Quantification of metabolically active biomass using Methylene
Blue dye Reduction Test (MBRT): Measurement of CFU
in about 200 s
Prashant Bapat1, Subir Kumar Nandy1, Pramod Wangikar, K.V. Venkatesh*
Department of Chemical Engineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India
Abstract
Quantification of viable cells is a critical step in almost all biological experiments. Despite its importance, the methods
developed so far to differentiate between viable and non-viable cells suffer from major limitations such as being time intensive,
inaccurate and expensive. Here, we present a method to quantify viable cells based on reduction of methylene blue dye in cell
cultures. Although the methylene blue reduction method is well known to check the bacterial load in milk, its application in the
quantification of viable cells has not been reported. We have developed and standardized this method by monitoring the dye
reduction rate at each time point for growth of Escherichia coli. The standard growth curve was monitored using this technique.
The Methylene Blue dye Reduction Test (MBRT) correlates very well with Colony Forming Units (CFU) up to a 800 live cells
as established by plating. The test developed is simple, accurate and fast (200 s) as compared to available techniques. We
demonstrate the utility of the developed assay to monitor CFU rapidly and accurately for E. coli, Bacillus subtilis and a mixed
culture of E. coli and B. subtilis. This assay, thus, has a wide applicability to all types of aerobic organisms.
Keywords: Colony Forming Units (CFU); Viability; Methylene blue dye; Dye decoloration; Cell membrane enzymes; Escherichia coli
1. Introduction
Viability is an essential analytical measurement for
quantifying cell culture. However, quantifying viabi-
lity in bacterial cultures is a challenge. The various
methods for evaluating viable cell count can be
grouped into three broad categories. The first category
represents microbiological estimation such as viable
cell count, drop plate count and membrane filtration
methods. Although these have been the preferred
methods, they are known to be labor and time intensive as well as error prone (Colwell, 2000; Auty et al.,
2001; Stentelaire et al., 2001; Herigstad et al., 2001).
108
The second category of cell viability estimation is
based on specific enzymatic activity prevalent in the
cell. These methods are usually species or genus
specific (Rompre et al., 2002; Callaway et al.,
2004). The third category is based on microscopic
examination of stained cells of an organism. Typically
stains such as methylene blue, acridine orange, propidium iodide or rhodamine are used (Miller et al.,
1993; Vaija et al., 1993).
Among commercially available kits, the LIVE/
DEAD BacLight viability kit (Molecular Probes
Inc., Eugene, Oreg.) is available to differentiate live
and dead bacteria cells based on plasma membrane
permeability and has been used to monitor growth of
bacterial populations. Different fluorescence probes
are used to enumerate live and dead cells. Another
method to enumerate live and dead cells is 4V, 6diamidino-2-phe-nylindole (DAPI) staining methodology (Auty et al., 2001). Confocal scanning laser
microscopy (CSLM) is a microscopic technique
which is used extensively in cell biology (Wright et
al., 1993) to study viability of Escherichia coli and
Salmonella, where rhodamine 123 and propidium
iodide were employed to differentiate viable from
nonviable bacterial cells based on its membrane
potential. Conventional epifluorescence microscopy
is also widely used for viability staining of liquid
samples such as milk (Pettipher et al., 1980). Flow
cytometry can also yield valuable information regarding viability (Davey and Kell, 1996; Deere et al.,
1998), these methods are however instrumentation
intensive and expensive. On the other hand, the vital
stain method using a dye such as methylene blue
overestimates cell viability (Vaija et al., 1993).
These methods can be error prone as it is sometimes
difficult to differentiate living cell from dead during
microscopic observations (Walley and Germida,
1995). Other methods such as assays based on ATP
(Venkateswaran et al., 2003) and rRNA measurements
(Karner and Fuhrman, 1997) are also reported. Thus
problems in differentiating viable and non-viable cells
in a cell culture persist with limitations including lack
of sensitivity, expense, labor intensive and time consuming. Here, we present a rapid, sensitive and low
cost yet a simple quantification method to evaluate
viable count during a growth experiment. The method
is based on the Methylene Blue dye Reduction Test
(MBRT).
