DE2953720C2 - Method for the detection of bacteria in liquid samples - Google Patents

Description

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There is a need for the rapid detection of bacteria in liquids or fluids from various Sources. In particular, there is a need for rapid detection of pathogenic bacteria in physiological Liquids such as blood, urine and the like. There is also a need to quickly determine sensitivity of infectious bacteria.

Urine samples generally constitute the bulk of the workload in diagnostic microbiological laboratories. Urinary tract infections are by far the most common urological diseases. In many hospitals, bacteriuria are the most common type of hospital-specific infection often after the use of indwelling catheters and after various surgical procedures appear. The number of specimens that require bacteriuria testing is increased increases the need to repeat the tests to ensure a reliable diagnosis, if there is a risk that the reliability of the tests will be impaired by contamination of the samples during the Sampling has been decreased. Another problem in the diagnosis and treatment of bacteriuria is the fact that there is often a relationship between the patient's symptomatic response to an antimicrobial Treatment and successful treatment. To make sure that the prescribed Antimicrobial agents are actually effective, repeated tests need to be done during therapy will. This creates a need for a simple, rapid bacteriuria test. With regard to that frequent, unexpected, asymptomatic occurrences of urinary tract infections in children, pregnant women Women, diabetics and the elderly, for their diagnosis the acquisition and examination of several Samples may be required, bacteriuria tests must be so simple and inexpensive to carry out that they can be performed routinely can be made. This, too, results in the need for a quick, cheap one Test to help diagnose urinary tract infections and ensure that they are treated appropriately.

In view of the high mortality from septicemia and bacteremia, there is also a need for rapid Tests to find bacteria in the blood. The immediate detection of the disease allows early administration an appropriate antibiotic, which greatly improves the chances of survival.

According to conventional methods, bacterial infections are found in samples such as blood, urine, spinal fluid and the like, detected by diluting the samples with a culture medium and storing them at 36 ° C incubated. The appearance of cloudiness indicates bacterial growth. However, this requires relatively long incubation times required because clouding due to bacterial growth is difficult to separate from blood cells or impurities in the sample due to turbidity or from a turbidity caused by formation caused by precipitation is to be distinguished. A significant increase in turbidity after incubation periods of about 24 hours indicates bacterial growth.

Another important procedure in the clinical laboratory is the determination of antimicrobial susceptibility. Among the most important methods currently used to determine the sensitivity of a microorganism Against an antibiotic being applied include the dilution tests, for example the methods with fermentation tubes and agar plates; and the agar diffusion tests, in which those with an antibiotic impregnated discs are used. Such methods usually require incubation times from 16 to 18 hours before the inhibitory effect of an antimicrobial agent is precisely determined can. These tests are often time consuming and relatively expensive and must be performed by trained laboratory personnel will.

Annealing techniques are known in clinical microbiology, but are generally becoming more of a Staining of dried smears on microscope slides as used for liquid specimens. In practice it will with such a conventional staining process, a dried, bacterial smear on a slide treated with a reagent that

10

rien stains in such a way that a microscopic examination of the smear is possible. Thus, such Analyzes performed only by trained microbiologists using expensive equipment will.

In addition to a bacterial examination of body fluids, it is often necessary to determine the bacterial content of other fluid or liquid samples such as water and pharmaceutical products.

From DD-PS 101 759 a reagent for the detection of bacteria in liquids is known which a) determined Dyes reducible by bacteria, b) nutrients to achieve bacterial growth and c) citrate as Contains buffer substance. This reagent does not stain bacteria, rather it becomes the dye discolored by the bacterial growth.

In A. Jorgensen, Microorganisms for the fermentation industry, 7th edition, p. 730, Verlag Hans Carl, Nuremberg, 1956. are various dyes for coloring bacteria listed, but there is no mention of the fact that chelating bacteria are used for staining gram-negative bacteria Funds are required.

From R. Dickscheit, Handbook of Microbiological Laboratory Technology. Th. Steinkopf-Verlag. Dresden 1967, p. 180 it is known. Bacteria in liquid samples to colors. Chelating agents are not mentioned there.

