DE3427679A1 - Method for the investigation of microorganism population - Google Patents

Abstract

A culture solution with a content of a non-fluorescent 2,3-dicyanobenzene derivative is placed in a square-shaped vessel. Subsequently a particular volume of a test solution is introduced into the vessel and mixed. The resulting mixture is incubated at 37 DEG C for 120 minutes. During the incubation, the non-fluorescent reagent is converted by intracellular esterase into a form capable of fluorescence emission. The vessel is subsequently introduced into a fluorimeter. The reaction solution in the vessel is irradiated with exciting light of a wavelength of 380 nm. The fluorescence with a wavelength of 450 nm is measured in the fluorimeter. The microorganism population can be determined from the fluorescence intensity.

Description

description

Method for studying microorganism populations The invention relates to a method for measuring microorganism populations, in particular for measuring in blood, urine, living body tissue, food, water or the like contained microorganisms without their isolation (separation in single cells) is required.

In general, it is important to initiate a clinical investigation the exact test results are available as quickly as possible for a therapy to have.

These results serve as basic information about a possible early diagnosis and an accurate treatment plan. Bacterial Examinations are clinically hematological and clinically biological examinations clearly inferior in terms of speed and automation, since on this biological one Difficulties in the introduction of automatic examination methods.

A conventional, common method of determining effectiveness of antibiotics against microorganisms (susceptibility test) includes two basic Procedural stages. The first stage involves a microorganism in a sample in Colony shape grown. At this stage the microorganisms become what are known as Subject to isolation. Sodsnn became the isolated in the second stage Microorganisms treated with various antibiotics. It is found which antibiotics inhibit the growth of microorganisms. This assessment of the Effectiveness of antibiotics is carried out as follows: The isolated bacteria are inoculated and grown on a medium in a Petri dish. The judgment is done as soon as colonies appear. In general, the increase lasts which allows visual observation of colonies, approximately 24 hours. This method must be repeated 2 times to see if it is bacteria of a single tribe or not.

Thus, until the completion of the conventional process, the Determination of the effectiveness of antibiotics against microorganisms at least > 48 hours.

In some cases it happens during the period that it takes to perform of bacterial susceptibility testing is required leads to worsening or a change in the patient's condition. Hence, it is important as possible To be able to quickly determine which antibiotics are used against the microorganisms in question are effective.

There is also a strong need to remove impurities from Prevent food with microorganisms. For example, there are statutory Provisions that food such as fish pastes, meat products and the like must not contain bacteria such as E. coli.

Hence, in the production and distribution of food strict hygienic precautionary measures required. From this point of view it is desirable to have the time required to detect microorganisms decrease to avoid food marketing difficulties and the problems associated with microorganism contamination as quickly as possible to manage.

In recent years, for example, a process has been developed in which the change in the specific electrical Resistance (impedance) in a conductor with the accompanying multiplication of bacteria is measured; See, for example, US Pat. No. 3,984,766. According to this method, the verification time required by bacteria in clinical materials is shortened as follows be: If one compares, for example, a conventional culture method and a Baktometer method (a baktometer is a device that is used according to the aforementioned impedance measurement method works) with regard to the detection of bacteria in urine, the conventional culture method takes around 18 Hours for the detection of bacteria (if the cultivation is only carried out once), while the method in which the impedance change is tracked by means of the bactometer requires an average detection time of 2.5 hours, provided that the Bacterial population in urine is close to 104 cells / ml.

In such a method of measuring microorganism populations by means of the change in impedance, a bacterial colony growing on a medium becomes scraped off and examined for contamination. Then the bacteria cultured and then treated with a medicinal substance whose effect on bacterial growth Is evaluated. However, this growth cannot be determined before the bacteria have reached a high population density. Would it be possible with the evidence already starting with a small number of cells could be used for the detection reduce the time required accordingly. The degree of a drug exerted effect can only be assessed by determining whether or not the microorganisms (bacteria) may or may not have reproduced.

If a drug proves to be effective, its reproduction is inhibited, so that the number of cells does not increase.

The continuous detection of this bacterial multiplication means one high detection accuracy. Traditional methods of measuring population density of microorganisms by means of the change in impedance have the disadvantage of a slight Accuracy, since the microorganisms due to the im- pedance change can only be detected if they multiply considerably.

