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2 - Bacterial reproduction happens with a process called binary fission, in which a bacterium cell is divided in two different daughter cells. This process can be divided in the following steps. First, there is the duplication of the bacterial chromosome, then the elongation of the cell and the formation of a transverse septum centrally located. Finally, the subdivision of a copy of the chromosome and cytoplasm in the two daughter cells.The growth of a bacterial population can be divided in the following 4 phases.During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual cells are maturing and are not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.During the exponential phase, bacteria duplication happens. In this phase each bacterial cell divides in two different daughter cells and the time interval between two duplication processes is known as mean generation time. During the exponential phase, bacteria consume the nutrients present in the growth medium and produce waste compounds due to their metabolism. When there is the lack of an essential nutrient or the formation of an inhibitory product due to bacterial metabolism, the bacterial growth rate slows down and the Stationary phase begins.During the death phase, bacteria die. This could be caused by lack of nutrients, environmental temperature above or below the tolerance band for the species, or other injurious conditions.3 - The standard reference technique to measure the bacterial concentration is the Plate Count method.In the standard plate count technique, Petri dishes, shown in the figure, are filled with a nutrient medium to allow the growth of bacteria. Different nutrients can be used, depending on the particular bacterial species that must be detected. For the total bacterial concentration, the Lauria Bertani agar is used. If the coliforms concentration must be tested, then a suitable medium is Mc Conkey agar. In the case of staffilococci, a suitable medium is Mannitol salt agar.Serial dilutions of the sample under test are made using a saline buffer solution and 100 micro liters of the diluted samples are inoculated on the Petri dishes, that are then incubated at a temperature favoring bacterial growth. After incubation, the number of colonies growth on the Petri dish are counted and the bacterial concentration is calculated.The Plate Count method is the reference technique for bacterial concentration measurement and is reliable and accurate. However, long incubation times, in the range of 24 to 72 hours, are needed. Moreover, it is a laboratory method that must be carried out by trained personnel and it is not suitable to be implemented in automatic form.In the following, an alternative method for bacterial concentration measurements is presented. The method is based on the measure of the electrical parameters of the sample under test and is suitable to be implemented in automatic form.4 - When a liquid sample is placed in direct contact with a couple of electrodes, the system can be described with the electrical model in the figure, where R-m and C-m are the resistive and capacitive components of the bulk medium, while R-I and C-I are the components of the electrode electrolyte interface. The impedance technique is based on the principle that bacterial metabolism transforms uncharged or weakly charged compounds in highly charged compounds, thus changing the sample electrical parameters.When the frequency of the test signal applied to the electrodes is relatively low, the bulk capacitive component is negligible, and then the system can be modeled as the series of a resistance and a capacitance.5 -The resistive and capacitive components of the impedance, R-s and C-s, can be measured using the circuit presented in the figure. A sinusoidal test signal is applied to the electrodes and the current through the electrodes is converted to a voltage by means of the operational amplifier feedback resistor. The electrical parameters can be estimated by the analysis of both the input and output sine wave signals.6 - The working principle of the impedance technique for bacterial concentration measure is here presented. The sample under test is placed in a sensor vial that features a couple of electrodes to measure the electrical parameters. Then, the sensor is incubated at a temperature favoring bacterial growth and the electrical parameters are measured at time intervals of 5 minutes.In the figure, the sample bacterial concentration, plotted in blue, and the resistive parameter, plotted in red, are shown versus time. As can be seen, as long as the bacterial concentration is lower than a critical threshold in the order of 10 millions colony forming units for milliliter, the electrical parameters remain essentially constant at their baseline values. However, when this critical threshold is exceeded, the electrical parameters deviate from their baseline values.The time needed for the electrical parameter to deviate from its baseline value is thus the time needed for the bacterial population to reach the critical threshold of 10 millions colony forming units for milliliter. This time is defined as Detect Time.7 - The measured Detect Time is a linear function of the logarithm of the sample bacterial concentration. Highly contaminated samples are characterized by lower Detect Times than samples with low bacterial concentration. This is shown in the figure, where the resistive component of the impedance is plotted versus time for three different samples. Sample 1 features an higher bacterial concentration than sample 2, and sample 3 an even lower bacterial concentration than sample 2. 