W. Oberzill, Microbiological Analysis, Carl Verlag

20th

25 equals. A semi-qualitative analysis of stained bacteria can be carried out by washing with an organic acid with a pH of about 2.5 or 2.6, since complete discoloration occurs only in the case of gram-positive bacteria which are stained according to the method according to the invention.

If bacteria are incubated with an antimicrobial agent before staining, the sensitivity can be reduced determine the bacteria versus the active ingredient. The relative intensity of the coloring by doing this treated, stained, concentrated bacteria is related to the effectiveness of the used Means.

As the color intensity of the stained, concentrated Bacteria related to the number of bacteria in a sample can, as already mentioned, be a semi-quantitative one Bacterial analysis can be made by the color development of the stained, concentrated bacteria with a nomogram or a compares other known standard. When the process of concentration of bacteria is due to deposition of the Bacteria carried on a semipermeable membrane cannot be associated with the bacteria Dye that could interfere with the accurate detection and quantitative determination of bacteria Washing with an organic acid having a pH in the range of about 2.7 to 4.0 can be removed. Become bacteria before coming into contact with the stain briefly with an antimicrobial agent

tion of bacteria with membrane filters. This publication does not contain any details on the staining of bacteria.
The object of the invention is to provide a quick, simple.

Hanser, Nürnberg 1967, p. 95 describes the concentrate when incubated, the sensitivity of the bacteria

n..i ..........., - ,. -. ,. . rien compared to the active ingredient by determining the

Color intensity of the colored, concentrated bacteria compares with a control sample. A distinction between the Gram stain and bacteria can be carried out by "

cheap and accurate method of evidence and 35 den. by looking at the stained bacteria with an orga analysis of bacteria in body fluids and andean acid with a pH of around 2.5 to 2.6

washes. Gram-positive bacteria are such Washing process completely discolored, while this is not the case with gram-negative bacteria.

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to provide ren fluid or liquid samples.

DE-PS 29 38 511 relates to a preparation for coloring bacteria at a pH above 7.0 based on a dye that also contains a salt of ethylenediaminetetraacetic acid or from citric acid. These salts are hereinafter referred to as chelating agents designated.

The invention relates to a method for the detection of bacteria in liquid samples by staining of bacteria at a pH value above 7.0 with a preparation according to patent 29 38 511, which is characterized is to concentrate the bacteria after staining.

Surprisingly, it was found that both gram-negative and gram-positive, live bacteria easily colored and made visible by the method according to the invention. According to the invention Bacterial concentrates obtained can thus also be used by personnel who have not been specially trained for this purpose without microscopic Investigation can be recognized. In addition, according to the present invention, the antimicrobial sensitivity can be determined of bacteria quickly and easily. Furthermore, the dyeing process according to the invention can be converted into ·· f> o Carrying out cheap, simple and quick quantitative analysis of bacteria. In the end it is possible according to the invention to differentiate between gram-negative and gram-positive bacteria.

A semiquantitative analysis method for bacteria b5 rien can be carried out in such a way that the color gradation of colored, narrowed Bakieria can be obtained with a nomogram or another calibrated standard. The methods according to the invention can in particular for the detection and analysis of bacteria in physiological fluid samples, especially urine samples, use. According to the invention, a quick and cheap detection and consequently an expedient one Treatment of bacterial infections possible.

Safranin-O and toluidine blue are particularly suitable as dyes in the process according to the invention. Methylene blue, Crystal violet and neutral red. Safranin O is particularly preferred.

The di- and tetra-sodium saltsc are used as chelating agents of ethylenediaminetetraacetic acid (EDTA) and the di- and trisodium salts of citric acid are preferred. Trasodium EDTA is particularly preferred as a chelating agent.

The amounts of chelating agent and dye required to stain bacteria are about 0.001 to about 0.1M chelating agent and dye at a dilution of 1: 1,000 to 1: 300,000. These amounts refer to the final concentrations, with dilutions made by the material, in which the bacteria are present, caused, are taken into account.

