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ISSN 0026-2617, Microbiology, 2016, Vol. 85, No. 4, pp. 509–513. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © V.K. Plakunov, S.V. Mart’yanov, N.A. Teteneva, M.V. Zhurina, 2016, published in Mikrobiologiya, 2016, Vol. 85, No. 4, pp. 484–489.
A Universal Method for Quantitative Characterization of Growth
and Metabolic Activity of Microbial Biofilms in Static Models
V. K . Plaku nov1, S. V. Mart’yanov, N. A. Teteneva, and M. V. Zhurina
Winogradsky Institute of Microbiology, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia
1e-mail: plakunov@inmi.ru
Received Februa ry 18, 2016
DOI: 10.1134/S0026261716040147
According to the results of worldwide research in
medical microbiology, many microbial infections are
caused by microorganisms organized as biofilms. Lab-
oratory simulation of these communities in vitro is
therefore necessary for investigation of the patterns of
their formation and for testing the effect of antimicro-
bial preparations. For these studies, the correlation
between in vitro and in vivo activity of biocidal agents
is of primary importance. Standard criteria of antimi-
crobial activity used for planktonic cultures, such as
the minimal inhibiting concentration (MIC), at which
growth of planktonic cultures is suppressed completely
(growth is not registered by optical techniques), or
minimal bactericidal concentration (MBC), which
causes the death of 99.9% of microorganisms (deter-
mined as the number of surviving cells), are not appli-
cable to biofilm microbial communities. The proper-
ties of biofilm microbial populations, which contain
high numbers of persister cells insensitive to antimi-
crobial agents (Verstraeten et al., 2016), and of viable
nonculturable cells (Lee et al., 2007; Li et al., 2014),
prevent the application of these techniques.
The new pharmacodynamic parameters proposed
in order to overcome these difficulties include biofilm
prevention/inhibiting concentration (MBPC or
MBIC) (Sabaeifard et al., 2014), minimal biofilm bac-
tericidal concentration (MBBC) (Macià et al., 2014),
and minimal biofilm eradication concentration
(MBEC) (Takei et al., 2013). Since they cannot be
determined using the standard techniques for biofilm
testing, new approaches are required, including the
application of metabolic indicators (Peeters et al.,
2008).
Existing methods for the reconstruction and mod-
eling of microbial biofilms belong to several major
types (McBain, 2009; Coenye and Nelis, 2010; Leb-
eaux et al., 2013): (1) “closed” or static models based
on the application of microtiter plates (usually with 96
wells) and colony biofilms (Vandecandelaere et al.,
2016); (2) “open” or dynamic systems with constant
flow of fresh medium (Palmer, 1999; McBain, 2009);
and (3) microcosms, i.e., multispecies biofilms
formed on a surface similar to the one occurring in the
environment and simulating the in situ situation
(Rudney et al., 2012). Approaches based on microflu-
idic techniques and combining the flow method with
the possibility of continuous microscopic monitoring
of the process of biofilm formation became popular
recently (Coenye and Nelis, 2010; Kim et al., 2012;
Hassanpourfard et al., 2014).
Due to their relation to the topic of this article, we
will discuss the most widespread models of the first
type in more detail.
The main shortcoming of microtiter plates (which
are made of polystyrene, polypropylene, of polycar-
bonate) is disordered growth of microbial biofilms.
They may form at the bottom of the well, on its wall,
or on the surface of the medium. In the latter case,
separation of the biofilm and the planktonic culture is
almost impossible. Limited volume of the wells and
poor mixing of the medium result in oxygen and nutri-
ent limitation, i.e., in starvation stress, which in com-
bination with an antimicrobial agent may have a syn-
ergistic or antagonistic effect on microbial growth.
Moreover, conditions in which pathogenic microor-
ganisms exist within a host are different from starva-
tion conditions. Biofilm growth in the wells is usually
determined by staining the cells attached to the walls
and bottom of the well with bacteriological stains, usu-
ally crystal violet (CV). Since CV stains both the living
and dead cells, as well as the extracellular polymer
matrix (EPM) in which the cells are embedded, deter-
mination of the number of metabolically active bacte-
ria is possible only after additional staining by the dyes
which can be metabolized: resazurin, fluorescein dia-
cetate, or tetrazolium salts (Peeters et al., 2008).