8 - The impedance technique has many advantages compared to the standard Plate Count method. First of all, its response time is shorter. The impedance technique can estimate the bacterial concentration with a time response in the range from 2 to 12 hours, depending on the sample bacterial concentration, while the Plate Count technique needs from 24 to 72 hours. Moreover, the impedance technique can be easily implemented in automatic form as a portable system or embedded as part of an industrial machine, while the Plate Count is a laboratory method that needs to be carried out by trained personnel.The impedance technique, however, presents also some critical points. To estimate the sample bacterial concentration from the measured Detect Time, a calibration equation is needed. Thus, a calibration with samples of known bacterial concentration must be carried out before any measurement. The measured electrical parameters are also strongly affected by temperature, thus the measurement must be performed in a temperature controlled environment where maximum temperature variations of 0.25 Celsius degree are allowed.9 - Different commercial systems exist that measure the bacterial concentration using the impedance technique, such as the Bactometer by Biomerieux and the Bac Trac by Sy-Lab.These are benchtop systems that are intended to be used for rapid bacterial concentration measure in a laboratory environment.10 - Our research group has built a portable biosensor system for bacterial concentration measure using the impedance technique. The system is intended for in situ measurements in industrial environments.The biosensor system is composed of the following parts. An electronic board, hereafter defined as Impedance Measure Board, is the main board of the system and is used to measure the sample electrical parameters, calculate the detect time by means of an ad hoc designed algorithm and estimated the sample bacterial concentration. The second electronic board, hereafter defined as the Thermoregulation board, is used to control the incubation chamber heating system to maintain the sample temperature stable at the target value. The incubation chamber hosts the sample under test. It integrates a couple of stainless steel electrodes for the electrical measurements, power resistances to heat the sample by Joule effect and a temperature sensor for thermoregulation. The system features also a serial port for communication with a PC and a Telit module for wireless data transmission. 11 - Here the procedures for the realization of the incubation chamber are presented.First, a copper foil is used as heat diffuser to transmit heat to the sample under test.The heat diffuser is applied with a couple of adhesive power resistances that heat the sample by Joule effect.A cylindrical support is drilled and thenThe stainless steel electrodes, the heat diffuser and the temperature sensor are inserted in it.Finally, all the chamber parts are sealed with thermosetting resin.12 - Here a schematic of the biosensor system is shown. The measure with the proposed system is carried out according to the following steps.At the beginning, the incubation chamber is filled with the sample under test.Then, thermoregulation is enabled and a time delay of 30 minutes is waited to allow the sample temperature to stabilize.Once the sample temperature is stable at the target value, the electrical parameters are measured at time intervals of 5 minutes, until a deviation in the measured baseline is detected.Then, the detect Time is calculated and the bacterial concentration is estimated based on a previously built calibration equation.13 - The system has been successfully tested for different types of liquid and semi liquid media.In the case of ice cream samples the target temperature has been set to 35 Celsius degree. The measures have been carried out on the untreated sample, without any dilution in nutrient media.For each sample tested, a measure of bacterial concentration has been carried out using the Plate Count technique to study how well the measured Detect Time correlates with the reference method.As can be seen from the figure, to achieve good correlation between the biosensor data and the reference technique, sample with significant different composition must be separately calibrated, since the different chemical composition affects the bacteria generation time and thus the measured detect time for a given bacterial concentration.14 - The biosensor system was also used to measure the bacterial concentration in raw milk samples. In this case two different incubation temperatures were tested, 18 Celsius degree and 30 Celsius degree. Also in this case, the samples have been tested without any dilution in nutrient media.As can be seen from the figure, the incubation temperature of 30 Celsius degree results in faster response time than the incubation temperature of 18 Celsius degree.However, the incubation temperature of 18 Celsius degree is characterized by much better correlation with the results of the reference Plate Count technique.This shows how the choice of the incubation temperature is critical in achieving a good correlation with the reference technique and that a trade off between response time and accuracy must be made.15 -The biosensor system was also used to measure the bacterial concentration in water samples. The incubation temperature in this case was set to 37 Celsius degree. In this case, since water have not enough nutrients to allow fast bacterial growth, the samples were diluted in suitable enriching media. The chosen media were Lauria Bertani Broth to test the total bacterial concentration and two different media to test the coliforms concentration, Mc Conkey Broth and Lactose Broth.As can be seen from the figure, Lauria bertani broth and Lactose Broth are characterized by comparable accuracy, while in the case of Mc Conkey Broth the correlation with the reference Plate Count technique was lower. If you want to know more, please read: [1] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2008). Detection of microbial concentration in ice-cream using the impedance technique. Biosensors & Bioelectronics, 23, 