Modified resazurin microtiter assay for in vitro assessment of different antimicrobials against laboratory pathogen

1
Modified resazurin microtiter assay for in vitro assessment of
different antimicrobials against laboratory pathogen.
Objectives:
Development of microtitre plate-based
antibacterial assay incorporating
resazurin as an indicator of cell growth,
and its application in the in vitro
antibacterial screening of laboratory
pathogens.
1) To standardise the assay using
standard strains of bacteria, different
concentrations of dye, incubation
conditions (time).
2) To compare the disc diffusion assay
methods with the microtitre resazurin
reduction test in determining
susceptibility.
3) To validate the assay in clinical
samples.
Introduction:
Antibiotics have revolutionised
mankind’s health status, allowing
treatment of life threatening infections.
But, resistance has been observed to
virtually most of the antimicrobial
agents approved for use in human and
veterinary clinical medicine. Moreover,
there is a limited number of
antimicrobials agents currently
available. This makes the selection of an
appropriate antimicrobial agent an
increasingly more challenging task. And
hence, it is essential to employ an in vitro
antibacterial assay that is simple, rapid,
efficient, reliable, sensitive, safe and
cost-effective. There are a variety of
formats for invitro susceptibility, most
common being disc diffusion, agar
dilution, broth macro dilution, broth
micro dilution and concentration
gradient test that are available for
antibacterial screening, can be a limiting
factor sometimes. As, all this
conventional methods requires solid
agar plates, and occupies space and
reagents and could be cumbersome and
tedious. Although disc diffusion assay is
the gold standard used in hospitals for
determining the suitable antimicrobial
agent against the microorganism causing
particular infection. We propose a
modified resazurin microtiter assay for
invitro assessment of different
antimicrobials against laboratory
pathogens. There are no reports in
literature where microtitre format using
discs and resazurin dye for evaluating
antimicrobial susceptibility has been
attempted. There is considerable
opportunity for improvement in the area
of rapid, economical detection of
bacterial resistance /susceptibility to
antibiotics.
Outcomes and benefits of the study:
This method, with incorporated
resazurin as indicator, would allow the
detection of microbial growth in
extremely small volumes of solution in

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microtiter plates faster than conventional
method using solid /liquid Medias.
An advantage of the microtiter format is
that it allows the screening of several
isolates in a short period of time.It could
visually determine the resistance
/susceptibility of the pathogen to a panel
of antibiotics, requiring less incubation
space. The economyof reagents and less
space constraints due to the
miniaturization of the test is another
advantage. This would result in the
development of a diagnostic test that is
simple, accurate, rapid and economical
in detecting susceptibility of laboratory
pathogens including multidrug resistant
organisms which would be especially
ideal in low resource setting and in low
income countries.
Methods:
A) Disc diffusion assay(Kirby-Bauer
method):
Preparation of bacterial inoculum (1-
2×108 cfu/ml. Applying it on the agar
surface (Mueller-Hinton) with sterile
swab stick. Commercially-prepared
fixed concentration, paper antibiotic disc
are placed on inoculated agar surface.
Incubate the plates at 37˚C for 16-
24hrs.The diameter of the zones in
millimetre showing complete inhibition
would be measured. The zone diameter
of each drug are interpreted using the
clinical and laboratory standards
institute (CLSI) guidelines.
The result of the disc diffusion test are
“qualitative” and categorised as
susceptible, intermediate, resistant.
B) Broth method with incorporation
of resazurin dye.
 Preparation of bacterial culture:
Inoculate 3-4 similar looking colonies in
to MH broth (Mueller Hinton) and
incubate at 37˚C for 1-2 hour until light
to moderate turbidity develops.
Compare the inoculum with that of
standard 0.5 McFarland. Dilute or else
incubate further as necessary to attain
desired turbidity. Alternatively the
desired density can be obtained by
taking OD at 625nm around (0.08-0.13)
and diluted to 1:100 with MH broth.
 Preparation of resazurin solution:
The resazurin solution will be prepared
by dissolving resazurin due in D/W.The
solution would be filter sterilized
through 0.22µm and used. This ready to
use prepared solution is stable fora week
if stored at 4˚C.
 Resazurin reduction test:
Preparation of 96 well microtiter plates
are done with antimicrobial discs of
particular known potencyand pre-placed
in the well under aseptic condition.100µl
of MH broth added in to the wells.
Finally bacterial inoculum is added of
concentration 5×10^6 cfu/ml.To all
wells 50µl of desired concentration of
dye is added. A set of control is kept,
positive control with all solutions and
bacterial inoculum and a negative
control with all solutions except
bacterial inoculum. And a sterility
control of media.
Incubate the plate at 37˚C and record the
change in colour from blue to pink for
growth development. Gram negative
panel results can be read in 4-8hrs and
gram positive 8-16hrs.The plate would
be further read in ELISA reader at the