The Methylene Blue dye Reduction Test is widely
used in dairy industry to determine the microbial
load in the milk. This test involves the addition of
methylene blue into a milk sample and measuring the
time required for decoloration. The disappearance of
the color in a short time indicates a high microbial
load. The disappearance of the color is due to the
removal of oxygen from milk and formation of
reducing substances during bacterial metabolism
(Atherton and Newlander, 1977; APHA, 1978;
Impert et al., 2003).
Methylene blue in a sample containing microorganisms gets reduced to dleukoT or colorless form
of the dye at the cell surface via reductase enzymes
present in the cell membrane. This colorless form
of methylene blue (MBH) is uncharged, lipophilic,
and enters cells by diffusion across the plasma
membrane where it is re-oxidized and thus sequestered within the cells (May et al., 2003). If oxygen
is available, reduced MB can be oxidized by the
mitochondrial electron transport system. This will
result in the reappearance of the blue color.
Although the exact mechanism of dye reduction
is not clearly known, some reports available suggest that MB is reduced by transmembrane reductases (Bongard et al., 1995; Merker et al., 1997).
This mechanism is applied to evaluate the microbial
load in a liquid medium. The shorter time required
for the disappearance of the blue color is indicative
of a higher microbial load. It is assumed that
greater the number of microorganisms, more the
oxygen demand and lesser the oxygen concentration in the medium resulting in the faster disappearance of the color. This fact has been used as a
broad indicative test of a microbial load representing microbial quality of milk (Atherton and Newlander, 1977). However; methylene blue reduction
has not been used for quantification of viable count
in cell cultures.
Thus, the main focus of this study is to develop a
protocol to relate MBRT to optical density measurement (OD) and viable plate count as CFU. The MBRT
test was applied to the growth of E. coli and Bacillus
subtilis. We demonstrate that the MBRT can be used
as a direct measure of viable count of bacterial cell
cultures. Further, the method was extended to enumerate the CFU in a mixed culture broth of E. coli and
B. subtilis.
109
strain was obtained from MTCC, IMTECH Chandigarh, India. The strains were maintained on Luria
agar (LA) slants (Kirkpatrick et al., 2001). A loopful of the culture from the slant was sub cultured
before each test into 100 ml of sterile Luria broth
(LB) and grown for 6 h at 37 8C at 240 rpm. The
2. Materials and methods
2.1. Microorganism
E. coli k12 (MTCC 1302) and B. subtilis168trpC2 was used throughout this study. The
One loopful
culture
10 % seed (v/v)
6 – 8 hrs
6 – 8 hrs
LB agar
slant
LB medium
37°C / 240 rpm
LB medium
37°C / 240 rpm
Centrifuge
10,000 rpm / 10 min
Reconstitute with
PBS (pH : 8)
Discard
supernatant
Serial
dilutions
Up to 1 : 1024
6 ml
Centrifuge
10,000 rpm / 10 min
0.1 ml
0.5 ml
Sample
couvette
1 ml
Discard
supernatant
Add 1 ml
PBS (pH : 8)
0.5 ml
Optical density
At 600 nm
Spread
plate
Viable count
Reference
couvette
Serial
dilutions
AUTOZERO
Methylene Blue
3.3 µL
MBRT
CFU/ml
MBRT
at 700 nm
OD
MBRT
Fig. 1. Schematic representation of MBRT protocol: determination of relationship between optical density, CFU/ml and MBRT. Fresh slant is
inoculated with E. coli stock culture. After incubating overnight, one loopful of culture is transferred to LB medium. Ten percent of the same
was used as a seed to inoculate 100 ml LB medium. Whole broth was centrifuged when cells were in exponential phase. These cells were
resuspended in 100 ml PBS and further used for estimation of optical density, colony forming units (CFU/ml) and MBRT slope.
