ПРИБОРЫ И ТЕХНИКА ЭКСПЕРИМЕНТА, 2013, № 4, с. 112-116
ФИЗИЧЕСКИЕ ПРИБОРЫ ДЛЯ ЭКОЛОГИИ, МЕДИЦИНЫ, БИОЛОГИИ
A SIMPLE LASER-BASED DEVICE FOR SIMULTANEOUS MICROBIAL CULTURE AND ABSORBANCE MEASUREMENT
© 2013 г. X. C. Abrevaya", E. Cortón4, O. Areso", P. J. D. Mauas"
aInstituto de Astronomía y Física del Espacio (IAFE), UBA-CONICET, Ciudad Universitaria, Ciudad Autónoma de Buenos Aires (1428), Argentina E-mail: abrevaya@iafe.uba.ar bLaboratorio de Biosensores y Bioanalisis, Departamento de Química Biologica, Facultad de Ciencias Exactas y Naturales, UBA and IQUIBICEN-CONICET, Ciudad Universitaria, Ciudad Autonoma de Buenos Aires (1428), Argentina Received April 25, 2012; in final form June 28, 2012
In this work we present a device specifically designed to study microbial growth with several applications related to environmental microbiology and other areas of research as astrobiology. The Automated Measuring and Cultivation device (AMC-d) enables semi-continuous absorbance measurements directly during cultivation. It can measure simultaneously up to 16 samples. Growth curves using low and fast growing microorganism were plotted, including: Escherichia coli, and Haloferax volcanii, a halophilic archaeon.
DOI: 10.7868/S0032816213030178
INTRODUCTION
Microbial cell growth quantification is essential in different areas of research involving microbiological work. To implement it, several methodologies have been developed, which differ mainly in the physical or chemical properties employed to take the determinations [1].
Traditionally, growth curves are obtained manually, sampling culture aliquots, which are measured using a spectrophotometer or a similar optical instrument, thus obtaining a few experimental points that are used to plot a continuous curve. This could lead to errors in the determination of the growth parameters [2, 3]. In particular for microorganisms that grow slowly (generation time longer than 2 hours) this method could be very demanding, and to perform studies in this kind of microorganisms results unfeasible. In particular, it is not easy to detect this kind of microorganisms or their communities in environmental samples using conventional turbidimetry or plating methods.
A diversity of instruments were built to automatically measure the growth of different kind of microorganisms, not only on Earth but also in space [4—10]. Most are simple, inexpensive and automatic devices designed according to specific needs or applications [11-13].
Similar automated devices are commercially available, like the Turbidity Transmitter Trb8300® (Metller Toledo GmbH, Switzerland) which only allows instantaneous measurements. On the other hand, Bio-screen C® (Oy Growth Curves Ab Ltd, Finland) allows automatically obtaining growth curves, but it is an expensive instrument. Later, Brewster [4] developed a
device with very similar characteristics to Bioscreen C, but with lower cost, including the use of microwell plate technology.
In this paper we present an inexpensive alternative to Bioscreen C, the Automated Measuring and Cul-turing device (AMC-d), which enables continuous absorbance measurements by photometry on-line at real time, directly in the culture (in situ), running several samples simultaneously and consistently, which helps to perform duplicates or to test different conditions in parallel (multi-factorial experiments). The information is acquired, processed, and exported to a PC for the generation of microbiological growth curves, plotting turbidity vs. time, and the results are available remotely via the internet.
We demonstrate that the device works in a wide linear range where Lambert-Beer's law is applicable and that the measurements obtained using it has high re-producibility. As an example, we present growth curves for microorganisms from two different domains: the commonly used bacterium Escherichia coli (generation time around 20 min in optimum conditions) and Haloferax volcanii, a halophilic archaeon which is an extremophilic microorganism that grows at high salt concentrations (generation time around 3 hours in optimum conditions).
DESIGN AND CALIBRATION
The device basically consists of a structure built in acrylic glass which supports a rotating wheel made in aluminum with 16 slots where the samples can be placed using standard polymethyl methacrylate spectrophotome-ter cuvettes (1 cm optical path, total volume of 5 mL). A
Top view
g
Motor
Ps r>
/
' RW
V
VI
Slot
u
Pr
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Beam splitter
Laser view
RW
Laser
H I
Motor
Screw
Ring
Optic path
Slots
Fig. 1. Schematic diagram of the device: (Ps) sample photodiode, (RW) rotating well, (Pr) reference photodiode.
ring made of Delrin® is fixed on top of the rotating well using 4 stainless steel screws (Fig. 1). The wheel is moved (typically at 4.3 rpm) by a 12 VCc motor (model MR6-4.3, IGNIS, Buenos Aires, Argentina). This rotating wheel allows alternatively measuring each ofthe cuvettes, and mixing the cultures, providing aeration if an air chamber is left inside the cuvettes.
