RU2286565C2 - Method and device for determining microbiological pollution of water medium - Google Patents

Abstract

FIELD: microbiology; ecology.

SUBSTANCE: method and device can be used for monitoring of water pools and ground waters, as well as for bacteriological inspection of water solutions and suspensions in medical, food and other branches of industry. Method of quantitative determination of microbiological pollution of water and water media is based upon selection of samples of tested medium, on letting it go through bactericide with formula R₄NI_n(n-1)/2)H₂O, maximizing pH of sample to values of 5-6 units and determination of microbiological pollution of medium from concentration of iodine, extracted after interaction of sample of bactericide. Microbiological pollution of medium can determined as from iodine concentration in restored form of J^-, and in oxidized form of J₂. Device for realization of method has bactericidal filter connected with reaction chamber at output of which chamber the registrar is mounted.

EFFECT: improved sensitivity; improved truth of quantitative determination; reduced time of measurement.

6 cl, 3 ex, 4 tbl, 1 dwg

Description

Application area

The invention relates to microbiology and ecology, in particular to the field of determination of microbiological pollution of aqueous media, and can be used to monitor surface and groundwater, as well as bacteriological control of aqueous solutions and suspensions in medicine, food and other industries.

State of the art

Evaluation of the quantitative presence of bacteria in water and liquid aqueous media, as well as the degree of infection are the most important environmental safety requirements. Particularly important is the control of bacterial infection of aquatic environments in epidemiological situations when such control should be carried out as quickly as possible.

Currently generally accepted is the method of control screening with subsequent counting of the formed colonies.

Optical devices, such as a microscope, are used as a recording device for direct quantitative counting of microbial bodies in water and aqueous media.

This method is quite accurate, but it takes a considerable time - from 48 to 72 hours.

There are other methods of controlling the degree of bacterial infection, which can be divided into two large groups.

1) Direct methods consisting in direct counting of microbial cells. Direct methods are known, in particular, such as direct counting of microbial cells having their own color (see application for invention of the Russian Federation No.2000122909, IPC C 12 Q 1/04).

2) Indirect methods using certain specific properties of bacteria, for example, electrical conductivity (see application for invention of the Russian Federation No. 2001132198, IPC C 12 Q 1/06), light scattering (see patent for invention GB No. 2386946, IPC C 12 Q 1 / 04), the ability to fluorescence (see patent for invention CA No. 2264272, IPC C 12 Q 1/04).

Indirect methods are more diverse. For example, methods are known that use color indicators for staining bacterial vital products (see International Application No. WO 0218625, IPC C 12 Q 1/04; US Patent No. US 6,632,632, IPC C 12 Q 1/04; US Patent No. US 2003203422 , IPC C 12 Q 1/04) or using color reactions in the interaction of bacteria with antigens and antibodies (see European patent No. EP 1356080, IPC C 12 Q 1/04).

For indirect determination of microbiological contaminants, instruments for measuring light scattering and fluorescence are used as recording devices.

Recently, the so-called "molecular" methods have become widespread, consisting in the preparation of bacterial preparations with labeled nucleic acids that can be detected by physical methods (see US patent No. US 6630302, IPC C 12 Q 1/68).

These "molecular" methods are quite accurate in assessing bacterial infection, however, even the fastest of them (if you do not include preliminary preparation of drugs), require at least 4 hours.

Among other things, all of the above methods require compliance with sterile conditions, which is difficult to achieve in the field.

Closest to the proposed device for recording microbiological contaminants is a detector of mobile microorganisms designed to control water quality using the optical method, containing a cuvette for the test fluid in the form of a segment of a hollow optical waveguide, an optical radiation source, and two or more photodetectors (see patent for invention of the Russian Federation No. 2143487, IPC C 12 M 1/34).

Disclosure of invention

The objective of the invention is to increase the sensitivity and reliability of the method of quantitative determination of microbiological pollution of water and aqueous media while reducing the time of determination.