110
2.2. Chemicals
LB (Hi-media, Mumbai, India) was used throughout the experiments to grow the organism. LA (Himedia, Mumbai, India) was used for culture maintenance on slant and for viable count using spread plate
method. Methylene blue dye was obtained from E.
Merck, India.
2.3. Methylene Blue dye Reduction Test: protocol
development
2.3.1. Cuvette volume standardization
Methylene blue dye was prepared by dissolving
10 g of the dye in 1-l double distilled water. The
solution was filtered through Whatmann filters to
avoid interference from undissolved matter. The
same dye concentration was used throughout this
study. The ratio of methylene blue dye to the culture
was standardized to 6.7 ml per 1 l of the culture by
trial and error. Our analysis indicated that a higher
ratio will result in a non-linear relationship between
MBRT and OD, while a lower ratio will increase the
rate of decoloration resulting in lower sensitivity.
During time course measurement, cuvettes were covered with a plastic lid to prevent fresh oxygen dissolution into the culture. This was done in order to
keep the concentration of dissolved oxygen same in
the headspace of the cuvette. Dead bacterial cells
were used as negative control in dye reduction test.
The quartz cuvettes were used instead of plastic to
minimize error caused by the adhesion of methylene
blue dye over a period of time. Further, the MB dye
concentration and the blue color intensity was also
linear in the range 0.1–1.0 g l 1 of methylene blue
indicating that any concentration in this range can be
used for this assay.
(around 100 ml) was aseptically centrifuged at 10,000
rpm (Remi C-24 centrifuge, India) for 10 min. Cells
were resuspended in an equal amount of phosphate
buffer saline (PBS, pH 8). This 10 ml sample was
used for making serial dilutions. PBS was used as a
diluent. From each test tube out of 15 employed in
this calibration experiment, 1 ml of sample was used
for optical density measurement (at 600 nm, ModelV530, Jasco Corporation, Japan) while 0.1 ml was
taken for viable count. It should be noted that for
viable count 3 to 4 different dilutions were used to
avoid error. Spread plate method was employed to
enumerate the bacteria. The LB agar plates were
incubated at 37 8C for 24 h. For sensitivity analysis
of MBRT, around 6 ml sample volume from each testtube was centrifuged at 10,000 rpm for 10 min and
resuspended in one ml PBS. This was done to concentrate the bacteria and to reduce the MBRT test
duration. From this sample 0.5 ml was loaded into
reference cuvette (capacity, 1 ml) and another 0.5 ml
was loaded into sample cuvette. After doing autozero,
MB dye was added to sample cuvette and MBRT
slope was calculated using time course measurement
mode at 700 nm. Fig. 1 shows a detailed protocol for
the method to evaluate the sensitivity of the method.
Fig. 2 shows the time profile of the decoloration of
methylene blue as an output of spectrophotometer at
700 nm. The initial slope of decoloration was evaluated as shown in the figure.
1.3
1.0
Absorbance
10% (v/v) of this seed was then added to 100 ml
sterile LB and grown for another 24 to 36 h for
kinetic analysis.
b
0.5
0
2.3.2. Standard calibration curve and sensitivity
analysis of MBRT
To obtain the relationship between optical density,
viable cells and methylene blue decoloration rate,
E.coli and B. subtilis were propagated as mentioned
earlier. After 6 to 8 h of incubation, the whole broth
a
0
100
200
300
Time[sec]
Fig. 2. Estimation of MBRT slope of decoloration. A typical time
profile graph generated by spectrophotometer is shown. Solid curve
represents decrease in optical density due to decoloration of the dye.
Slope was calculated by taking linear range of the decoloration (that
is from point daT to point dbT, as shown in the figure).
111
2.3.3. Effect of pH on initial rate of methylene Blue
dye Reduction
Phosphate buffer saline (PBS) of different pH was
prepared in a pH range of 4–9. Ten percent (v/v) E.
coli culture in exponential phase was added to different shake flask containing PBS of different pH.