The specific concentration of dye and chelating agent may be partly dependent on the condition of the bacteria, that are brought into contact with the staining preparation. For example, if there is a coloring of Bacteria carried out that are present in a relatively concentrated form or free of interfering substances, so

competing chemical or physical reactions usually occur to a lesser extent and it more concentrated preparations can be used. On the other hand, if the bacteria are in a fluid medium, that contains other materials in suspension, so it may be necessary to adjust the concentration of dye and / or chelating agent up or down to correct reactions with these additional Compensate materials. For example, reduced dye concentrations should be set in urine samples continues to prevent the formation of precipitates with urine compounds that cause dye dilution of 1: 1000 occur to prevent. In general is a dye dilution of the order of 1: 2500 or more is sufficient to achieve such a low! 5 to prevent the formation of blows. Dilutions of 1: 10,000 or more are particularly preferred. in the In general, bacteria can be stained particularly effectively if preparations with a content of about 0.05 m chelating agent and a dye dilution of 1: 1000 or more if relatively pure or concentrated bacteria can be used. If the bacteria are present together with interfering materials, so preferably the chelating agent in a concentration of 0.05M and the dye in one Dilution of 1: 10000 or more is used.

In practice, the staining preparations can be stored in concentrated form. For example, sterile Safranin-0 and EDTA can be stored at the concentrations Safranin = 1: 1000 and EDTA · Na ₄ = 0.5 m. In use, this mixture is diluted to the desired concentration. For example, when 1 ml of this preparation is added to 9 ml of test material, a final concentration of 1: 10,000 safrunin and 0.05 m EDTA is obtained. The storage stability of the staining preparation is increased if the dye used for the production of the preparation has been brought into solution in undiluted organic media.

The degree of coloration depends to a certain extent on the dye concentration and the contact time. The contact time can be reduced at higher concentrations. Conversely, longer contact times are required for lower dye concentrations. Furthermore, there is an inverse relationship between contact time and contact temperature. For example, requires an optimal staining bacteria in liquid samples at a dye dilution of 1: 5000, contact times of 45 minutes at 4 ⁰ C, 15 minutes at 25 ° C. 5 minutes at 37 ° C and 1 to 2 minutes at 50 ⁰ C. In general, is required at room temperature for a contact time of at least 15 minutes to reach a maximum Bukterienfärbung in urine samples. No further coloration is observed after 30 minutes. However, if the bacteria are concentrated on semipermeable membranes before staining, contact times of only 15 to 60 seconds are required, since staining preparations with a dye dilution of 1: 1000 can be used. Bacteria stained according to the invention can easily be detected if they are present in concentrated form. If the dyeing process has been carried out on concentrated bacteria in such a way that the bacteria are not dispersed in the liquid phase, the presence of the bacteria can be determined immediately. Colored bacteria that are dispersed in the liquid phase are easily visible after concentrating.

The bacterial concentration that can be detected with the method according to the invention depends to a certain extent on the type of bacteria. In general, gram-negative bacteria can be ^{detected in concentrations of 10 5} CFU ml, while a concentration of 10 ⁶ CFU ml may be required when detecting gram-positive bacteria. Of course, lower bacterial concentrations can be detected by concentrating larger amounts of liquid.

Bacteria can be concentrated or concentrated, for example, by sedimentation or filtration will. When sedimentation is carried out, the bacteria present in the sample make themselves up noticeable as a precipitate, the color of which corresponds to the color of the dye used. In the application By filtration techniques, the bacteria are deposited on semipermeable membranes, taking their presence visible through the coloring that develops on the membrane and is attributable to the dye will.

Conventional methods, for example centrifugation, can be used to carry out sedimentation serve. For example, bacteria can be found in a physiological fluid sample with a volume sediment of 100 ml by centrifugation for 15 to 30 minutes at 3000 rpm. Then they will brought into contact with the dye preparation. The presence of bacteria is indicated by a color on the pellet displayed according to the color of the dye.