Numerous recent modifications of this method ame-
liorate some of its shortcomings to a certain degree
(Chavant et al., 2007; Lyamin et al., 2012).
Quantitative determination of live cells in biofilms
is a serious challenge. The standard staining technique
(live/dead) is practically inapplicable to biofilms due
SHORT COMMUNICATIONS
510
MICROBIOLOGY Vol. 85 No. 4 2016
PLAKUNOV et al.
to interaction of the dyes with the extracellular poly-
mer matrix (EPM) containing high levels of DNA,
which produces a screening effect (Netuschil et al.,
2014).
Since quantitative separation of the cells and EPM,
even with harsh homogenization techniques affecting
microbial viability, is almost impossible for many bio-
films, especially for mature ones, enumeration of col-
ony-forming units (CFU) results in determination of
the minimal probable number of culturable cells.
Moreover, the presence of viable nonculturable cells
mentioned above also prevents accurate determina-
tion of the number of live bacteria in biofilms.
We developed a method of simultaneous analysis of
the biofilm and planktonic cultures, which is an alter-
native to microtiter plates and makes it possible to
characterize biofilm growth in general, EPM synthe-
sis, metabolic activity, and the minimal probable
number of viable microbial cells in the biofilm.
Two variants of static models for biofilm cultivation
were used: on Teflon cubes in test tubes and on glass
fiber filters in petri dishes with the growth medium.
Experimental subjects were typical gram-positive
(Staphylococcus aureus, S. epidermidis) and gram-neg-
ative (Pseudomonas aeruginosa, P. c h lo ro ra phis, Esche-
richia coli, and Chromobacterium violaceum) bacteria.
The methods described below were shown to be appli-
cable to all these subjects, including oil-oxidizing bac-
teria isolated from oil-contaminated soil (Rhodococcus
equi), and from stratal waters of oil fields (Kocuria rhi-
zophila, Dietzia natronolimnaea).
Application of Teflon Cubes
Staining with CV (which stains both live and dead
cells, as well as the EPM), 1,9-dimethylmethylene blue
(DMMB) (a more specific dye for the EPM acidic poly-
saccharides), and metabolized stain 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) were used for analysis of planktonic cultures and
biofilms. The latter dye is commonly used for detection
of viable, metabolically active cells, acting as an electron
acceptor in the mitochondrial respiration chain; in the
process it is converted to water-insoluble blue formazan
(Berridge and Tan, 1993). It is presently used in microti-
ter plate methods for detection of metabolically active
cells in bacterial biofilms (Grare et al., 2008; Wang et al.,
2010).
Teflon (polytetrafluoroethylene, PTFE) has been
often used (mainly as plates) as a surface for biofilm
formation (Kondoh and Hashiba, 1998; Planchon
et al., 2006; Fuchslocher Hellemann et al., 2013). We
used the standard 4-mm PTFE cubes (Ftoroplastovye
tekhnologii, Russia). The sort of PTFE is unimport-
ant for the purpose. Prior to the experiment, the cubes
were treated with the standard bichromate mixture
(5% potassium bichromate in concentrated sulfuric
acid) for 24 h at room temperature or for 1 h in a boil-
ing water bath, and washed with distilled water to
achieve the neutral reaction of rinsing water. The
cubes may be used repeatedly. Washed cubes (3 g, 21–
22 pieces) were transferred into the standard 20-mL
bacteriological test tubes with 3 mL of the nutrient
medium (usually LB medium). The surface area of the
cubes (~20 cm2) in a test tube corresponded approxi-
mately to the area of eight wells of a standard 96-well
plate, providing for stability and reliability of the
experimental results. Sterilization was carried out at
1 atm. After addition of the tested compounds (e.g.,
antimicrobial agents) and inoculation (usually with 5–
50 μL of a 24-h culture), the tubes were incubated on
a shaker at 150 rpm at a required temperature value.