Further, the slope of decoloration was evaluated for
the same cell concentration. Fig. 3 shows the slope at
different pH values.
2.3.4. Relating OD and CFU with MBRT
The sensitivity analysis discussed in Fig. 1 was
carried out to relate OD and CFU to slope of decoloration obtained from MBRT. The analysis was carried out in PBS at pH 8. Fig. 4a shows the linearity
between the MBRT slope and OD measurement in a
OD range of 0–5. Fig. 4b shows the linearity between
the MBRT slope and CFU as obtained by plating. In
this case, the linearity was observed for log(MBRT
slope) and log(CFU/ml) in a range of CFU between
105 and 1016. Similar linearity was established for B.
subtilis and a mixed culture of E. coli and B. subtilis
(results not shown).
2.3.5. E. coli growth experiment
Sterile LB medium was inoculated with 10% (v/v)
inoculum and incubated at 240 rpm at 37 8C.
Samples were taken at regular intervals for optical
0.012
MBRT slope
It should be noted that the color of the dye
emanates only from the broth. The dye does not
penetrate into the biomass until the dye is in the
reduced state. After the decolorization due to reduction, the dye enters the cell and remains in a reduced
state inside the cell. The lid on the cuvette prevents
the diffusion of oxygen in the short time of the assay.
This ensures that the dye remains in a reduced state
in the cell leading to the estimation of blue color
intensity from the broth alone. Further, the concentration step involved in the sensitivity analysis may
change the environmental dynamics and alter the cell
metabolic state. MBRT was performed before and
after concentrating the biomass. The test demonstrated that there was no difference in the CFU.
However, it should be noted that the MBRT demonstrated a lower slope before concentrating the broth.
Therefore, a better measure was estimated from the
concentrated broth.
0.008
0.004
0
0
2
4
6
8
10
pH
Fig. 3. Effect of pH on initial rate of dye decoloration. MBRT was
carried out at different pH values with the same initial biomass
concentration. The figure demonstrates that MBRT is valid in a pH
range of 5.8–9.0. Symbol D represents measured value of MBRT
slope. The error bars indicate standard deviation of three true
replicates.
density measurement, viable plate count and MBRT.
The slope of the decoloration was evaluated in a
200 s period for each sample from the spectrophotometer. Experiments were conducted as three true
replicates and the average is reported. Similarly,
growth experiments were carried out to relate
MBRT to CFU for B. subtilis and a mixed culture
of E. coli and B. subtilis. The linearity obtained
from the sensitivity analysis and MBRT yielded
estimates of CFU, further CFU were measured
independently at these time points to validate the
CFU measurement.
3. Result and discussion
MBRT depends on measuring the rate of decoloration of methylene blue in a cell culture. The
protocol discussed in the previous section (see Fig.
1) was used to estimate the OD of metabolically
active cells and CFU for E. coli. The MBRT can be
used in a range of 6–8 pH values for E. coli (refer
to Fig. 3). Determination of the pH range of the
MBRT test for any organism is important as the test
involves reduction by an active biological enzyme.
Therefore, phosphate buffer saline (with a pH 8) was
used in the test to avoid any errors caused due to
pH. Similar experiments indicated that a pH range of
112
a
MBRT slope (decol/sec)
0.012
0.008
0.004
0
0
2
4
6
OD (at 600 nm)
b
Log (slope), (decol/sec)
6
4
2
0
0
5
10
15
20
Log (VC per ml)
Fig. 4. Evaluation of linearity (a) linear relationship between optical density and MBRT slope. Symbol D represents experimental points. Solid
line represents best fit with R 2 = 0.98. Best-fit equation is given by Y = 0.0018 X. (b) Linear relationship between CFU and MBRT slope. Symbol
D represents experimental points. In this case, log(MBRT slope) and log(CFU) were fitted as a straight line. Solid line represents best fit with
R 2 = 0.97. Best-fit equation is given by Y = 0.2056 * X + 5.5242.
