American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

ISSN: 1553-3468© 2014 T. Srimongkon et al., This open access article is distributed under a Creative Commons Attribution(CC-BY) 3.0 license
doi:10.3844/ajbbsp.2014.81.87 Published Online 10 (1) 2014 (http://www.thescipub.com/ajbb.toc)

DEVELOPMENT OF A BACTERIAL CULTURE SYSTEMUSING A PAPER PLATFORM TO ACCOMMODATE MEDIAAND AN INK-JET PRINTING TO DISPENSE BACTERIA
1
2
1

Tithimanan Srimongkon, 2Takuya Ishida,
Kiyohiko Igarashi and 1Toshiharu Enomae

Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan
2
Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

Received 2014-03-05; Revised 2014-04-05; Accepted 2014-04-26

ABSTRACT
Generally, bacterial culture is performed manually and is subject to error. Here, we created a novel, wellordered and reliable system for dispensing bacteria microscopically by using paper and an ink-jet printer forcontrolled patterning. For paper to accommodate a culture medium, hydrophobic/hydrophilic patterns wereincorporated onto the paper by immersing paper in a toluene solution of polystyrene and drying forcomplete hydrophobization, followed by etching discrete, small areas of hydrophilicity by ink-jet printing
with toluene. Agar was hydrolyzed with sulfuric acid for appropriate viscosity and dispensed with an ink-jet
printer. In a separate experiment, bacterial cells were sequentially printed on a medium and colonies wereobserved microscopically. The results of this experiment ensured the successful dispensing of bacteria using
ink-jet printing. An almost constant number of particles per droplet were ejected using a polystyrene latex
as a model of bacterial dispersion. Consequently, we expect this technology to be adapted for thedevelopment of a paper-based bioassay system.
Keywords: Paper Platform, Bacterial Cells Printing, Hydrophobic/Hydrophilic Patterning

1. INTRODUCTION
Bacteria are present everywhere: In soils, deep inrocks, in all water bodies and in the atmosphere,
including on and inside other living organisms (Kaláb,
2008). Several kinds of bacteria are commercially
important in industries such as pharmaceutical and foodindustries (Hossain et al., 2012). However, some bacteria
are harmful. To extensively research bacteria species,
microbiologists isolate pure cultures and further analyze
them. To culture bacteria, a nutrient medium is required
in a container, such as a Petri dish or test tube. If growthunder different conditions is needed, several sets ofcontainers are required. To culture bacteria, a nutrient
medium is required as well as a Petri dish or test tube asa container. In preparation for nutrient media, an
autoclave is always required for sterilization at so high atemperature and pressure that high electric power isconsumed. Then, bacterial cells are transferred to a
medium plate in a clean bench. In the same way, an
autoclave was used again for disposing bacteria. In caseof observation under different conditions, several sets ofcontainers are additionally required. Therefore, we camewith an idea to use paper that is easy to handle as acontainer instead of a Petri dish. The study container
would use ethylene oxide gas for sterilization andcombustion for disposal.
Recently, various novel paper-based devices havebeen presented. Paper is ubiquitous, inexpensive andrecyclable; it is easy to store, transport, manipulate anddispose. Attempts have been made to fabricate paperbased medical sensors to analyze clinical body fluids
and metabolic substances, such as blood, urea
(Martinez et al., 2008; Abe et al., 2008) and glucose

Corresponding Author: Tithimanan Srimongkon, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan
Science Publications

81

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

examine the pattern dispensed on a substrate. Processing,such as hydrophilic/hydrophobic patterning, with an inkjet printer is a time-saving, low-cost and controllable
technique that is an alternative to manual operation.
Through this technology, researchers can avoid
harmful or pathogenic bacteria from directly touchingthem as a concomitant effect.
In this study, we present a new method to create awell-ordered and reproducible system for dispensing
bacteria on a microscopic scale by using advantages of
paper and ink-jet technology. We used paper as a media
platform for bacterial culture and ink-jet printing
technology to dispense small amounts and for
manageable patterning of bacterial suspension. Bacterial
growth on a nutrient medium contained in a Petri dish issometimes evaluated viaually by positive/negative
judgement followed by a statistical analysis(Overdevest et al., 2010), counting the number ofcolonies (Tortoli et al., 2002), color of colonies(Glupczynski et al., 2007) and more semi-quantitatively,scores based on the number of quadrants in which growth
was observed (Huang et al., 2010). The system we are
creating aims to quantify bacterial growth with amicroscope and image analysis as the future work, which
will be useful to routine laboratories using agar plates.