1616-1623. https://www.researchgate.net/publication/5498134_Detection_of_microbial_concentration_in_ice-cream_using_the_impedance_technique [2] Grossi M., Pompei A., Lanzoni M., Lazzarini R., Matteuzzi D., Riccò B. (2009). Total bacterial count in soft-frozen dairy products by impedance biosensor system. IEEE Sensors Journal, 9 (10), 1270-1276. https://www.researchgate.net/publication/224586474_Total_Bacterial_Count_in_Soft-Frozen_Dairy_Products_by_Impedance_Biosensor_System [3] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2010). An embedded portable biosensor system for bacterial concentration detection. Biosensors & Bioelectronics, 26, 983-990. https://www.researchgate.net/publication/46219514_An_embedded_portable_biosensor_system_for_bacterial_concentration_detection [4] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2011). A portable biosensor system for bacterial concentration measurements in cow’s raw milk. 4th IEEE International Workshop on Advances in Sensors and Interfaces, 132-136. https://www.researchgate.net/publication/229458394_A_portable_biosensor_system_for_bacterial_concentration_measurements_in_cow's_raw_milk [5] Grossi M., Lazzarini R., Lanzoni M., Pompei A., Matteuzzi D., Riccò B. (2013). A portable sensor with disposable electrodes for water bacterial quality assessment. IEEE Sensors Journal, 13 (5), 1775-1781. https://www.researchgate.net/publication/236018061_A_Portable_Sensor_With_Disposable_Electrodes_for_Water_Bacterial_Quality_Assessment
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A Portable Biosensor System for Bacterial Concentration Measurement in Liquid and Semi-Liquid Media Marco Grossi Department of Electrical Energy and Information Engineering University of Bologna, Italy http://www.researchgate.net/profile/Marco_Grossi
Bacterial reproduction happens by means of binary scission. • duplication of the bacterial chromosome • elongation of the cell • formation of a transverse septum centrally located • subdivision of a copy of the chromosome and cytoplasm in the two daughter cells (A) Lag Phase (B) Exponential Phase (C) Stationary Phase (D) Death Phase
The standard reference technique for bacterial concentration measurement is the Plate Count method. Advantages Drawbacks • It is the reference method • Long response times • Accurate and reliable results • It is a laboratory method
A system composed of a liquid medium in direct contact with a couple of electrodes can be modeled with the following circuit. Rm : medium resistance Cm : medium capacitance Ri : interface resistance Ci : interface capacitance When the frequency of the applied signal is lower than 1MHz Rs = Rm + Ri Cs = Ci
a sine wave voltage Vin(t) is applied to the electrodes • the current through the electrodes Iin(t) is measured • the electrical parameters Rs and Cs are calculated
The liquid sample is placed in a sensor in direct contact with a couple of electrodes. The electrical parameters Rs and Cs are measured at regular time intervals of 5 minutes. Conc. < CTH: the electrical parameters Rs and Cs are almost constant. Conc. > CTH: the electrical parameters Rs and Cs deviate from baseline value. Detect Time (DT): time needed for the bacterial population to reach the critical concentration CTH = 107 cfu/ml
Low contaminated samples feature high values of DT. Higher contaminated samples feature lower values of DT.
The impedance technique has advantages and drawbacks if compared with the reference Plate Count method. Advantages Drawbacks • Shorter response time (2−12 hours vs 24−72 hours) • Need of calibration for the different sample types and different bacterial strains • The technique can be easily automated as part of an embedded system • The electrical parameters are very sensitive to temperature variations
Different commercial systems have been produced that measure the bacterial concentration with the impedance technique. • Bactometer • (Biomerieux, France) • 2ml sensor cell • up to 128 samples • weight 130.2 kg • Bac Trac • (Sy-Lab, Austria) • 20ml sensor cell • up to 64 samples • weight 20 kg
A portable biosensor system that measures bacterial concentration using the impedance technique has been built. • Electronics boards: impedance measures and thermoregulation • Incubation chamber to store the sample • Both serial and wireless communication
The sample is loaded in the sterile incubation chamber • Thermoregulation is enabled • Electrical parameters are measured every 5 minutes • At the end of the assay, DT is calculated and the bacterial concentration is estimated
Bacterial concentration measure in ice-cream samples Incubation temperature 35 °C
Bacterial concentration measure in raw milk samples Two incubation temperatures (a) 18 °C and (b) 30 °C Shorter response time when incubated at 30°C Better correlation when incubated at 18°C A trade off between accuracy and measure time is needed
Bacterial concentration measure in water samples Incubation temperature 37 °C
Bibliography [1] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2008). Detection of microbial concentration in ice-cream using the impedance technique. Biosensors & Bioelectronics, 23, 1616-1623. [2] Grossi M., Pompei A., Lanzoni M., Lazzarini R., Matteuzzi D., Riccò B. (2009). Total bacterial count in soft-frozen dairy products by impedance biosensor system. IEEE Sensors Journal, 9 (10), 1270-1276. [3] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2010). An embedded portable biosensor system for bacterial concentration detection. Biosensors & Bioelectronics, 26, 983-990. [4] Grossi M., Lanzoni M., Pompei A., Lazzarini R., Matteuzzi D., Riccò B. (2011). A portable biosensor system for bacterial concentration measurements in cow’s raw milk. 4th IEEE International Workshop on Advances in Sensors and Interfaces, 132-136. [5] Grossi M., Lazzarini R., Lanzoni M., Pompei A., Matteuzzi D., Riccò B. (2013). A portable sensor with disposable electrodes for water bacterial quality assessment. IEEE Sensors Journal, 13 (5), 1775-1781.