When performing filtration techniques, a semi-permeable membrane with a pore size that sufficient to hold back the bacteria required. In general, membranes with a pore diameter of about 0.2 to 1.0 μm can be used. The membrane can be made of conventional materials such as glass fibers, epoxy resins, nitrocellulose. Cellulose acetate, asbestos, or combinations thereof. Epoxy fiberglass filters with good flow velocities are favorable and a depth such that clogging is minimized. at Examination of samples that are very cloudy, such as urine, play the flow velocity and the depth of a membrane play a particularly important role.

Furthermore, such membranes are used in particular, which do not have substantial amounts of the dye that is not associated with the bacteria. hold back. The retention of free dye by the membrane should be as low as possible that between the colorations, which arise on the membrane when, on the one hand, only free dye is present and, on the other hand, in addition colored bacteria are present, can be distinguished. In general, membranes must be used that do not have an electrostatic negative net surface charge exhibit. The relative suitability of membranes can be determined by simply looking at the dye to be used in the appropriate !. Directs concentration through the various membranes and compares the intensities of the colorations formed.

Membranes that do not adsorb any free dye or only small amounts of it are favorable. But it is the combination of dye and membrane is such that the free dye partially covers the membrane surface colors, according to the invention the presence of colored bacteria can be exactly detected by one subtracts the intensity of this partial coloration as the background. To provide accurate evidence of To ensure bacteria, a control experiment is carried out, with the at. Using a liquid sample, which a staining preparation contains but no bacteria, the color developed compared with the color

that is being used in a similar experiment the stain preparation and a pathological amount of bacteria.

Membranes stained by free dye, can be partially or completely decolorized by washing with an organic acid. Upon contact Certain membranes with these organic acids are affected by the staining preparation in the absence surfaces stained by bacteria are at least partially freed from the adsorbed free dye without that the dye associated with the bacteria deposited on the membrane is removed. By such a Acid washing will thus increase the amount of free dye trapped by the membranes and thereby the accuracy with which bacteria are detected on such membranes can. \ improved.

The degree of discoloration obtained by washing with an acid depends on a number of factors. among other things on the type of membrane used Acid, the pH of the acid and the material in which the dye is dissolved. Due to the free dye on the membranes containing various Materials, such as glass lasers. Nitrocellulose. Cellulose acetate. Asbestos and epoxy resins, leaves made dye generally to some extent by washing remove with an organic acid. Organic acids such as citric acid and acetic acid generally work removal of free dye from membrane surfaces. without the dye associated with the jo Connected to bacteria. is removed provided the pH of the acid is in the range of about 2.7 to 4.0. pH values below about 2.7 should be avoided, as this will also result in discoloration of the stained bacteria can. Acetic acid with a pH of about 3 becomes preferred as washing medium.

The degree of adhesion of the free dye to the membranes can be decreased and the removal of the dye by washing with an acid can be increased if the dye used in the dye preparation is dissolved in organic media. Basic color Miiffe. those in VWi »er or in the presence of inorganic ones Salts are dissolved, tend to adhere to the membranes and are generally washed away not discolored with organic acids.

A particularly effective free dye removal is achieved in those cases where a basic dye is completely in a medium with a high organic fraction is brought into solution. The degree of dissolution can be determined by taking a test solution of the basic dye via a cation exchange erb ;: rz «ibt and then you? Eluate through a Membrane suitable for adsorbing the dye. filtered. The degree of resolution results from the at the Membrane adsorbed amount of dye. Is a dye stalk gone completely into solution, no dye is visible on the membrane, whereas with lower ones Degrees of resolution an increase in the color intensity developed on the membrane can be observed.

The dissolution of basic dyes can be done in Me- bC dia with a high organic content, such as those used for the cultivation of bacteria will. Examples of such media are trypticase soy broth. Tryptose phosphate broth. Glucose solution or brain-heart infusion. Preference is given to undiluted Tr. '. Pticase soy broth. Tryptose phosphate broth. Brain-heart infubion or media of a similar nature, since such media are not just a minimum cause false positive results, but also give dye preparations that even after longer Show a reduced tendency to precipitate or cloudiness and thus a higher storage stability exhibit.

