for normality. Report if the data has been transformed or
normalised for example, using a standard curve, log transformation,
square root or any other appropriate normalisation method.
4. Describe statistical tests and rationale for use (i.e. parametric, non-
parametric, small sample, paired etc.) performed and any post-hoc
tests. Provide information on the test parameters, descriptive statis-
tics such as significant differences, standard errors, standard devia-
tion, variance and confidence intervals. Additionally, include
descriptive statistics for the controls used in the experiment. If a high-
throughput screening assay is being reported, it is recommended to
include the calculation of the screening windows coefficient, or Z’
[18].
5. Ensure that the appropriate graph types and data visualizations are
used. Figures should provide all the essential and relevant informa-
tion necessary for a full understanding of the results [19]. We suggest
the use of scatter plots or box and whisker plots instead of line graphs
or bar charts, which often do not portray all the necessary information
in a dataset (Fig. 2). For instance, many different normal, skewed or
bimodal data distributions can lead to the same mean and standard
deviation values [19]. Summarizing data as a mean with standard
deviation can also conceal unequal sample sizes and outliers [19,20].
Plotting all measurements in tandem with means and standard de-
viations provides transparency and allows readers to evaluate data for
themselves (Fig. 2).
6. Provide details of the statistical package used and its version. If more
than one was used, they all need to be mentioned. Additionally, if any
open source systems such as R packages were used, provide a refer-
ence or a link to it.
“Raw absorbance data can be found in our supplementary data section. To
evaluate the within plate variability, the mean  1 standard deviation
(STDEV) of all the technical replicates for each sample were calculated
J. Allkja et al. Biofilm 2 (2020) 100010
Table 2
Simplified checklist for minimum information guideline spectrophotometric
methods of biofilm assessment.
01. Experimental design
Aim of the experiment/hypothesis presented ✓
Type of experiment
Biological and technical replicates
Control replicates and descriptions
Reference to original article containing protocol (If applicable)
Supplementary information (If applicable)
02. Biofilm formation
Microorganism description
Inoculum preparation protocol
Treatment description (If applicable)
Microplate description
Plate layout i.e. sample distribution (Optional)
Incubation conditions for microplate
03. Biofilm assessment method
Fig. 2. Show the dots on plots: scatter graphs allow readers to evaluate Planktonic suspension removal
data distributions for themselves. Biofilm formation was measured for Pseu- Washing description
domonas aeruginosa strains CF39S and CF39, which express functional and Staining description
mutant alleles of the thermosensory diguanylate cyclase (tdcAþ and tdcA), Additional steps: fixing, drying, buffer solutions (if applicable)
respectively. Each condition has 48 replicates, representing sixteen technical Absorbance/Fluorescence measurement
replicates from each of three independent biological replicates. (a) Line graph. 04. Statistical assessment and data interpretation
Datum points represent means and standard deviations. (b) Scatter plot. Each
point denotes a replicate datum point and lines and bars represent means and Raw data handling
Outliers
standard deviations, respectively. (Data courtesy of Joe J. Harrison). Normality testing
Appropriate data presentation
Statistical test with post-hoc and descriptive stats
Statistical programme used
05. Bioinformatics (Optional)
Standardised terminology
Data formatting according to data submission guidelines
Submission to online databaseand are summarised in table [1]. The means of all the different samples
were corrected by subtracting the corresponding negative control (TSB
only) values. The data from all three replicate experiments were analysed
using a one-way ANOVA test with a Levene’s post-hoc analysis to compare
the absorbance values. These results were represented in Fig. 1 and a more
detailed description can be found in the figure legend. [Statistical Pro-
gramme; version] was used to perform all tests.”05. Bioinformatics (optional)
1. Use standard terminology. In the coming years ontology guidelines
for biofilm terminology are expected to be developed. A starting
guide can be found on the MIABiE website [21].
2. The data should be formatted in a way that makes it easier to submit
and extrapolate it to existing databases such as BiofOmics (http://
www.biofomics.org/) or other databases currently in development
[22].
As illustrated above, the amount of information necessary to fully
characterise a complex system such as a biofilm experiment is significant.
Therefore, a simplified checklist of the guideline has been included in
this paper (Table 2). This checklist can assist authors during their writing
process as well as reviewers during the peer-review process. In fact,
complementary fields such as ecology and evolution have very recently
started to make checklists available in their field of knowledge [23].