(Ornatska et al., 2001). Paper sensor aiming at thedetection of mesothelin as a cancer biomarkers waspresented (Lei et al., 2013). A multiplex detection
system of Escherichia coli (referred-to E. coli hereafter)
was developed using an enzyme-based lab-on-paper test
strip (Martinez et al., 2008). Furthermore, fabrication
methods and design techniques of paper-based
microfluidic channels using wax for portable bioassay
were discussed (Lu et al., 2009). Paper can be divided
into multi-channels for liquid-transporting sensor tests byusing
apatterningtechniquetoseparatehydrophobic/hydrophilic areas. We considered that wewould apply this useful technique to bacterial cultures
under various conditions. Hydrophilic areas were tried to
be built on a small sheet of hydrophobized filter paper inorder to accommodate dispensed agar and then bacteria.
In addition, paper is a combustible substrate, therefore
grown bacteria could be simply disposed with the paper
substrate after combustion. Thus, no autoclave isrequired in the disposal process.
There is another factor for inconvenience caused bymanual operation. Bacterial cells are commonlytransferred by manually in culture tests. The
transferred volume cannot be constant or wellcontrolled. To solve the instability problem due tomanual procedure, we proposed an ink-jet dispensing
method. However, (John et al., 1983) observed, under
microscope, that rod-shaped cells like E. coli grew sosimply that freshly-divided cells elongated with little orno increase in girth. Eventually a transverse wall waslaid down near the center of the cell, When the cellreached approximately twice its original size, it separated
into two cells nearly equal in size. Therefore, E. coli
cells transfer was tested as a model of bacterial cells byusing ink-jet technology. Because ink-jet technology is
no longer only an office printing technology (Abe et al.,2008); it has gradually become a versatile tool in various
fields for accurately dispensing very small quantities of
fluids on substrates, such as human organs or cells(Mironov et al., 2009; Guillemot et al., 2010; Xu et al.,2005; Cui and Boland, 2009). Futhermore, numerical andexperimental investigation of a fluid flow through porous
media is important for a wide range of applications
varying from environmental analyses to inkjet printing
technology (Mohd Irwan et al., 2010). Therefore, ink-jet
technology is an essential technique for transferring
bacterial cells in a small, stable and controlled volume.
In addition, ink-jet ejection is easily controlled using
software that can print any designed patterns and acharge-coupled device camera, which is usuallyequipped with high-performance ink-jet printers to
Science Publications

2. MATERIALS AND METHODS
2.1. Preparation of the Hydrophilic/Hydrophobic
Pattern
We selected paper as a potential new substrate tofacilitate efficient observation of bacterial growth undervarious conditions. Previous research (Abe et al., 2008)
presented a method to modify paper to form hydrophobic
and hydrophilic areas in the same substrate by using Poly
Styrene (PS) and toluene. In the present study, we used
PS to serve as a hydrophobic barrier to block spreadingof the aqueous medium that was printed on hydrophilic
areas. In addition, filter paper was chosen as a substrate
to settle the medium because of its high absorbency.
First, the filter paper was soaked in a 3.0 wt% toluene
solution of PS for 1 h and then allowed to dry at room
temperature for 15 min, to allow the entire filter paper tobecome hydrophobic. Squares (10×10 mm) were etchedto make hydrophilic areas by printing toluene with
Solvent Blue 35 at 0.015 wt% (blue toluene), forvisualization, with 3 head nozzles and overprinting thearea twice for the best organized patterns with distinctive
borders. Finally, hydrophilicity was tested by dropping 2µL of water on the etched areas and the degree ofpenetration was observed and compared.
82