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two wavelengths of 575(blue) and
600(pink) nm.
Preliminary studies:
 Standarization of the test:
Standardization of the test was carried
out using standard bacterial strains like
E.coli, P.aeruginosa and S.aureus.
For standardization the parameters
considered where various concentration
of resazurin dye and various
concentration of bacteria
simultaneously.
Concentration of resazurin dye: 0.5mM,
1mM, 1.5mM, 2mM, 2.5mM and 3mM.
Concentration of bacterial inoculum:
108, 107,106,105,104,103,102,101 cfu/ml.
Incubation temperature: 37˚C.
Controls:
Positive =all solutions with one
concentration of dye and bacteria
Negative =all solutions except bacteria.
 Standardisation of test using
antibiotic disc:
The second step was to standardize the
findings with a resistant and sensitive
antibiotic for the standard strains. As, all
the above strains are standard and are
expected to besensitive to all antibiotics.
Concentration of culture:108,107,106,105
cfu/ml.
Concentration of resazurin dye: 0.5mM,
1mM, 1.5mM, 2mM, 2.5mM and 3mM.
Antibiotics used-Amikacin (E.coli)
Oflaxain (P.aeruginosa).
Gentamicin (S.aureus).
The test was put with and without
antibiotic for all the respective cultures
such that without antibiotic plate would
be considered as control to observe the
effect of antibiotic on them.
 Note: From all the above trial 106
was considered as appropriate
concentration of culture for gram
negative bacilli but gram positive
being a slow grower conclusion
were not made for them.
 Standardization ofdye concentration.
From the above experiments we were
able to deduceappropriate concentration
of the culture i.e. 106cfu/ml (1:100)
dilution of 108 cfu/ml.
Therefore, to decidethe concentration of
the dye to be used, concentration of
bacterial inoculum is kept constant with
varied concentration of dye and
appropriate antibiotic.
To know the sensitivity and resistant
pattern simultaneously Kirby Bauer
method was put.
Antibiotic used:
 E.coli
Cotrimox, vacomycin.
 P.aeruginosa
Oflaxacin, vancomycin.
 S.aureus
Vancomycin.

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 Carry out the test with clinical
isolates.
After obtaining concentration of dye
and bacterial inoculum, the test was
put for clinical isolates and results
were compared with Kirby Bauer
method.
 Results:
Standardization of the test:
Standardization of the test using
antibiotic:
Standardization of dye concentration:
Clinical isolate results:

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20 E.coli isolates-1)Kirby Bauer and
2)Resazurin reduction test.
Amikacin Amox-
clav
Cefotaxi
me
piperacill
in
cotrimox norfloxac
in
Nalidixic
acid
Nitrofura
ntoin
meropenem
1 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 16mm(R) 10mm(R)
pink pink pink pink pink pink pink BLUE BLUE
3 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 20mm(S) 11mm(R)
4 18mm(S) 6mm(R) 10mm(R) 10mm(R) 14mm(R) 6mm(R) 6mm(R) 18mm(S) 9mm(R)
7 16mm(R) 6mm(R) 6mm(R) 13mm(R) 6mm(R) 19mm(S) 6mm(R) 19mm(S) 20mm(R)
blue pink pink pink pink pink pink BLUE BLUE
10 20mm(S) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 25mm(S) 16mm(R) 18mm(S) 25mm(S)
Blue pink pink pink pink pink pink BLUE BLUE
11 19mm(S) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 26mm(S) 6mm(R) 19mm(S) 17mm(R)
12 20mm(S) 6mm(R) 26mm(S) 9mm(R) 6mm(R) 6mm(R) 6mm(R) 18mm(S) 15mm(R)
Blue pink blue pink pink pink pink BLUE BLUE
17 15mm(R) 14mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 18mm(S) 14mm(R)
blue blue pink blue pink pink pink BLUE BLUE
pink pink pink blue pink pink pink BLUE BLUE
19 19mm(S) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 6mm(R) 16mm(R) 11mm(R)
20 21mm(S) 15mm(R) 24mm(S) 23mm(S) 6mm(R) 19mm(S) 6mm(R) 15mm(R) 22mm(S)
pink blue pink blue pink pink pink BLUE BLUE