Moreover, certain sections of this guideline can be applied to other bio-
film assessment methods in microplate experiments, such as when viable
plate counts are used to assess biofilm density and treatment efficacy.
Discussion
Microplate-based spectrophotometric and fluorometric methods of
biofilm assessment have led to the generation of a vast amount of data
throughout the years. However, while these data have provided essential
information on biofilm biology and experimental therapeutic strategies
to tackle biofilms, biofilm experiments have often been difficult to
reproduce. Furthermore, most of the time it is not possible to compare
data between studies, which means that attempting to draw conclusions5
by combining data from different studies is not feasible. To minimize this
problem, we suggest that a minimum information guideline should be
adopted by researchers.
Lack of data comparability can in part be attributed to the high
variability of protocols used for these types of methods. Table 3 illus-
trates this phenomenon of variability in protocols of the crystal violet
assay for three common organisms: Staphylococcus aureus, Pseudomonas
aeruginosa and Candida albicans. It contains the range of parameters (low
to high) for different conditions of inoculum preparation, biofilm growth
and biofilm assessment for each microorganism. Major differences in the
inoculum preparation and biofilm growth parameters, are expected as
the parameters of these steps are largely dictated by the physiology of the
microorganism being investigated and the type of experiment being
performed. However, Table 3 shows that large differences are also pre-
sent among the biofilm assessment parameters such as dye concentra-
tions and absorbance wavelengths. Taken together, this information
means that comparing different datasets at this stage is not possible for
different studies and that the guidelines can only facilitate reproduc-
ibility and comparison to a certain degree. On the other hand, it is
important to note that the variability in protocols used in the biofilm area
is often due to the differences in the subject of the investigations.
Hence, a consensus regarding certain aspects of the methodology is
necessary to improve reproducibility. On this matter, there are already
standardised biofilm methods approved by the American Society for
Testing and Materials (ASTM) which could serve as a starting point for
this process, such as the E2647-08 Standard Test Method for Quantifi-
cation of a Pseudomonas aeruginosa Biofilm Grown Using a Drip Flow
Biofilm Reactor with Low Shear and Continuous Flow [24], the E2562-17
Standard Test Method for Quantification of Pseudomonas aeruginosa
Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm
Reactor [25] and the E2799-17 Standard Test Method for Testing
J. Allkja et al. Biofilm 2 (2020) 100010
Table 3
Example of the variability in protocol conditions of crystal violet assays for three
different example microorganisms.
Condition Organism
Staphylococcus Pseudomonas Candida
aureus spp. aeruginosa spp. albicans
Inoculum preparation
Media TSB, TSB wS*, TSB, TSB wS*, LBb*, YNB*, YPD*,
LBb*, Water [8, LB*, BHI*, MHI*, T- RPMI-1640*,
11,18,36–41] broth*, AB* [8, SDB* [8,
42–49] 50–57]
Inoculum 35-37 [8,11,18, 25-37 [8,42–49] 30-37 [8,
incubation 36–41] 50–57]
temperature
(
C)
Incubation time 0**-24 [8,11,18, 0**-24 [8,42–49] 12-24 [8,
(hours) 36–41] 50–57]
Inoculum 0–200 rpm/min 0–250 rpm/min [8, 0–200 rpm/
shaking [8,11,18,36–41] 42–49] min, Roller
conditions drum [8,
50–57]
Inoculum 103 108– CFU/mL, 10–108 CFU/mL, 104 108– CFU/
concentration/ 0.5 McFarland, OD600nm¼0.0025, mL,
OD/growth OD600nm¼0.1 [8, OD595¼1.5 [8,42–49] OD600nm¼1 [8,
phase at 11,18,36–41] 50–57]
harvest
Biofilm growth
Media TSB, LB*, BHI* [8, TSB, T-broth*, AB*, YNB*, YPD*,
11,18,36–41] BHI*, MHI* [8, RPMI-1640*,
42–49] ASM*, SDB*,
PBS* [8,50–57]
Incubation 35-37 [8,11,18, 25-37 [8,42–49] 37 [8,50–57]
temperature 36–41]
(
C)
Incubation time 18-48 [8,11,18, 2-48 [8,42–49] 2-48 [8,50–57]
(hours) 36–41]
Shaking 0–200 rpm/min 0–180 rpm/min [8, 0–120 rpm/
conditions [8,11,18,36–41] 42–49] min [8,50–57]
Biofilm Assessment
Washing agent Water, Saline, Saline, Water, PBS* PBS*, Water,
PBS*, MilliQ [8,42–49] Saline [8,
water [8,11,18, 50–57]
36–41]
Washing (x 1-3 [8,11,18, 1-3 [8,42–49] 1-3 [8,50–57]
times) 36–41]
Crystal violet 0.01–2.3% [8,11, 0.1–2% [8,42–49] 0.02–1% [8,
concentration 18,36–41] 50–57]
Staining time 1–20 min [8,11, 5–30 min [8,42–49] 5–45 min [8,
18,36–41] 50–57]
Solubilisation 33% acetic acid, 30–33% acetic acid, 30–33% acetic
agent 95–100% ethanol 95–100% ethanol, acid, 95%