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

2.2. Modification of Agar

2.4. Ejection of Escherichia coli on Medium

A common culture medium for bacteria is an agar
medium that contains necessary nutrients. We
attempted to print a nutrient agar medium by using the
ink-jet printer for dispensing small quantities of themedium on hydrophilic areas of the filter paper.
However, the medium quickly solidified at room
temperature, which is not conducive to printing.
Therefore, we modified the molecular structure of agarby hydrolysis. Agar contains agarose as a major
component, which is a linear polymer made up of therepeating unit of agarobiose-a disaccharide made up of
D-galactose and 3, 6-anhydro-L-galactopyranose.
Glycosidic bonds between each agarobiose are easilyhydrolyzed by acids, such as Sulfuric Acid (H2SO4),
that shorten polysaccharide chains. The optimumhydrolysis of agar was sought to generate agar
polymers conducive to printing. First, 0.5 g of agar
powder was dissolved in 20 mL of 0.05, 0.1 and 0.02%
H2SO4 aqueous solutions and then the solutions were
heated at 100°C for 10, 15, 20, 25, 30, 40, 45 and 50
min. To stop the acid hydrolysis, the agar solutions
were cooled on ice water for 5 min. Sodium Hydroxide(NaOH) was added to neutralize the solutions forbacterial culture. Sodium Sulfate (Na2SO4) produced
during neutralization was removed by electrodialysis(Microacilyzer S1 with a membrane cartridge AC11010, ASTOM, Japan). Viscosity of each hydrolyzed agar
solution was measured by using a Cannon-Fenske
Routine Viscometer. Finally, the hydrolyzed agar was
mixed with yeast extract, Bacto tryptone and Sodium
Chloride (NaCl). The agar mixture was loaded into an
ink cartridge wrapped with a film-type heater and wasprinted on rectangular hydrophilic areas (5×5 mm). The
drying speed of printed hydrolyzed agar was recorded
to evaluate water retention.

E. coli cells were printed with a testing ink-jet printer(Dimatix DMP-2831, Fujifilm, Japan), with apiezoelectric actuator, on a standard culture medium toconfirm bacterial cell applicability and viability. First, a
5-mm-thick square sheet of culture medium was
prepared and placed on the stage of the ink-jet printer.
Next, E. coli cells were loaded into a cartridge andprinted following patterns designed using a specialized
application. The pattern was considered to be a rectangleof 5×10 dots arranged on a sheet at intervals of 5 mm.
The printed E. coli cells were incubated at 37°C for 24 hand the resultant colonies were examined.

2.5. Preparation of Emulsified Particulates
Polystyrene acrylate hollow latex particles with adiameter of about 1 µm (latex particles; PAT8125;
ZEON Corporation, Japan) were selected as abacterial cell model to ensure the ability to print a
constant number of particles. The stock latex
emulsion, consisting of 26.5% solids, was diluted to
0.001% and then mixed with a small amount ofglycerol to obtain an appropriate viscosity for ink-jet
printing. Latex particles were subsequently printed ona sheet of photo-grade ink-jet printing paper (Super
photo grade; Kassai, Fujifilm, Japan) using anapplication to manage printing conditions such as
printing frequency and number of droplets in 1 dot on
the paper. A square pattern of 11×11 dots was printed
only once or overprinted 3 or 5 times on the paper andphotographs of the printed dots were taken by ascanning electron microscope (S-4200, Hitachi, Japan)
to count the number of latex particles.

2.6. Confocal Laser Scanning Microscopy

2.3. Bacterial Culture on Paper

Automatic and selective monitoring of bacterial
growth by a Confocal Laser Scanning Microscope(CLSM 700; AxioObserver, Carl Zeiss Microscopy) was
performed to develop this bioassay system. A dried pieceof filter paper with a bacterial colony on the agar
medium was subjected to fluorescence microscopic
observation with an excitation wavelength (ex) of 555
nm (green) and an emission wavelength (em) ranging
from 500-630 nm (blue to red) for the agar and ex of 639
nm (red) and em>640 nm (red) for the hydrophilic area.
CLSM was used to obtain a regular light transmission
image showing the fiber network and the composite
image was created from the 3 images.