The object of the invention is to provide an examination method which makes it possible to determine the population density in a sample with high accuracy to be measured even if the population density in the sample is low.

The invention is based on the experimental finding that bacteria and common fungi, such as yeast, molds and the like, 2,3-dicyanobenzene derivatives, for example non-fluorescent 1,4-diacetoxy-2,3-dicyanobenzene of the formula resorb into the cells, decompose these compounds with the esterase present in the cell and release a fluorescent substance to which the cell wall is impermeable (2,3-dicyano-1,4-hydroquinone). The fluorescence intensity of the incubation medium is proportional to the general viable Cell population.

Thus, according to the invention, there is a method for measuring microorganism populations provided in which (a) a culture solution for microorganisms and a Reagent containing a 2,3-dicyanobenzene derivative are mixed, (b) the resulting mixture at a predetermined temperature for a predetermined time is incubated and (c) the incubated reaction mixture with suitable for excitation Irradiated light and the intensity of the fluorescence emitted by the reaction mixture is measured.

Fig. 1 is a schematic diagram showing the construction of a Analysis device explained.

Fig. 2 is a diagram for explaining the proportional relationship between the fluorescence intensity and the microorganism population in a reaction mixture.

The phrase "generally viable bacteria means primarily live aerobic bacteria and real fungi found in processed foods, perishable food, water, pharmaceuticals, and clinical test solutions (Patient samples) are available for testing for microorganisms. About these in general Viable bacteria include, for example, gram-negative rods and cocci of the genera Pseudomonas, Flavobacterium, Achromobacter, Alcaligenes, Acetobacter, Escherichia, Proteus, Salmonella, Serratia, Erwinia, and the like; gram-positive Bacteria, ctäbehen and cocci of the genera Brevibacterium, Corynebacterium, Streptococcus, Micrococcus, Bacillus, Staphylococcus, Lactobacillus and the like; Actinomycetes and related microorganisms of the genera Arthrobacter, Nocardia and the like; Microorganisms such as yeasts of the genera Saccharomyces, Candida, Torulopsis, Pichia, Hansenula and the like; and molds of the genera Aspergillus, Penicillium and the same.

The term "Enterobacteriaceae" in the examples below means gram-negative bacteria that do not form endospores, lactose with gas formation decompose, represent aerobic or optionally anaerobic rods and to the aforementioned Genera of microorganisms Escherichia, Erwinia, Serratia, Proteus, Salmonella and belong like that.

Examples of 2,3-dicyanobenzene derivatives according to the invention are 1,4-diacetoxy-2,3-dicyanobenzene of the formula 1,4-Dibutyryloxy-2,3-dicyanobenzene of the formula and 1,> 4-di- (tert-butyloxycarbonyl-1-alanyloxy) -2,3-dicyanobenzene of the formula Peptone liquid, heart infusion broth, nutrient broth and the like for examining generally viable bacterial populations, and Czapek Dox agar, malt medium, potato dextrose medium and the like for the selective detection of yeasts can be used as nutrient media. These nutrient media are generally used in accordance with the methods customary in the investigation of microorganisms. If only special microorganisms, eg Enterobacteriaceae, are to be measured in a sample, it is sufficient to inhibit the growth of microorganisms that do not belong to the Enterobacteriaceae by adding sodium deoxycholate to the nutrient media.

When carrying out the method according to the invention, first the 2,3-dicyanobenzene derivative added to the test solution.

The mixture obtained is incubated at 20 to 50 C. If in the reaction mixture Microorganisms are present in a range of 102 4 to IC cells / ml or more, if the aforementioned substrate is broken down by the esterase contained in the cells, whereby a fluorescent substance is released, its fluorescence intensity can be measured by a conventional method. As the fluorescence intensity is proportional to the microorganism population in the reaction mixture, as in -Fig. 2, the cell population in the sample can be determined quickly after one beforehand the relationship between the fluorescence intensity and the number of cells Has been established. The effectiveness of an antibiotic added to the reaction mixture can be assessed by following the growth of the bacteria. If a sample is grown in a liquid culture medium, the measurement time can be reduced by adding the 2,3-dicyanobenzene derivative to the medium beforehand.

In this way, microorganisms can be measured which> 4 present in the reaction mixture in a myriad of 10 3 to 10 4 cells / ml or more are. In this way, the test for microorganisms can be done in 1 to 2 hours be performed.