Attached to the structure, there are one 5 mW red laser diode module which emits at 655nm (M655-5, US Lasers Inc., CA, USA) and two silicon pin photodiodes (BPW34, Vishay Electronic GMBH, Germany) with a radiant sensitive area of 7.5 mm2, one located behind the wheel (Ps, sample photodiode) and the other at 90° from the laser (Pr, reference photodiode). The laser beam is split by a semi-transparent mirrored glass: one beam is directed to Ps and the other to Pr. Ps measures the light from the laser after passing through the cuvette with the sample to be studied (or the corresponding blank/reference cuvette), and Pr measures the laser light passing through air, and is used to control the laser output (avoiding possible absorbance errors, originated in laser radiant power variations). The absorbance A is calculated as the logarithmic relationship between the radiant power passing through a cuvette containing a blank or reference solution (P0) and the one containing the sample (P), by using the light detected by Ps photodiode, as shown in Equation
A = log10(PoP-1).
(1)
In some experiments, the concentration of a given molecule can be calculated by using the well know Lambert-Beer's law, which establish that between a given concentration range, the absorbance is proportional to the molar absorbance of the analyte (s), the optical path (b), and the concentration (c):
A = sbc. (2)
Microcontroller and Programmation
External to the instrument, there is a command box containing an 8-bit AVR microcontroller, with 10-bit Analog to Digital Converter (Atmel, USA), an LCD
Display (16 character x2 lines converter ATmega16, Atmel, USA) and a numerical keyboard for operation. The command box is connected to a computer through an RS-232 port for data storage and to configure several parameters related to the measuring process and the shaking of the samples.
These parameters are: the frequency of the measurements (in the 1—250 min range), the interval of time between two measurements, the timeout (which defines how much time is employed to stabilize the optical reading, in the 1—250 s range), the number of cuvettes to be measured (up to 15 samples plus a blank cuvette simultaneously) and the number of measurements (i.e. the length of the experiment, up to 3500 measurements). Automatic blank subtraction is possible, using a value registered in one cuvette, to be subtracted from the other values. Between measurements, the AMC-d works shaking the samples turning N times with a time interval t, where both N and t are parameters that can be adjusted.
For each measurement, the date is registered (in format day, hour and minute). There is also a sensor which registers the temperature, and it is possible to set an alarm, which will sound if the temperature in the incubator goes outside a pre-specified range. The data is acquired and processed by the device and sent to the computer where it is stored in tabular form in a text file. At the same time, a web interface allows to plot the results or to see them in tabular form both locally or via the internet, by mean of Apache HTTP Server 2.2.
Linearity
The linearity predicted by Lambert-Beer's law was checked with a calibration curve (absorbance vs. concentration) using a standard solution of CuSO4. We prepared standard solutions of known concentrations (0.004 to 0.8 M), which were transferred to polymethyl methacrylate spectrophotometer cuvettes with lids that were placed into the AMC-d. The absorbance of each solution was measured and recorded. The same dilutions were also measured in a spectropho-
Fig. 2. E. coli growth curve obtained using the AMC-d. The average of fourteen culture measurements is shown (continuous line). There is a mistake here, the residual values are not more in the plot, please, delete this sentence.
tometer (UVIKON 710, Kontron, USA) at a wavelength of 655 nm. The calibration curves obtained were plotted and adjusted to a linear regression.
Application in Microbiology Studies
We assayed the ability of the AMC-d to obtain growth curves using two different microorganisms, with very different generation times: Escherichia coli (K-12 strain), a facultative anaerobic bacterium, and Haloferax volcanii (DS70 strain), an aerobic halophilic archaeon. H. volcanii DS70 strain was kindly provided by Dr. R.E. de Castro, Universidad Nacional de Mar del Plata, Argentina and E. coli K-12 strain was obtained from ATCC.
E. coli was grown aerobically at 37°C until an OD (600 nm) =1.0 was reached. Tryptic Soy Broth (DIF-CO) was used as culture media (30 g • L-1 in distilled water). H. volcanii was grown aerobically at 30°C, to an OD (600 nm) = 0.5. Growth medium Hv-YPC contains (g • L_1), yeast extract (5.0), peptone (1.0), casaminoacids (1.0), NaCl (144.0), MgSO4 ■ 7H2O (21.0), MgCl ■ 6H2O (18.0), KCl (4.2), CaCl2 (0.35), and Tris-HCl (1.9); the pH was adjusted to 6.8.
The cultures for E. coli and H. volca
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