Bacterial growth was examined by transferring asuspension of bacteria and comparing the growth on anormal agar plate and on hydrolyzed agar medium on
paper. First, bacteria floating in the air were taken
randomly from stationary phase and isolated for pureculture by the streak technique. At least 3 repetitions of
this technique were performed to confirm the purity ofthe bacteria and classify the morphology of the bacterialcolonies. Serial dilution was applied to reduce theconcentration of the bacterial suspension. Finally, abacterial suspension of 2.7×107 CFU/mL was transferred
onto hydrolyzed agar to observe the bacterial growth.
Science Publications

83

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

3. RESULTS AND DISCUSSION

observed for all printed dots. These results also suggestthat this ink-jet printing method is capable of dispensing
fairly regular arrangements of E. coli cells, althoughsome satellite colonies are visible next to the primarycolonies in the photograph (Fig. 3). This deviation in theregular arrangement was due to droplet split, which
occurred immediately as the droplets were released fromthe nozzle; deviation was solved by increasing viscosityand decrease surface tension by adding a small amountof glycerol to the bacterial solution.

3.1. Hydrophilic/Hydrophobic Patterning
Afterhydrophobic/hydrophilicpatternwaschecked by dropping water, the results show that 40
min printing method including 3 head nozzles andoverprinting twice was chosen for optimumhydrophobic/hydrophilic patterning.

3.2. Optimized Modification of Agar for Inkjet Printing

3.4. Stability in Number of Ejected Particles

As shown in Fig. 1, hydrolyzed agars were classified
into 3 groups, depending on the state of the material:
Gel, paste and aqueous states. Agar powder dissolved in
0.05% H2SO4 aqueous solution and heated for 30 min
attained an optimum hydrolysis level, which was aviscosity conducive to ink-jet printing. Additionally, a
film-type wrapping heater was used to change thehydrolyzed agar from the paste to aqueous state. As aresult, heating guaranteed smooth ejection of thehydrolyzed agar medium by maintaining the aqueous
state and avoided clogging the nozzle heads. However,
50 µL aliquots of the hydrolyzed agar medium dried soquickly that the moisture content of the agar wasmaintained only for 16 h. Because we developed aculture system for bioassays that will be processed
within a few hours, the drying time of 16 h is sufficient
to maintain a suitably moist environment.
To confirm the survival of bacterial cells on paper,
the isolate originally from air-floating bacteria weretransferred on the hydrolyzed agar medium manally.
Colonies were not observed clearly on the hydrolyzed
agar medium on paper as shown in Fig. 2.

After we confirmed the ability to dispense viablebacterial cells by ink-jet, we verified that the ink-jet
printer could dispense regular arrangements of non-ink
liquid dots and eject consistent numbers per droplet oflatex particles, a model for bacterial cells. The ink-jet
printer is designed to eject droplets with volumes of 1-10
pL. Although, the average diameter of the latex particles
is approximately 1 µm and latex particles were expected
to print smoothly, we sought to avoid clogging of thehead nozzles or aggregation between particles. A small
amount of glycerol was added to latex particles toincrease the viscosity and decrease the surface tension(Segur, 1953). Figure 4 shows printed dots of latex
particles at certain intervals on photo-grade ink-jet paper,
which resulted in the formation of well-dispersed
particles for easy counting and a potentially desirable
inoculation level of bacterial cells. For themeasurements, the number of primary particles with adiameter of approximately 1 µm was counted andcoexisting particles much smaller in size were excluded
from counting. We randomly selected 10 out of 121 dots
for counting. The stability of number of latex particles
per droplet was tested by using the Analysis Of Variance(ANOVA) method. The statistical data are listed inTable 1. When F-values critical at α = 0.95 wascompared with the F-values calculated from ANOVA,
the result showed that number of latex particles per
droplet was not siginificantly different among different
overprinting times. In addition, the average number oflatex particles per droplet was consistently
approximately 15. This finding suggests that liquids
containing bacterial cells can be dispensed evenly andregularly onto a culture medium. In addition, the circularshape of 1 dot evenly formed from 5 droplets (Fig. 4),
indicating that overprinting several times can be applied
to adjust the number of cells per dot.