The invention is explained in more detail below with the aid of examples.

Example 1 Fig. 1 shows the structure of the for measuring the microorganisms used device.

The reaction area of this device has a vessel supply area 42, a thermostated water bath 44 and a vessel receiving area> 46. The reaction vessels 40 are made of a translucent material such as glass or transparent Plastic, and are rectangular shaped so that light escaping from the side is easy can be caught. A microcomputer 10 controls a printer 12, a sample plate 20, a pipetting device 30, a reagent supply device 50 and a mechanism (not shown) for transporting the reaction vessels to connect the individual Work steps.

The pipetting tube 32 of the pipetting device 30 moves up and down down and between a sample suction position 34, a sample discharge position 35 and a waste container 36 for the pipette tips. At the front end of the pipette tube 32 is a removable tube consisting of a tube with a predetermined volume Top. This tip is attached to the pipette tube using a device not shown attached, but can also be attached by hand.

The tip is removed from a sample container prior to aspirating a sample 22 attached. After the sample has been dispensed from the pipette tube 32 into a reaction container > 40 the tip is thrown into the waste container 36. The top will be there removed by means of a device not shown. The distance can, however can also be done by hand.

The sample plate 20 is filled with sample vessels 22 containing human urine included, loaded. When the vessel 22 reaches the sampling position 34, it lowers the pipetting tube 32 is in the vessel 22, the tip dips into the urine sample, sucks up 5.0 ml of urine into the tip and holds it in place. Then moved the pipette 32 to the sample delivery position 35 and gives the urine in the tip to the reaction vessel> 40. The pipette tube 32 then moves to waste chamber 36 for the tips and discards the attached pipette tip.

The reaction vessel 40, which extends from the vessel supply area 42 to a reagent addition area 52 has been transported by means of the reagent supply mechanism 50 with FIG ml of liquid heart infusion medium containing 1,4-diacetoxy-2,3-dicyanobenzene in a concentration of 10 m.

As a liquid heart infusion medium, a solution is used which by adding distilled water to 500 g of beef heart infusion, 10 g of Bacto-Tryptone, 5 g NaCl and 15 g Bacto agar up to a total volume of 1000 ml has been. The pH of this solution is 7.0.

The reaction vessel containing the dicyanobenzene derivative and the culture solution the sample is added to the sample delivery point 35. The thermostated Bath 44 is kept at 37 ° C. The reaction vessel located in the sample delivery position 35 becomes intermittent with constant immersion in the thermostated water bath transported and reaches the irradiation site 65 after 120 minutes.

The irradiation site 65 represents part of a fluorimeter 60 The fluorimeter has an excitation spectroscope 64 and a fluorescence spectroscope 66 on. Light from a light source 62 results in a spectrum in the excitation spectroscope 64. Exciting light with a wavelength of 380 nm is extracted. That in the radiation position 65 located reaction vessel is with the stimulating light beam directly from above irradiated so that the beam is perpendicular to the surface of the solution. Through the side wall of the reaction vessel 40 in the irradiation position 65 enters the fluorescence spectroscope 66 Light in the horizontal direction.

The fluorescence spectroscope 66 directs the fluorescence radiation a wavelength of 450 nm on a light detector 68. The by the light detector 68 analog signals converted into electrical signals are processed in an A / D converter 70 converted into digital signals and fed to the microcomputer 10.

The microcomputer 10 calculates the proportion of microorganisms and displays the result in a display device having the printer 12.

In the method according to the invention, the result is based on the measurement the fluorescence intensity has a low background.

Therefore, a high S / N (signal / noise) ratio can be achieved, so that a high measurement accuracy is achieved.

Example 2 Escherichia coli populations according to the invention Method and according to a conventional method using agar plate cultures certainly. The values are compiled in Table I.