[8,11,18,36–41] DMSO* [8,42–49] ethanol, 0.1%
Triton-X [8,
50–57]
Absorbance 540-595 [8,11,18, 550-595 [8,42–49] 540-595 [8,
wavelength 36–41] 50–57]
(nm)
*wS - with Supplement (i.e. added yeast and/or glucose); TSB- Tryptic Soy Broth;
LBb – Luria Bertani broth; BHI- Brain Heart Infusion; LB – Lysogeny broth;MHI –
Mueller-Hinton broth; T-broth – Terrific broth; AB – minimal growth media;
YNB – Yeast Nitrogen Base; YPD – Yeast Peptone Dextrose; SDB – Sabauraund
Dextrose Broth; RPMI-1640 - Roswell Park Memorial Institute–1640 medium;
ASM – Artificial Saliva Medium; PBS - Phosphate buffered saline; DMSO -
Dimethyl sulfoxide.
**0 – Inoculum prepared directly from agar culture.Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the
MBEC Assay [26]. They can also provide an excellent example on how to
identify critical steps and describe the methodology in order to ensure
reproducibility.
In addition, many investigations aim at optimising and modifying
spectrophotometric and fluorometric methods to increase their effi-
ciency, reliability and their applications. For example, Skogman et al.
proposed the use of consecutive staining with resazurin, WGA and crystal6
violet to improve the assessment of antimicrobial effectivity against
biofilms [17]. More recently, Junka et al., developed a way to assess
wound dressing effectiveness in 24-well plates using crystal violet and
TTC analysis [27]. This means that with time, as new steps are introduced
or more robust ways of performing certain steps are developed, the
methods will evolve. Minimum information guidelines have the advan-
tage of remaining applicable to the methods despite these changes.
As science evolves, we will be able to measure new parameters and
conditions which affect reproducibility. For example, even when manu-
factured from the same base polymer, microplates can have different
surface properties depending on the production process, resulting in
differences in cell adhesion [28]. As the methods to characterise surface
properties becomemore accessible, parameters such as surface roughness
might be used in future. Since guidelines are often part of an online
database such as MIBBI, they can be updated when necessary and evolve
together with the methods.
As is the case with compliance to MIABiE and other guidelines,
compliance to the new guideline presented here will be difficult as it
needs to be endorsed by both authors and journals [29,30]. To improve
compliance a balance needs to be obtained between the level of detail
asked, and the ability of most labs to be able to provide such data. As an
example, many studies have shown that oxygen availability influences
biofilm formation and can lead to different physiological features being
expressed [31]. Therefore, understanding the oxygen availability within
a well and across different wells in a microplate might be useful. How-
ever, most laboratories lack the kind of system needed to assess this
environmental parameter and it would be very difficult to implement this
reading routinely. Hence, the oxygen profile within the microplate is not
a requirement in the guideline.
We are convinced that the implementation of minimum information
guidelines will contribute to solving the reproducibility crisis and thus
improve the use that the research community makes of data and ulti-
mately advance science.Methodology
To create the minimum information guideline, we conducted a
literature review using three different databases: Pubmed, Google
Scholar and Web of Science. The research was separated into literature
related to the methods and literature related to biofilm properties and the
various factors affecting them. For the former, very broad search terms
such as, “Biofilm AND microtit* plate”, “Biofilm AND Spectro*” and
“Biofilm AND Fluor*” were used as a starting point. These resulted in
thousands of hits from all three databases, and to further refine this
output more specific terms such as “Crystal violet”, “Resazurin Or Alamar
Blue”, “XTT”, “TTC”, “MTT”, “FDA”, “Syto9” and “WGA” were used. The
results were ordered according to number of citations (most to least) and
publishing date (newest to oldest). 180 papers were selected to be used as
references to write the guideline. These were categorised into papers
evaluating the methods and highlighting critical factors or steps, and
papers that used the method in a specific investigation. The latter were
used to create an understanding of what is commonly reported in sci-
entific articles. Approximately 30 of the papers in this category were
discarded from the literature review, as the only description of the
method was a reference to a previously published paper.