3.3. Ejection of E. coli on Agar Medium
After a suspension of E. coli cells was printed, thenormal agar medium sheet was immediately observed
microscopically. Individual E. coli cells were notvisible; however, mature E. coli colonies appeared as
white spots after culture for approximately 30 h andwere photographed (Fig. 3).
These results suggest that E. coli cells that wereejected through narrow nozzles were still viable,
although they were subject to high shear stress in thenozzles. Previous reports discuss problems of low
survival rates in such kinds of ink-jet bio-dispensing
systems (Harrigan, 1998; Hossain et al., 2012).
However, the survival rate obtained using this system
was satisfactory, considering that bacterial growth was
Science Publications

84

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

Fig. 1. Viscosity of agar hydrolyzed at three different concentrations of H2SO4

Fig. 3. Arrangement of cultured E. coli colonies after
printing on a sheet of agar medium

Fig. 2. Colonies of bacteria growing on modified agar medium

3.5. Fluorescence Monitoring of E. coli Growthand Agar Location

The combined image (Fig. 5) clearly indicates that theagar medium spread and settled within the hydrophilicarea and the dark area. Unfortunately, the bacterialcolonies could not be distinguished by CLSM,
because both of bacteria and medium contains protein
so the auto-fluorescence was same. However, CLSM
is a promising technique to distinguish mixed
components in the paper. This technique would beincorporated in the bioassay system as an automatic
evaluation of bacterial growth.

Light transmission and fluorescence optical
micrographs were captured by CLSM. Figure 5 shows
a combined image of an overall paper structure
consisting of fibers of the filter paper with a dark area,
agar medium with a yellow and toluene blue adsorbed
by fibers in red, respectively. It should be noted thatthe false-colors appearing in the images are not thesame as those visible to the human eye.
Science Publications

85

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

Fig. 4. Latex particles in 1 dot observed by scanning electron microscope; (a) 1 time overprinting, (b) 3 times overprinting and (c) 5times overprinting

Fig. 5. Combined image of agar medium on hydrophilic areas of filter paper as observed by CLSM
Table 1. ANOVA result of latex particles per droplet at α = 0.95
Source of variation df
SS
MSF
Treatments
9
1.73
0.19
0.0256Error
20
149.75
7.49
Total variation
29
151.48evaluate
bacterial
growthrates.
The
hydrophilic/hydrophobic patterning provided a paper
substrate suitable for accommodating an ink-jet printed
culture medium. Agar was hydrolyzed to adjust theviscosity, making it conducive to printing on paper andbacterial growth. CLSM was performed to detect thelocations of the agar, blue dye and fibers. The discrete
locations on the paper substrate were discernible in the
fluorescence images. In a separate experiment, E. coli
cells were printed on a sheet of agar medium and thegrowth of colonies with high survival rates were

4. CONCLUSION
The ink-jet printer-assisted bioassay system we are
currently developing in combination with a paper
substrate is a promising technique to more efficiently
Science Publications

86

AJBB

Tithimanan Srimongkon et al. / American Journal of Biochemistry and Biotechnology 10 (1): 81-87, 2014

Kaláb, M., 2008. Microscopy and Higene. Infocus.
Lei, Z., L. Dan, W. Su, Y. Liu and Y. Chen et al., 2013.
Detectionofcancerbiomarkers
withnanotechnology. Am. J. Biochem. Biotechnol., 9:71-89. DOI: 10.3844/ajbbsp.2013.71.89
Lu, Y., W. Shi, L. Jiang, J. Qin and B. Lin, 2009. Rapid
prototyping of paper-based microfluidics with wax
for low-cost, portable bioassay. Electrophoresis, 30:
1497-1500. DOI: 10.1002/elps.200800563
Martinez, A.W., S.T. Phillips, E. Carrilho, S.W. Thomasand H. Sindi et al., 2008. Simple telemedicine fordeveloping regions: Camera phones and paper-based
microfluidic devices for real-time, off-site diagnosis.
Analytical
Chem.,
80:
3699-3707.
DOI: 10.1021/ac800112r
Mironov, V., R.P. Visconti, V. Kasyanov, G. Forgacs
and C.J. Drake et al., 2009. Organ printing: Tissue
spheroids as building blocks. Biomaterials, 30:
2164-2174.
DOI:
10.1016/j.biomaterials.2008.12.084
Mohd Irwan, M.A., A.M. Fudhail, C.S. Nor Azwadi and
G. Masoud, 2010. Numerical investigation ofincompressible fluid flow through porous media in alid-driven square cavity. Am. J. Applied Sci., 7:1341-1344. DOI: 10.3844/ajassp.2010.1341.1344
Ornatska, M., E. Sharpe, D. Andreescu and S.
Andreescu, 2001. Paper bioassay based on ceria
nanoparticles as colorimetric probes. Analytical
Chem., 83: 4273-4280. DOI: 10.1021/ac200697y
Overdevest, I.T.M.A., I. Willemsen, S. Elberts, C.
Verhulst and J.A.J.W. Kluytmans, 2010. Laboratory
detection of extended-spectrum-beta-lactamaseproducing enterobacteriaceae: Evaluation of two
screening agar plates and two confirmation
techniques. J. Clin. Microbiol., 49: 519-522. DOI:
10.1128/JCM.01953-10
Segur, J.B., 1953. Physical Properties of Glycerol and its
Solutions. In: Glycerol, Miner, C.S. and N.N.
Dalton, (Eds.)., Reinhold, New York, pp: 238-334.
Tortoli, E., M. Benedetti, A. Fontanelli and M.T.
Simonetti, 2002. Evaluation of automated BACTEC
MGIT 960 system for testing susceptibility of
mycobacterium tuberculosis to four major
antituberculous drugs: Comparison with theradiometric bactec 460tb method and the agar plate
method of proportion. J. Clin. Microbiol., 40: 607610. DOI: 10.1128/JCM.40.2.607-610.2002
Xu, T., J. Jin, C. Gregory, J.J. Hickman and T. Boland,
2005. Inkjet printing of viable mammalian cells.
Biomaterials,
26:
93-99.
DOI:
10.1016/j.biomaterials.2004.04.011

