Apparatus for the analysis of microorganisms growth and procedure for the quantification of microorganisms concentration

• US 20040009572A1
• Filed: 03/10/2003
• Published: 01/15/2004
• Est. Priority Date: 03/11/2002
• Status: Abandoned Application

• Current Assignee: YPF SA (Repsol YPF SA), Universidad Nacional De Cordoba
• Original Assignee: YPF SA (Repsol YPF SA), Universidad Nacional De Cordoba
• Inventors: Madrid, Rossana Elena, Felice, Carmelo Jose
• Application Number:
  US10/383,538
• Publication Number:
  US 20040009572A1
• Time in Patent Office: Days
• US Class Current: 435/243
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00

Abstract

An apparatus and a procedure to detect and quantify the microorganisms concentration in anaerobe ecosystems, for instance, sulfate-reducing bacteria in oil producing systems or industrial or urban waste-waters production systems, as well as in aerobe microbial ecosystems, for instance in the industrial, clinical fields, etc. The apparatus analyzes the growth of microorganisms in cells provided with a culture medium by conducting automatic, continuous, and simultaneous measurements of the impedance components between at least two electrodes immersed in the culture medium and the turbidity measurements of the inoculated medium. The use of two incubators makes it possible to conduct simultaneous analysis at two different temperatures. The determination of the growth Threshold Detection Time (TDT) makes it possible to quantify the microorganisms concentration in an unknown sample.

Claims

1. An apparatus for the analysis of microorganisms growth in cells having a culture medium and at least two metal electrodes measuring the impedance between them at two frequencies, and measuring the turbidity of the medium, wherein the process comprises:

Means to produce sine currents to be applied to the culture cells;

Means to obtain interface resistance (Ri) and interface reactance (Xi), medium resistance (Rm), and absorbance or transmittance growth curves as a function of time based on the analog processing and the periodical digitalization of the voltage between the electrodes and the light detectors, as well as the respective serial resistance which can be stored and/or printed;

Means to reduce the non-microbial shifts produced by the measuring circuits at the interface reactance curves;

Means to maintain the culture cells at a constant temperature comprised within a range going from 10 to 75°

C. at two different temperatures;

Means to produce signals that are proportional to the optical variations;

Means to obtain different wavelengths;

Means to perform measurements in cells provided with three or four electrodes.

2. An apparatus, according to claim 1, wherein the means to generate sine currents include programmable square waves and analog filters, both controlled by the computer, to produce pure low and high frequency sine signals.

3. An apparatus as claimed in claim 1, wherein the means to obtain interface resistance (Ri), interface reactance (Xi), medium resistance (Rm), and absorbance or transmittance growth curves as time functions, comprises:

Means to select and measure voltages in each culture cell, light detectors and the respective serial resistances;

Means to analogically amplify and filter the voltages measured;

Means to digitalize the previously conditioned voltages.

4. An apparatus, according to claim 1, wherein the means used to reduce the non-microbial shifts comprises:

A stage with analog multiplexer made up of reed-relays that select any of the 200 culture cells and remain open between measurements;

Operational amplifiers having a very low bias polarization current (<

0.4 pA) used as input buffers for the signal coming from each measurement cell.

5. An apparatus, according to claim 1, wherein the means used to maintain the culture cells at a constant temperate ranging from 10 to 75°

C., at two different temperatures, include a controller, Peltier cells for cooling, heaters for heating, temperature sensors and fans for temperature homogenization in two air ovens having approximately 100 cells each, which can operate at two different temperatures.

6. An apparatus, according to claim 1, wherein the means used to obtain the Ri, Xi, Rm growth curves and the absorbance or transmittance curves as a function of time includes:

Means to apply constant low and high frequency sine currents to each cell in a sequential manner;

Means to apply constant low frequency sine currents to the light detectors of each cell in a sequential manner;

Means to process the voltage and current values for each culture cell, and to obtain the total resistance and the total reactance at low frequency as well as the total resistance at high frequency for each of them;

Means to obtain the interface and medium values based on the equations;

Ri=(Rib−

Ria)/2;

Xi=Xib/2 and Rm=Ria;

Means to process the voltage values from the light detectors to obtain absorbance or transmittance curves;

7. An apparatus, according to claim 1, wherein the additional means used to generate signals that are proportional to the optical variations include light emitting sources and detectors transducing optical signals into electrical ones which are then analogically processed by the apparatus.

8. An apparatus, according to claim 1, wherein the additional means used to obtain different wavelengths include cell supports with different wavelength emitters, where the use of a single white light source having a wavelength selector and fiber optics to transmit the light beam to each cell has been foreseen.

9. An apparatus, according to claim 1, wherein the additional means used to perform measurements in the cells provided with three or four electrodes include outputs for the connection to additional selection boards (usually not included) which allow making measurements using three or four electrodes.

10. A procedure to selectively quantify the concentration of microorganisms using the apparatus according to any one of claims 1 to 9, wherein the procedure comprises the following stages:

Preparing the suitable culture medium;

Inoculating microorganisms in the culture medium;

Incubating such inoculated culture media;

Determining the Threshold Detection Time (TDT) in any of the curves measured by the apparatus, which can be then stored and/or printed;

Quantifying the concentration of microorganisms in samples of an industrial origin.

11. A procedure, according to claim 10, wherein the microorganisms are sulfate-reducing bacteria.

12. A procedure, according to any one of claims 10 or 11, wherein the preparation of the suitable culture medium comprises the following stages:

Preparing the Postgate C culture medium;

Adjusting the salinity thereof by adding NaCl, depending on the characteristics of the sample extraction point;

Packing, sealing, and sterilizing the culture tubes.

13. A procedure, according to any one of claims 10 to 12, wherein the inoculation of the microorganisms in the culture medium comprises taking the sample and keeping it at a low temperature until incubation is carried out.

14. A procedure, according to any one of claims 10 to 13, wherein the incubation of the inoculated culture media comprises placing the samples in the incubators for a period and at a temperature which are to be determined based on the type of microorganism under analysis.

15. A procedure, according to any one of claims 10 to 14, wherein the stage whereby the Threshold Detection Time is determined for any of the curves measured by the apparatus that can be stored and/or printed, comprises:

Calibrating the resistance measurements of the light detectors based on the absorbance or transmittance;

Obtaining the threshold detection time (TDT) for turbidity (T), interface reactance (Xi) and medium resistance (Rm) growth curves measured, with an initial known concentration;

Entering the initial concentration values (Ci) for the curves mentioned in the foregoing paragraph and obtaining a set of Ci points versus Threshold Detection Times to subsequently obtain a calibration curve derived therefrom;

Obtaining, based on the measurements mentioned above;

turbidity, interface reactance, interface resistance and medium resistance calibration lines.

Obtaining the Threshold Detection Time from a microbial sample having an unknown concentration and determining the concentration thereof based on such calibration line or lines.