When researching the literature on biofilm properties and what af-
fects them, terms such as “impact”, “influence”, “effect or affect”,
“changes or differences” were used. These helped in creating an under-
standing of the different parameters that should be reported for a biofilm
experiment. Additionally, other minimum information guidelines were
used as templates in the initial drafting process.
The final guidelines are the result of a dialog among biofilm experts
familiar with microplate methods. These experts are included in the
authors list and contributed throughout the drafting process of the
manuscript.
J. Allkja et al. Biofilm 2 (2020) 100010Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
We would like to thank Prof. Dr. Mark E. Shirtliff for his contribution
in the development of this guideline and the review of early manuscript
drafts prior to his untimely death. We would also like to thank Jill Story
(Montana State University, Center for Biofilm Engineering) for designing
Fig. 1.
This project has received funding from the European Union’s Horizon
2020 research and innovation program under the Marie Sklodowska –
Curie grant agreement No 722467, as part of the Print-Aid consortium.
The information and views set out in this article are those of the authors
and do not necessarily reflect the official opinion of the European Union.
Neither the European Union institutions and bodies nor any person
acting on their behalf may be held responsible for the use which may be
made of the information contained therein.
This work received additional financial support by: project UID/
EQU/00511/2019 - Laboratory for Process Engineering, Environment,
Biotechnology and Energy – LEPABE funded by national funds through
FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” –
NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional
Operational Programme (NORTE 2020), under PORTUGAL 2020 Part-
nership Agreement, through the European Regional Development Fund
(ERDF).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https
://doi.org/10.1016/j.bioflm.2019.100010.
References
[1] Baker M. Reproducibility crisis? Nature 2016;533:26.
[2] Dirnagl U. Rethinking research reproducibility. EMBO J 2019;38:e101117.
[3] Grimes DR, Bauch CT, Ioannidis JP. Modelling science trustworthiness under
publish or perish pressure. R. Soc. Open Sci. 2018;5:171511.
[4] Taylor CF, et al. Promoting coherent minimum reporting guidelines for biological
and biomedical investigations: the MIBBI project. Nat Biotechnol 2008;26:889–96.
[5] Anon. A checklist for our community. Nat. Ecol. Evol. 2018;2:913.
[6] Lourenço A, et al. Minimum information about a biofilm experiment (MIABiE):
standards for reporting experiments and data on sessile microbial communities
living at interfaces. Pathog. Dis. 2014;70:250–6.
[7] Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the Natural
environment to infectious diseases. Nat Rev Microbiol 2004;2:95–108.
[8] Peeters E, Nelis HJ, Coenye T. Comparison of multiple methods for quantification of
microbial biofilms grown in microtiter plates. J Microbiol Methods 2008;72:
157–65.
[9] Lopes SP, Azevedo NF, Pereira MO. Emergent bacteria in cystic fibrosis: in vitro
biofilm formation and resilience under variable oxygen conditions. BioMed Res Int
2014;2014.
[10] Machado I, Graça J, Sousa AM, Lopes SP, Pereira MO. Effect of antimicrobial
residues on early adhesion and biofilm formation by wild-type and benzalkonium
chloride-adapted Pseudomonas aeruginosa. Biofouling 2011;27:1151–9.
[11] Stepanovi
c S, et al. Quantification of biofilm in microtiter plates: overview of
testing conditions and practical recommendations for assessment of biofilm
production by staphylococci. APMIS 2007;115:891–9.
[12] Alonso B, Cruces R, P
erez A, S
anchez-Carrillo C, Guembe M. Comparison of the XTT
and resazurin assays for quantification of the metabolic activity of Staphylococcus
aureus biofilm. J Microbiol Methods 2017;139:135–7.
[13] van Hengel IAJ, et al. Selective laser melting porous metallic implants with
immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-
resistant Staphylococcus aureus. Biomaterials 2017;140:1–15.
[14] Honraet K, Goetghebeur E, Nelis HJ. Comparison of three assays for the
quantification of Candida biomass in suspension and CDC reactor grown biofilms.
J Microbiol Methods 2005;63:287–95.