Table 1 Patient Conventional method According to the invention (cells / ml) Method ~ ~~~~ ~~~~~~~~~~~~~ (cells / ml) 1 3.5 x 105 2.8 x 105 2 3.6 x 106 4.0 x 106 3 1.3 x 107 1.5 x 107 4 6.5 x 106 6.3 x 106 The measurement is based on the following Way done. 5.0 ml urine each from 4 patients with infectious diseases have been diagnosed in the urinary tract add to each 5.0 ml of cardiac infusion medium with a content of 1,4-diacetoxy-2,3-dicyanobenzene in a concentration of 10 to given. The resulting mixture is incubated at 37 ° C. for 120 minutes. After centrifugation of the mixture for removing insoluble matter becomes the fluorescence intensity the reaction solution measured. The measurement of the Escherichia coli populations according to The conventional agar plate method is using serial dilution plates Use of bouillon agar media carried out under> 48 hours of cultivation. In the case of the method according to the invention (cf.

Table I) values obtained are values obtained with the aid of a calibration curve previously established for colon bacilli (3rd coli) was obtained are.

Thus, according to this example, it is possible to determine the cell population of Measure bacteria in a shorter time than the conventional method.

Example 3 In Table II are the results of measurement of sensitivity (minimum inhibitory concentration, g / liter) against various antibiotics. In this example, Colon Bacillus (ATCC 25922) is used as the test microorganism. Heart infusion medium is used as the medium.

Table II Antibiotic Literature value Measurement result (g / l) (g / l) Ampicillin 0.002 0.002 Cephalothin 0.008 0.008 Nitrofurantoin 0.008 0.035 Tetracycline 0.002 0.003 Similar results are also obtained for other selective media.

The procedure of this example for the detection of microorganisms under Use of fluorescence analysis to measure extremely low esterase activities can be used in numerous areas, e.g. for food, cosmetics, Drugs, clinical microorganism samples, and the like.

Example 4 The water contained therein is made from 10 ml of river water by centrifugation contained bacteria. The bacteria are in a reaction vessel with brought a content of a culture solution. The culture solution is a phosphate buffer solution containing 1,4-diacetoxy-2,3-dicyanobenzene at a concentration of 10 3 m. The volume the culture solution in the reaction vessel is 10 ml. The mixture obtained is 120 minutes at 370C incubated. After incubation, the mixture is centrifuged. The fluorescence intensity the overhang is measured.

Bacteria obtained from the same water sample are used for comparison added to a standard agar medlum and cultured according to a conventional method. The general bacterial population is examined. The results of the investigation are in Table III both for the process according to the invention and for the conventional procedures compiled.

Table III Sample Conventional method According to the invention (cells / ml) Method (cells / ml) 1 3.8 x 106 3.7 x 106 2 5.1 x 107 5 o x 107 3 8.2 x 108 8.4 x 108 Fig. 2 shows the relationship between the relative fluorescence intensity and the cell population.

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Claims (4)

Claims 1./6 experience for the investigation of microorganism populations, D a d u r c h e k e n n n z e i c h n e t that one (a) a culture solution for the Microorganism, a reagent containing a 2,3-dicyanobenzene derivative and a test sample mixed in a vessel, (b) the mixture obtained in a incubated at a predetermined temperature for a predetermined time and (c) the incubated The reaction solution is irradiated with light suitable for excitation and the intensity of the measures fluorescence emitted by the reaction solution. 2. The method according to claim 1, d a d u r c h g e k e n n -z e i c It should be noted that at least one component is selected as the 2,3-dicyanobenzene derivative the group 1,4-diacetoxy-2,3-dicyanobenzene, 1,4-dibutyryloxy-2,3-dicyanobenzene and 1,4-di- (tert-butyloxyearbonyl-1-alanyloxy) -2,3-dicyanobenzene is used. 3. Methods for studying microorganism populations, d a d u r c h e k e n n n z e i c h n e t that one (a) a culture solution with a content of a 2,3-dicyanobenzene derivative in a reaction vessel, (b) a test solution containing microorganisms in the culture solution Brings containing reaction vessel, (c) the 2,3-dicyanobenzene derivative with an esterase of the microorganisms with the formation of a substance capable of emitting fluorescence decomposed and (d) the solution containing the substance capable of fluorescence emission irradiated and measures the intensity of the fluorescence. 4. The method according to claim 3, d a d u r c h g e k e n n -z e i c h n e t that there is at least one component from the 2,3-dicyanobenzene derivative Group 1,4-diacetoxy-2, 3-dicyanobenzene, 1,4-dibutyryloxy-2,3-dicyanobenzene and 1,> 4-di- (tert-butyloxycarbonyl-1-alanyloxy) -2,3-dicyanobenzene used.