6–7 could also be used for B. subtilis. The test
depends on the number of active bacteria and oxygen concentration. To reduce the variation of oxygen
concentration, the loading volume was standardized
to 2 ml in a 3 ml cuvette; and the same was covered
with a lid to prevent fresh oxygen entry during
analysis. This fixed volume of 2 ml ensured the
reproducibility of the results. Further, MBRT on
dead cells and broth containing no cells demonstrated no reduction of the dye indicating that only
metabolically active cells are quantified by the
method.
The sensitivity analysis using E. coli demonstrated
that slope of decoloration obtained from MBRT can
be used to correlate the OD of metabolically active
cell culture and the CFU of the cells for a order of
magnitude greater than 800 cells. The key step in
establishing the sensitivity of the test for determination of CFU was the concentration of the cells by 6
fold to give a reliable slope of MBRT. This step in the
MBRT reduced the test time to 200 s from 2 h. The
correlation of MBRT slope to OD and viable count (as
shown in Fig. 4a and b, respectively) was established
to be linear and sensitive. Linearity of MBRT slope
113
was observed up to an OD of 7 and in the range of
104–1016 cells for the viable count.
The MBRT slope for a growing culture of E. coli at
different time points was measured. Fig. 5a shows the
variation of slope at different time point with progression of cell growth. The slope increases with time
indicating an increase in the active biomass. The slope
reaches a maximum value of 0.008 decoloration/s in
about 12 h before decreasing. The phase representing
a decrease in the slope characterizes the lowering in
the activity of the enzyme indicating decrease in the
number of metabolically active cells. Log of MBRT
slope was plotted versus time (see Fig. 5b) to evaluate
the maximum specific growth rate. The analysis
shows that l max for E. coli was 0.5 h 1 (slope of
the line indicated in Fig. 5b).
Fig. 6a shows the OD measurement at various
growth phases of E. coli and also the equivalent OD
obtained indirectly from the MBRT slope (see Fig.
4a). The OD matches very well in the exponential
growth phase, but deviates once the stationary phase
is reached. The OD indicating turbidity cannot compare the decrease in the metabolic state of the cells.
However, the equivalent OD obtained from the
MBRT captures the drop in the metabolic activity. It
can be noted from the figure that the drop in OD
obtained from MBRT slope is steep as compared to
the actual observed OD after the stationery phase.
Further, the MBRT was also used to correlate the
viable count as CFU. Fig. 6b shows the comparison
between the actual viable count obtained from plating
and that derived from the MBRT slope (see Fig. 4b).
a
0.01
MBRT slope
0.008
0.006
0.004
0.002
0
0
10
20
30
Age (hrs)
b
0
0
5
10
15
20
ln (MBRT slope)
-2
-4
-6
-8
Age (hrs)
Fig. 5. Evaluation of E. coli growth curve (a) variation of MBRT slope in time. The curve represents a typical growth pattern with a steep drop in
activity at the end of fermentation. Symbol D represents measured slope of decoloration. (b) Estimation of maximum specific growth rate using
MBRT slope. Ln (slope) was plotted against age and l max was calculated from the slope of exponential phase, which was evaluated to be 0.51
h 1. Symbol D represents ln (MBRT slope).
114
a
5.0
ln (OD at 600 nm)
4.0
3.0
2.0
1.0
0.0
0
10
20
30
Age (hrs)
b
20
Log (CFU per ml)
16
12
8
4
0
0
10
20
30
Age (hrs)
Fig. 6. Comparison of optical density and CFU as established by MBRT from that obtained through direct measurement. (a) Plot of ln(OD) at
various time. Symbol D represents equivalent OD at 600 nm as determined by MBRT; + represents actual OD measured. (b) Plot of log10(CFU)
at various time. Symbol D, actual CFU measured by plating and continuous line represents equivalent CFU as determined by MBRT.
The prediction of CFU using MBRT correlates very
well with the actual viable count obtained through
plating. Even at the end of the fermentation cycle,
MBRT is able to correlate well with the plate count.