A preferred combination for the most complete possible removal of adsorbed on the membrane surface free dye is given below: Glass fiber epoxy filter with a net positive surface charge. where the pore and flow properties are particularly those of the G-2 series from Finite F'ilter Corp. (Detroit. Michigan). Acetic acid of pH about 3 and a basic dye, preferably Safranin-O. dissolved in undiluted growth medium. When using this combination can be essentially the cesamte free dye on a membrane surface decolorize.

The acid washing carried out for the purpose of decolorization can be carried out by you simply bring the colored membrane surface briefly into contact with the acid and then the Sucks off washing liquid or in some other way removes it from the membrane.:. The cheapest duration and number of Washing processes can be determined empirically. In general, pH 3 is 1 to 3 acid Washes of less than 5 minutes each are sufficient.

The presence of bacteria can be semiquantitative using the method according to the invention be detected. Such a quantitative analysis is performed by testing the bacteria on the stains and concentrates as explained above. The color intensity of the colored, concentrated bacteria, which is related to the bacterial population can then be compared to a standard, its Calibration has been done using known amounts of bacteria. To carry out quantitative analysis, conventional methods such as nomographic colorimetric and photometric, be applied. Bacterial growth in liquids can be traced back to what has been explained above Measure method by measuring the intensity of the in samples, which have been taken from the liquid at different times, compares the developed color.

The method according to the invention can also be used to distinguish between the Gram staining will. Organic acids used for washing with a pH below about 2.7 tend to both to decolorize the stained bacteria as well as the free dye on the membrane surface. However, if the pH of the acid is kept at a value of about 2.5 to 2.6, it becomes gram-positive Bacteria completely discolored. At a pH below about 2.5, both gram-positive and gram-negative bacteria are decolorized. In this way it is possible distinguish between gram-negative and gram-positive bacteria. Used for washing an organic acid of the kind used to decolorize the free dye on the membrane however, the pH of the acid is only about 2.5 to 2.6, a semi-qualitative analysis of stained with the preparation according to the invention can be carried out Bacteria carry out.

According to the invention it is also possible to use the antimicrobial To determine the sensitivity of bacteria. Treating bacteria prior to their contact with the inventive Staining preparation with an antimicrobial

bial agent to which the bacteria are sensitive, there is a reduction in the number of Bacteria. As a result, the staining of thus treated stained, concentrated bacteria lower than with resistant cultures or untreated control samples. The reduction in color intensity runs roughly parallel to the degree of sensitivity towards the antimicrobial agent. Bacteria thus become before they come into contact with the Dye preparation with an antimicrobial agent is ίο the color intensity of the colored, concentrated bacteria is related to the sensitivity the bacteria versus the active ingredient. If bacteria are exposed to an antimicrobial agent before staining, that has a bacteriostatic or bactericidal effect is treated, the result is a lower color intensity of colored, concentrated bacteria versus colored, concentrated bacteria that are none Treatment with the agent. If one compares the developed coloration of bacteria, treated with different antimicrobial agents or with different amounts of a single agent the relative inhibitory effect of these agents can be assessed.

Treatment of bacteria with an antimicrobial agent can be done by having simply bacteria either concentrated or suspended in liquid generally 1 to 3 at most Bringing hours into contact with the agent. The method can be carried out with bacteria in a liquid sample or with Bacterial colonies were carried out from a culture plate suspended in an organic broth will. The amount of active ingredient used in this process corresponds to known standard values, for example the FDA approved standard values for antimicrobial discs.

Optionally, a bacterial sample can be incubated prior to treatment with the antimicrobial agent. Incubation increases the accuracy with which the sensitivity to the agent is increased can determine what is due to the fact that the culture has reached the logarithmic growth phase. There Bacteria grow rapidly when incubated at 35 to 36 ° C, samples infected with bacteria only need about 30 minutes incubated for up to 1 hour to ensure very accurate results. Such an incubation is desirable when the relative inhibitory effects of different antimicrobial agents with similar Activities to be determined.