To solve the problem, in accordance with the proposed method for the quantitative determination of microbiological contamination of water and aqueous media, conduct sampling of the test medium, which is passed through a bactericide of the formula R ₄ NI _n ((n-1) / 2) H ₂ O, adjust the pH of the sample to 5-6 values and determine the microbiological pollution of the medium by the concentration of iodine released after the interaction of the sample with the bactericide.

The specified bactericide is known as a drug for creating effective disinfectants for water and aqueous solutions (RF patent No. 2213063, 09.27.2003). In it, n is an integer from 3 to 9, R is an organic radical, N is nitrogen, I is iodine.

Microbiological pollution of the medium can be determined both by the concentration of iodine in the reduced form of J ^- and in the oxidized form of J ₂ .

A solution to the problem is directed to a device for implementing the method, comprising a recording device including an optical radiation source, a cuvette for the test medium and a photodetector, a bactericidal filter with a bactericide of the formula R ₄ NI _n ((n-1) / 2) Н ₂ О and a reaction chamber interconnected. In this case, a recording device is installed at the outlet of the reaction chamber.

The reaction chamber is made of corrosion-resistant material and has a neck for introducing reagents, a mixing unit.

The invention is illustrated by the drawing, which shows a block diagram of a device with which the inventive method can be implemented.

The positions in the drawing indicate: 1 - a bactericidal filter, 2 - a reaction chamber, 3 - a neck for introducing reagents, 4 - mixing unit, 5 - an outlet pipe with a valve, 6 - a recording device.

The proposed method is based on the use of an iodine-containing bactericide, which has the useful property of isolating a positive iodine radical "by signal" when live microorganisms appear in water that carry an excess of electrostatic charge. The positive iodine radical (I ^{* +} ) released by the bactericide interacts only with the electrostatic charge of microorganisms, and not with the functional groups of their shells. This quantitative interaction, which is the basis of the proposed method, is used to count microbiological contaminants. This interaction is illustrated by the following formula:

where Q ^{* -} is a microbiological object that carries a negative electrostatic charge;

R ₄ NI _n ((n-1) / 2) Н ₂ О - iodine-containing bactericide;

(Q ^* I ^* ) ⁰ - iodine radical attached to a microbiological object;

I ^- - iodine in reduced form.

The degree of microbiological contamination, such as bacterial infection, is determined by the number of formed iodide ions (I ^- ) by any of the known methods: weight, titrometric, colorimetric, etc.

The most convenient is the quantitative colorimetric determination of iodine formed after the conversion of iodide ions into molecular iodine as a result of an oxidative reaction in an acidic medium (pH 5-6), for example, by the formula

The experimental results showed that the iodine concentration is proportional to the degree of microbiological contamination, i.e. the number of microbial bodies per unit volume.

A device for the quantitative determination of microbiological contamination of water and aqueous media consists of a bactericidal filter 1 connected to a reaction chamber 2 having a neck 3 for introducing reagents. To accelerate the reaction, the chamber can be equipped with a mixing unit 4. The chamber has an outlet pipe with a valve 5. The device also contains a recording device 6. A photoelectric colorimeter can be used as a recording device.

The bactericidal filter 1 is a container in which an iodine-containing bactericide of the formula R ₄ NI _n ((n-1) / 2) H ₂ O is used as a backfill. The reaction chamber 2 is a housing made of a corrosion-resistant material, for example polypropylene.

The best embodiment of the invention

The determination of microbiological contamination is carried out by passing a water sample through a bactericidal filter 1, in which an iodine-containing bactericide quantitatively interacts with microbiological objects. The iodide ions released into the solution enter the reaction chamber 1, where, for example, phosphate-citric acid buffer is added to the solution to create a medium with a pH of 5-6. Then, an oxidizing agent, for example, ammonium sulphate, is added to the resulting buffer solution, as a result of which the iodide ions oxidize to iodine with a color change in the initial solution. The reaction can be accelerated by stirring.

When using the colorimetric method, the concentration of iodine in an aqueous solution can be easily converted to units of bacterial infection (the number of microbial bodies in cm ³ , bw / cm ³ ). As a comparison solution, distilled or deionized water was used.