[15] Tunney MM, Ramage G, Field TR, Moriarty TF, Storey DG. Rapid colorimetric assay
for antimicrobial susceptibility testing of Pseudomonas aeruginosa. Antimicrob
Agents Chemother 2004;48:1879–81.
[16] Stiefel P, Schmidt-Emrich S, Maniura-Weber K, Ren Q. Critical aspects of using
bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.
BMC Microbiol 2015;15:36.7
[17] Skogman ME, Vuorela PM, Fallarero A. Combining biofilm matrix measurements
with biomass and viability assays in susceptibility assessments of antimicrobials
against Staphylococcus aureus biofilms. J Antibiot (Tokyo) 2012;65:453.
[18] Sandberg M, M€aa€tt€anen A, Peltonen J, Vuorela PM, Fallarero A. Automating a 96-
well microtitre plate model for Staphylococcus aureus biofilms: an approach to
screening of natural antimicrobial compounds. Int J Antimicrob Agents 2008;32:
233–40.
[19] Weissgerber TL, Milic NM, Winham SJ, Garovic VD. Beyond bar and line graphs:
time for a new data presentation paradigm. PLoS Biol 2015;13:e1002128.
[20] Anon. Show the dots in plots. Nat. Biomed. Eng. 2017;1:0079.
[21] Sousa AM, Pereira MO, Azevedo NF, Lourenço A. Designing an ontology tool for the
unification of biofilms data. In: 8th international Conference on practical
Applications of computational biology & bioinformatics (PACBB 2014). Cham:
Springer; 2014. p. 41–8. https://doi.org/10.1007/978-3-319-07581-5_5.
[22] Lourenço A, et al. BiofOmics: a web platform for the systematic and standardized
collection of high-throughput biofilm data. PLoS One 2012;7:e39960.
[23] Parker TH, et al. Empowering peer reviewers with a checklist to improve
transparency. Nat. Ecol. Evol. 2018;2:929–35.
[24] ASTM E2647-08. Standard test method for quantification of a Pseudomonas
aeruginosa biofilm grown using a Drip flow biofilm reactor with low shear and
continuous flow. West Conshohocken PA: ASTM Int.; 2008.
[25] ASTM E2562-17. Standard test method for quantification of Pseudomonas
aeruginosa biofilm grown with high shear and continuous flow using CDC biofilm
reactor. ASTM Int. West Conshohocken PA; 2017.
[26] ASTM E2799-17. Standard test method for testing disinfectant efficacy against
Pseudomonas aeruginosa biofilm using the MBEC assay. ASTM Int. West
Conshohocken PA; 2017.
[27] Junka AF, Zywicka A, Szymczk, Dziadas M, Bartoszewicz M, Fijalkowski K.
A.D.A.M. test (Antibiofilm Dressing’s Activity Measurement) — simple method for
evaluating anti-biofilm activity of drug-saturated dressings against wound
pathogens. J Microbiol Methods 2017;143:6–12. https://doi.org/10.1016/
j.mimet.2017.09.014.
[28] Zeiger AS, Hinton B, Van Vliet KJ. Why the dish makes a difference: quantitative
comparison of polystyrene culture surfaces. Acta Biomater 2013;9:7354–61.
[29] Deutsch EW, et al. Minimum information specification for in situ hybridization and
immunohistochemistry experiments (MISFISHIE). Nat Biotechnol 2008;26:305–12.
[30] Anon. Time for leadership. Nat Biotechnol 2007;25:821.
[31] Xu KD, Stewart PS, Xia F, Huang C-T, Mcfeters GA. Spatial physiological
heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen
availability. Appl Environ Microbiol. 1998;64:5.
[32] Kragh KN, et al. The inoculation method could impact the outcome of
microbiological experiments. Appl Environ Microbiol 2018;84. e02264–17.
[33] Azevedo NF, Pinto AR, Reis NM, Vieira MJ, Keevil CW. Shear stress, temperature,
and inoculation concentration influence the adhesion of water-stressed
Helicobacter pylori to stainless steel 304 and polypropylene. Appl Environ
Microbiol 2006;72:2936–41.
[34] Erriu M, et al. Microtiter spectrophotometric biofilm production assay analyzed
with metrological methods and uncertainty evaluation. Measurement 2012;45:
1083–8.
[35] Anon. Standardizing data. Nat Cell Biol 2008;10:1123–4.