Biofilms were formed on PTFE cubes, while plank-
tonic cultures grew in the liquid medium in the same
test tube. The tubes were then cooled to room tem-
perature, the planktonic culture was decanted, and the
cubes were washed with 1% NaCl. Growth of the
planktonic culture was determined using relative opti-
cal density (absorption + light scattering) at 540 nm.
Biofilms were fixed with 96% ethanol for 15 min,
washed with distilled water, and stained. For staining
with CV and DMMB, the standard procedure (Peeters
et al., 2008) was used with some modifications. MTT
staining was carried out (without fixing) using our
modification of the published procedure (Wang et al.,
2010). This method was used successfully for investi-
gation of the effect of a number of antibacterial and
antibiofilm agents on formation of microbial biofilms
(Gannesen et al., 2015; Mart’yanov et al., 2015).
CV staining. CV solution (0.1%) was prepared by
dissolving 500 mg CV (Sigma) in 15 mL of 96% etha-
nol and diluting the mixture with distilled water to
500 mL. This solution may be stored at room tempera-
ture in the dark for several months. CV solution
(3 mL) was added to the test tubes with the cubes
washed of the planktonic culture. After 30-min incu-
bation, the solution was removed and the cubes were
washed with distilled water to remove the excess dye.
CV was extracted with 3 mL of 96% ethanol (the
results did not change significantly at extraction times
from 60 to 120 min). The experimental and control
variants were treated similarly. Optical density of the
extract (OD590) was measured against the relevant uni-
noculated control (a test tube with the cubes incu-
bated together with the experimental and control
tubes).
DMMB staining. DMMB (1,9-dimethyl-methy-
lene blue zinc chloride double salt, Sigma) was dis-
solved in 96% ethanol (80 mg/25 mL) and diluted with
100 mL 1 M guanidine chloride supplemented with
sodium formate (1 g) and 99% formic acid (1 mL).
The mixture was slightly acidic (pH 3). This solution
may be stored at room temperature in the dark for sev-
eral months. DMMB solution (3 mL) was added to the
test tubes with the cubes washed of the planktonic cul-
ture. After incubation for 60–120 min, the solution
MICROBIOLOGY Vol. 85 No. 4 2016
A UNIVERSAL METHOD FOR QUANTITATIVE CHARACTERIZATION 511
was removed and the cubes were washed with distilled
water to remove the excess dye. The dye was extracted
with 3 mL of the solution containing 1.64 g sodium
acetate, 100 g guanidine chloride, and 40 mL isopro-
panol in 400 mL distilled water (pH 6.8). Optical den-
sity was measured against the relevant uninoculated
control within the range from 620 to 670 nm (similar
to CV staining).
MTT staining. MTT dissolved in sterile distilled
water may be used for analysis of planktonic cultures.
For investigation of washed biofilms, MTT should be
dissolved in the relevant sterile medium, since the
amount of intracellular endogenous substrates may be
insufficient for quantitative MTT reduction.
Planktonic cultures were stained by adding 1 mL of
0.2% MTT solution to 1 mL of the culture (OD 1.0–
1.5), which was then incubated for 60–120 min at
30°C, depending on the rate of reduction. The mixture
was then cooled to room temperature and the cells
were precipitated by centrifugation for 15 min at
7000 rpm. The supernatant was discarded, and 3 mL
of chemically pure dimethyl sulfoxide (DMSO) was
added to the cell pellet. Extraction was carried out to
almost complete decoloration of the cells. The mix-
ture was then centrifuged, and OD of the supernatant
was measured within the range of 500–600 nm. Direct
proportionality between optical densities of the cell
suspension and of the extract (as well as the CFU val-
ues) was observed in the control cultures.
For biofilm staining, the cubes washed of plank-
tonic culture were incubated with 3 mL of 0.1% MTT
solution in the medium for 60–120 min at 30°C to the
maximal staining. In experiments with biocidal
agents, the biocide concentration in the mixture
should be the same as in the experimental test tubes.
Excess dye was removed, and DMSO (3 mL) was
added to the cubes. Optical density was measured
against the relevant uninoculated control within the
range from 500 to 600 nm (similar to CV and DMMB
staining).