Example 1:

Samples of sterile water with a volume of 50 cm ³ were prepared, then they were contaminated with E. coli bacteria at concentrations of 5000 and 10000 mt / cm ³ and sequentially passed through a bactericidal filter with a bulk volume of 100 cm ³ .

Then, the pH of the filtered solutions was adjusted by adding 10 cm of phosphate-citric acid buffer solution to values of 5-6, and the concentration of iodine released from the bactericide, proportional to the microbiological infection of water (the number of microbial bodies per unit volume), was determined by the weight method by deposition of 0.1 N AgNO ₃ solution. In parallel, a control experiment was conducted with a sample of sterile water.

The experimental results are presented in table 1.

Table 1 The concentration of iodine in the reduced form, g / DM ³ The degree of microbiological contamination of water, MT / cm ³ 0.0000 0 0.0020 5000 0.0039 10,000

The correlation between iodine concentration and microbiological water pollution is determined by the following empirical equation:

,

,

- концентрация йода в восстановленной форме,Where

- iodine concentration in reduced form,

C _J2 is the concentration of iodine in the oxidized form (molecular iodine),

Q is the number of microbial bodies per unit volume, bw / cm ³ .

Example 2

Samples of sterile water with a volume of 50 cm ³ were prepared, which were contaminated with E. Coli bacteria at concentrations of 500, 1000, 5000 and 10000 bp / cm ³ and subsequently subjected to operations similar to those described in example 1, to obtain a filtered solution with a pH of 5- 6 containing iodine in reduced form.

Then, I ^{- was} converted to the oxidized form of I _{2 by} stoichiometric oxidation of ammonium sulfide (NH ₄ ) ₂ S ₂ O ₈ by reaction (2).

The concentration of I ₂ proportional to the degree of microbiological contamination of water was determined colorimetrically by calibration, expressed by the equation

D = 16C _J2 ,

where D is the optical density.

The research results are presented in tables 2 and 3

table 2 The concentration of iodine, g / DM ³ The degree of microbiological contamination of water, MT / cm ³ 0.00, 0 0,0002 500 0,0004 1000 0.00180 5000 0,00391 10,000 Table 3 Optical density, D The degree of microbiological contamination of water, MT / cm ³ 0.0000 0 0.0064 500 0.0128 1000 0,0630 5000 0.1267 10,000

As a result of the experiments (see table 3), it was revealed that the dependence of microbiological contamination on optical density can be expressed by the following empirical equation:

D = 127 10 ^-7 Q

Example 3

The determination of microbiological contamination in a water sample with a volume of 50 cm ³ from an underground source 22 meters deep, located at a distance of 400 meters from the Volga River, was carried out analogously to example 2.

The results are shown in table 4.

Table 4 Optical density, D The degree of microbiological contamination of water, MT / cm ³ 0.5000 37000

Industrial applicability

The proposed method for the quantitative determination of microbiological contamination of aqueous media is very fast (up to 30 min), accurate (up to 400 mt / cm ³ ) and does not require special sterility.

Claims (5)

1. A method for determining the microbiological pollution of aqueous media, which consists in sampling the test medium, passing it through a bactericide of the formula R ₄ NI _n ((n-1) / 2) Н ₂ О, adjusting the pH of the sample to 5-6 and determining the microbiological pollution medium according to the concentration of iodine released after the interaction of the sample with a bactericide. 2. The method according to claim 1, characterized in that the microbiological pollution of the environment is determined by the concentration of iodine in the reduced form J ^- . 3. The method according to claim 1, characterized in that the microbiological pollution of the medium is determined by the concentration of iodine in the oxidized form J ₂ . 4. A device for implementing the method, comprising a recording device, characterized in that it further comprises a bactericidal filter with a bactericide of the formula R ₄ NI _n ((n-1) / 2) N ₂ O and a reaction chamber connected to each other, while registering the device is installed at the outlet of the reaction chamber. 5. The device according to claim 4, characterized in that the reaction chamber is made of a corrosion-resistant material and has a neck for introducing reagents, a mixing unit, an outlet pipe.