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This are the comparative result obtained
for 20 E.coli isolates from two methods
Kirby Bauer and resazurin reduction
test.The discrepant results are
highlighted in red colourand thoseresult
obtained for nitrofurantoin and
meropenem cannot be considered as
these two antibiotics being coloured
showed no distinct colour.
Discussion:
We explored the possibility of
evaluating antimicrobial sensitivity
using microtitre formatted antibiotic
discs panels incorporating resazurin dye
which could visually determine the
sensitivity of different pathogens. We
initially attempted to evaluate the assay
using standard strains of
microorganisms.
The objective was to develop a rapid
format for evaluation of MIC so to
enable the physician in choosing the best
appropriate antibiotic for individual
treatment. Attempts were made to
evaluate the time point for dye reduction
by visually comparing the microtitre
plate at different time intervals. It was
observed that the dye reduction depend
upon the number of organisms in the
inoculum and the metabolic activity of
the organisms. Organisms which had an
initial high inoculum and which was
metabolically active e.g. E.coli gave a
reduction in 4 hrs, whereas organisms
which were metabolically less active e.g.
S.aureus or those with low initial
inoculum took time to causereduction in
colour.
Hence, as used during standardization
various concentration of bacterial
inoculum i.e. 108,
107,106,105,104,103,102,101 cfu/ml, from
the result obtained 104,103,102,101
cfu/ml were ruled out as this
concentration had low inoculum to get
result in desired time.
Further, using appropriate antibiotics
like amikacin, oflaxacin and gentamicin
for E.coli, P.aeruginosa and S.aureus
respectively .106 cfu/ml was considered
appropriate concentration in comparison
with time taken for reduction in dye.
Further, to standardize concentration of
dye; bacterial inoculum was kept
constant (106 cfu/ml) and concentration
of dye was varied with combination of
antibiotic like amikacin & gentamicin,
oflaxacin & gentamicin and gentamicin
for E.coli, P.aeruginosa and S.aureus
respectively. Simultaneously Kirby –
Bauer method was also done for the
respective organisms with respective
antibiotics to compare the findings. The
results obtained were all sensitive and
both the method finding matched, this
did not helped to deduce the
concentration of antibiotic and hence it
was repeated with other antibiotic

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combination like cotrimox
&vancomycin, oflaxacin &vancomycin
and vancomycin for E.coli, P.aeruginosa
and S.aureus respectively. After
recording the readings at 575nm and 600
nm, we came to the conclusion that any
one concentration of dye could be used
as the colour change was observed even
at the lowest concentration and decided
to use 0.5mM.
Thus, after getting concentration of
bacterial inoculum and concentration of
resazurin dye to be used, the test was
carried out using clinical isolates for
about 20 E.coli isolates obtained from
urine samples. A 9 panel of antibiotics
were used (amikacin, amox-clav,
cefotaxime, norfloxacin, cotrimox,
pipercallin, nitrofurantoin, meropenem,
nalidixic acid).
The readings were recorded at 575 and
600 nm wavelength and it was observed
that the result obtained from both the
method were at 95% agreement. But for
those antibiotics which are coloured
didn’tgive a desired coloured and hence,
there was a limitation in interpreting
them.
Our study, thus can be assessed without
instrumentation .The 96 well micro titre
resazurin format could be employed to
determine antimicrobial sensitivity of
8samples with 12 antibiotics and vice
versa. A comparison of visual and
photometric reading of the microtitre
plates showed that the results could be
assessed without instrumentation.
Thus this modified resazurin method is
simple, sensitive, cost effective, rapid
and robust and reliable to assess
antimicrobial suspecibitlity. To support
our findings we would assess more
clinical isolates (900) and thus validate
the testsuchthat it can beused routinely.
References:
1. Microtitre plate-based antibacterial assay
incorporating resazuras an indicator of cell
growth, and its application in the
In vitro antibacterial screening of
phytochemicals
Satyajit D. Sarker, Lutfun Nahar, Yashodharan
Kumarasamy.
2. Development of resazurin microtiter assay
for drug sensibility testing of Trypanosoma
cruzi epimastigotes
Miriam Rolón Celeste Vega, Alicia Gómez-
Barrio José A. Escario.
3. Lactobacillus Bacteraemia, Species
Identiﬁcation, and Antimicrobial Susceptibility
of 85 Blood Isolates
M. K. Salminen, H. Rautelin, S. Tynkkynen,
T. Poussa, M. Saxelin, V. Valtonen, and A.
Jarvinen.
4. Spectrophotometric application of resazurin
reduction assay to evaluate boar semen quality
P. zrimsek, j. kunc, m. kosec and j. mrkun
Clinic for Reproduction and Horses,
Veterinary Faculty, University of Ljubljana.
6.Spectrophotometric analysis of resazurin
reduction test and semen quality in men
,Reddy KV,Bordekar AD ,Indian journal of
experimental biology (1999).

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