[36] Stepanovi
c S, Vukovi
c D, Jezek P, Pavlovi
c M, Svabic-Vlahovi
c M. Influence of
dynamic conditions on biofilm formation by staphylococci. Eur J Clin Microbiol
Infect Dis 2001;20:0502–4.
[37] Izano EA, Amarante MA, Kher WB, Kaplan JB. Differential roles of poly-N-
acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus
aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol 2008;74:
470–6.
[38] Zmantar T, Kouidhi B, Miladi H, Mahdouani K, Bakhrouf A. A Microtiter plate assay
for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen
peroxide supplementation. The New Microbiologica 2010;33(2):137.
[39] Xu Z, et al. Crystal violet and XTT assays on Staphylococcus aureus biofilm
quantification. Curr Microbiol 2016;73:474–82.
[40] Mohanty S, et al. An investigation on the antibacterial, cytotoxic, and antibiofilm
efficacy of starch-stabilized silver nanoparticles. Nanomed Nanotechnol Biol Med
2012;8:916–24.
[41] Kaplan JB, et al. Low levels of beta-lactam antibiotics induce extracellular DNA
release and biofilm formation in Staphylococcus aureus. mBio 2012;3. e00198-12.
[42] Sanchez CJ, et al. Biofilm formation by clinical isolates and the implications in
chronic infections. BMC Infect Dis 2013;13:47.
[43] Kim H-S, Park H-D. Ginger extract inhibits biofilm formation by Pseudomonas
aeruginosa PA14. PLoS One 2013;8:e76106.
[44] Palanisamy NK, et al. Antibiofilm properties of chemically synthesized silver
nanoparticles found against Pseudomonas aeruginosa. J Nanobiotechnol 2014;12:2.
[45] Friedman L, Kolter R. Two genetic loci produce distinct carbohydrate-rich structural
components of the Pseudomonas aeruginosa biofilm matrix. J Bacteriol 2004;186:
4457–65.
[46] Sabaeifard P, Abdi-Ali A, Soudi MR, Dinarvand R. Optimization of tetrazolium salt
assay for Pseudomonas aeruginosa biofilm using microtiter plate method.
J Microbiol Methods 2014;105:134–40.
[47] Jagani S, Chelikani R, Kim D-S. Effects of phenol and natural phenolic compounds
on biofilm formation by Pseudomonas aeruginosa. Biofouling 2009;25:321–4.
[48] O’Toole GA. Microtiter dish biofilm formation assay. J Vis Exp JoVE 2011;47.
https://doi.org/10.3791/2437.
[49] Bauer J, Siala W, Tulkens PM, Van Bambeke F. A combined pharmacodynamic
quantitative and qualitative model reveals the potent activity of daptomycin and
J. Allkja et al. Biofilm 2 (2020) 100010delafloxacin against Staphylococcus aureus biofilms. Antimicrob Agents Chemother
2013;57:2726–37.
[50] Marcos-Zambrano LJ, Escribano P, Bouza E, Guinea J. Production of biofilm by
Candida and non-Candida spp. isolates causing fungemia: comparison of biomass
production and metabolic activity and development of cut-off points. Int J Med
Microbiol 2014;304:1192–8.
[51] Negri M, et al. Crystal violet staining to quantify Candida adhesion to epithelial
cells. Br J Biomed Sci 2010;67:120–5.
[52] Monteiro DR, et al. Silver colloidal nanoparticles: antifungal effect against adhered
cells and biofilms of Candida albicans and Candida glabrata. Biofouling 2011;27:
711–9.
[53] Li Z, Sun J, Lan J, Qi Q. Effect of a denture base acrylic resin containing silver
nanoparticles on Candida albicans adhesion and biofilm formation. Gerodontology
2016;33:209–16.8
[54] Deveau A, Hogan DA. Linking quorum sensing regulation and biofilm formation by
Candida albicans. In: Rumbaugh KP, editor. Quorum sensing: methods and
protocols, vols. 219–233. Humana Press; 2011. https://doi.org/10.1007/978-1-
60761-971-0_16.
[55] Dovigo LN, et al. Susceptibility of clinical isolates of Candida to photodynamic
effects of curcumin. Lasers Surg Med 2011;43:927–34.
[56] Khan S, Alam F, Azam A, Khan AU. Gold nanoparticles enhance methylene
blue–induced photodynamic therapy: a novel therapeutic approach to inhibit
Candida albicans biofilm. Int J Nanomed 2012;7:3245–57.
[57] Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural
populations of Candida albicans. Microbiology 2003;149:353–62.