confirmed. We confirmed the regularity of the number ofejected cells in 1 droplet with latex particles as a cellsuspension model. Finally, we developed ink-jet assisted
paper-based substrate as a part of a bioassay system.

5. ACKNOWLEDGEMENT
This study was supported by JSPS Grant-in-Aid for
Scientific Research (B) No. 22380092 and Challenging
Exploratory Research No. 23658138. We would like tothank Ms. Yoko Saito of ZEON Corporation, Japan forproviding the latex sample and Mr. Nobuhiro Hayami ofCarl Zeiss Microscopy Co., Ltd., Japan for acquiring
fluorescence images.

6. REFERENCES
Abe, K., Suzuki, K. and D. Citterio, 2008. Inkjet-printed
microfluidic multianalyte chemical sensing paper.
Analytical
Chem.,
80:6928-6934.
DOI:
10.1021/ac800604v
Cui, X. and T. Boland, 2009. Human microvasculature
fabrication using thermal inkjet printing technology.
Biomaterials, 30: 6221-6227. PMID: 19695697
Glupczynski, Y., C. Berhin, C. Bauraing and P. Bogaerts,
2007. Evaluation of a new selective chromogenic agar
medium for detection of extended-spectrum βlactamase-producing enterobacteriaceae. J. Clin.
Microbiol., 45: 501-505. DOI: 10.1128/JCM.02221-06
Guillemot, F., A. Souquet, S. Catros, B. Guillotin and J.
Lopez et al., 2010. High-throughput laser printing ofcells and biomaterials for tissue engineering. Acta
Biomaterialia,
6:2494-2500.
DOI:
10.1016/j.actbio.2009.09.029
Harrigan, W.F., 1998. Laboratory Methods in Food
Microbiology. 1st Edn., Gulf Professional Publishing,London, ISBN-10: 0123260434, pp: 532.
Hossain, S.M., C. Ozimok, C. Sicard, S.D. Aguirre and
M.M. Ali et al., 2012. Multiplexed paper test strip
for quantitative bacterial detection. Anals
Bioanalytical Chem., 403: 1567-1576. DOI:
10.1007/s00216-012-5975-x
Huang, T.D., P. Bogaerts, C. Berhin, A. Guisset and Y.
Glupczynski, 2010. Evaluation of brilliance ESBL
agar, a novel chromogenic medium for detection ofextended-spectrum-beta-lactamase-producing
enterobacteriaceae. J. Clin. Microbiol., 48: 2091-2096.
DOI: 10.1128/JCM.02342-09
John, L. I., Ole, M. and C. N. Frederick, 1983. Growth of
the Bacterial Cell. 1st Edn., Sinauer Associates, Inc.,
Massachusetts. ISBN-10: 0-87893-352-2, pp: 227.
Science Publications

87

AJBB