Application of Glass Fiber Filters
Determination of metabolically active microbial
cells. The publications on the application of glass fiber
filters for investigation of biofilm formation are few
(Trémoulet et al., 2002; Guillier et al., 2008), primar-
ily due to the fact that the dyes used for biofilm stain-
ing bind to glass fiber, resulting in too high background
coloration of the extracts. MTT was used to stain the
biofilms formed on glass f iber filters, since formazan
forming in the reaction is localized inside the cells,
thus providing for no background staining of the filter.
This method makes it possible to determine the
number of viable, metabolically active cells in biofilms
and is therefore suitable for quantitative characteriza-
tion of the pharmacodynamic parameters mentioned
above (MBBC and MBEC).
Glass fiber paper (GF/F, Whatman) was used for
detection of metabolically active cells in developing
biofilms. It was cut into 2 × 2-cm squares, sterilized at
1 atm, and placed on the surface of solid nutrient
medium supplemented with the tested compounds. A
relevant amount of the solvent for the tested com-
pounds was added to the control plates. The studied
microorganism was applied to the filters as 50 μL of a
24-h planktonic culture. In the case of m otile bacteria,
the inoculum was immobilized in the medium with
0.3% agar. The density of planktonic cultures was
found to be important. Since the rates of MTT reduc-
tion by different microorganisms vary considerably, an
express method for determination of the optimal den-
sity was developed. Serial dilutions of the culture in
the medium were applied (50 μL) on a strip of regular
filter paper. Immediately after absorption of the sus-
pension, 50 μL of 0.1% MTT solution in the medium
was then added. The control spot (without suspen-
sion) contained only the MTT solution. After incuba-
tion for 30 min at 30°C, the color was assessed visually
and the dilution resulting in weak, albeit visible, color-
ation was chosen. The control spot should remain
uncolored.
Glass fiber filters were incubated at 30°С. Incuba-
tion time depended on the goal of the experiment.
After incubation, the filters were transferred into
weighing bottles with 0.1% MTT solution in the
medium and incubated at 30°С to complete develop-
ment of the stain (usually 15 to 30 min). Stained filters
were washed with distilled water and dried on strips of
filter paper. Quantitative measurement of the staining
was achieved by densitometry (e.g., on a Sorbfil densi-
tometer, Tekhnokom, Russia) or by photometry after
formazan extraction with DMSO, as was described
above. The results obtained by both techniques were
similar. Glass fiber filters may be used in test tube
instead of PTFE cubes. In this case, a f ilter (2 × 2 cm)
was placed into a test tube with 5 mL of the medium.
All procedures were similar to the experiments with
PTFE cubes, but only MTT staining was used.
CFU determination. As was mentioned above, close
association between microbial cells and EPM is the
main factor hindering CFU determination in biofilms.
Approaches used to separate the cells and EPM are
either ineffective (e.g., DNase treatment) or result in
partial disintegration of the cells (sonication). High-
speed centrifugation in a density gradient and
mechanical homogenization techniques proved to be
milder and more efficient (Toyofuku et al., 2012). The
glass fibers of the filters may act as an abrasive mate-
rial, and intense shaking may result in good homoge-
nization (Wang et al., 2010). For this purpose, a bio-
512
MICROBIOLOGY Vol. 85 No. 4 2016
PLAKUNOV et al.
film-covered filter was placed into a test tube with
5 mL of sterile medium, homogenized with a sterile
glass rod, and shaken on a Vortex-ZX3 mixer at the
maximal rate. After precipitation of the fragments of
the filter, tenfold dilutions in sterile medium were pre-
pared from the supernatant and then plated (50 μL) on
petri dishes with agar medium. The material was
spread with a sterile spatula, and the colonies were
counted after incubation for 48 h at 30°C.
While the results of CFU counts were shown to
correlate well with those of MTT staining, due to the
factors described above it is certainly a probabilistic
estimate of the minimal possible number of living cells
in a biofilm.
The work was supported by the Russian Science
Foundation, project no. 16-14-00028.
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Translated by P. Sigalevich