Also the l max obtained from OD and the plate count
matches that obtained by the MBRT slope which is
however not surprising due to the linear correlation
between the two.
Fig. 7a shows the comparison between the actual
viable count obtained from plating and that derived
from the MBRT slope for the growth of B. subtilis.
The maximum specific growth rate as evaluated by
the MBRT slope for the Bacillus strain was 0.63 h 1
that is comparable to that reported in literature. Sensitivity analysis using B. subtilis demonstrated that
MBRT could be used to obtain CFU for an order of
magnitude greater than 1200 cells. MBRT was also
used to predict the CFU in a mixed culture of E. coli
and B. subtilis. The plate count as CFU obtained for
the mixed culture was accurately predicted by the
MBRT test (See Fig. 7b). The maximum specific
growth rate as established by the MBRT slope for
the mixed culture was 0.58 h 1. The sensitivity
analysis on the mixed culture indicated that MBRT
could be used to enumerate viability for about 1000
cells. This demonstrated that the test as developed
115
a
18
Log (CFU per ml)
16
14
12
10
8
6
4
2
0
0
10
20
30
20
30
Age (hrs)
b
Log (CFU per ml)
20
16
12
8
4
0
0
10
Age (hrs)
Fig. 7. Comparison of CFU as established by MBRT from that obtained through direct measurement for B. subtilis and mixed culture. (a) Plot of
log10(CFU) at various time for the growth of B. subtilis. The linear equation of B. subtilis obtained to correlate log(MBRT slope) and
log10(CFU) is given by Y = 3.597 * X + 11.963 with a regression coefficient of 0.98. (b) Plot of log10(CFU) at various time for the growth of
mixed culture of E.coli and B. subtilis. The linear equation of B. subtilis obtained to correlate log(MBRT slope) and log10(CFU) is given by
Y = 4.7075 * X + 24.333 with a regression coefficient of 0.99. Symbol D, actual CFU measured by plating and line represents equivalent CFU
as determined by MBRT.
here could also be used to evaluate CFU for mixed
cultures.
4. Conclusions
In this study, reduction of methylene blue was used
as an indicator of metabolic activity in cells and was
further correlated to CFU and OD. As electron acceptors for the cellular dehydrogenase enzyme, the
methylene blue constitutes a good indicator for biological reducing system. Although the reduction of
methylene blue was used as a measure of microbial
load in liquid foods (such as milk) in the past, there
was no report of quantifying the decoloration rate as a
measure of active biomass. Stentelaire et al. (2001)
have reported the use of tetrazolium dye for enumeration of viable fungal spore. However, the methodology involved extraction of the dye before the
spectrophotometer analysis to further correlate to
viable spore count. Here, we present for the first
time such a quantification to quickly assess the physiological state of the cells. We also present a protocol
to measure the equivalent OD of metabolically active
cells and CFU using the slope of decoloration of
methylene blue.
116
The MBRT slope can thus be used to evaluate the
growth curve and the viability equivalent to plate
count. The advantage of using MBRT is that you
can obtain a plate count in about 200 s, instead of
greater than 24 h in case of actual plate count.
However, in such a case, the error associated with
plate count is also carried over to the MBRT test.
This may be the reason for deviations between CFU
and MBRT for counts less than about thousand cells.
The true advantage of MBRT is that it quantifies
metabolically active cells from the broth in a short
span without an intermediate state, such as plating or
microscopic evaluation. The successful demonstration of utilizing the MBRT assay protocol quantify
viability in organism like E. coli, B. subtilis and
mixed culture shows a greater promise. However,
the true advantage of the methodology can be
obtained only, if MBRT is correlated with a one
time accurate viable measure. Further, this methodology can easily be extended to online estimation of
viable cells in industrial reactors.
Acknowledgement
The authors thank Dr. Malkhey Verma for fruitful
suggestions and Ms. Alka Bikrol for helping in setting
up of initial experiments.
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