The method of the invention is particularly suitable for staining and analyzing bacteria in physiological Fluid samples. For example, urine. which has been resolved in the conventional manner described in Nutrient broth dissolved Safranin-0 in a dilution of 1: 10000 and 0.05 M tetrasodium ethylenediamine tetraacetate contains, are treated. The urine is then passed through a bacteriological filter with a positive Net charge, the stained bacteria can be easily recognized. If necessary, it can The filter can then be washed with acetic acid or citric acid at pH 3.

Another option is to pass the urine through the bacteriological membrane, causing sedimentation which carries bacteria from the urine to the membrane surface. Subsequently, the deposited bacteria with a sufficient amount of its mixture of 1: 1000 basic dye and 0.05 m-EDTA salt treated to cover the membrane surface.

After 15 to 60 seconds (or longer if necessary) the dye is sucked off through the membrane. Possibly the membrane can then be washed with acetic acid at pH 3.

In some cases, precipitate may be present in the urine of patients suffering from bacteriuria, which too lead to clogging of the membranes used according to the invention. Such urine samples are initially clarified, for example with a 5μΐτι-Κ1αΓνοΓ direction, to remove the precipitate and improve urine filtration. Occasionally can Urine containing gram-positive bacteria in the form of aggregates that are removed by the 5μm clarifier will. Without clarification, bacteriuria can be detected in urine samples of this type according to the method according to the invention Procedure not possible.

In order to increase the flow rate of urine through the filters used, urine can be present Sediments, such as urates, are brought into solution. Acetic acid is a suitable solvent for this purpose. Urine samples mixed with equal volumes of acetic acid with a pH of 2.0, 2.5, 3.0, 3.5 and 4.0, show an increased optical transmission at 540 nm only at a pH value of 2.5 or below. Further Mixtures of acetic acid at pH 2.5 and urine will in most cases give a final pH from 3.5 to 4.5 and do not prove to be disadvantageous in the staining reaction of the bacteria, i.e. the bacteria retain their ability to react with safranin.

The acetic acid used as a diluent increases the flow rate of the urine samples. In many In some cases, urine specimens diluted with acetic acid have a greater color intensity than corresponding ones Samples without acetic acid. This is presumably due to that suspended solids, which are brought into solution by the acetic acid, no longer arise the bacteria stuck on the filter hit and prevent staining.

Although, as mentioned above, acetic acid is used as a diluent the flow rate of urines containing solids improves greatly effect pigmented urines with a content of soluble organic components often blockage of membranes, which is due to the adsorption of pigments on the filters. For the removal of urine pigments anionic exchange resins can be used. The exchange resin is particularly favorable A109-D (Diamond Shamrock; Cl ~ -form), as this resin makes the urine almost colorless. The flow velocities of urines through 0,65μιτι-Ρί1ΐεΓ become strong increased if the urine is first given through the anionic exchange resin. The urine samples can be as follows be treated with such a resin: 3 ml of a urine sample are treated with a 5 g exchange resin given packed column. 2.5 ml of the sample are obtained in the resin filtrate. This filtrate is then mixed with an equal volume of acetic acid as a diluent and poured through the filter. Afterward is stained and washed as explained above.

Such a resin treatment facilitates a quick one Filtration of the sample through the filter. The average flow rate of such samples is 0.2 min Certain positive bacteriuria specimens that give negative results without such resin treatment pretend to show positive results in resin treatment. Also, urine samples pass without one such resin treatment causes clogging of the filters

after treatment with the resin lighter the filter.

In the method according to the invention, the treatment with the synthetic resin can be carried out as follows : Elkay fillers (serum separators), i.e. 10ml Plastic tubes with filters (30 to 40iim) am End of the tube and a rim protruding around the end, which forms a seal in the separator, are charged with 5 g of synthetic resin, which is suspended in water with a content of I: 250 formalin, and in Lömm test tubes, each containing 2.5 ml of acetic acid of pH 2.5 included, given. The liquid phase of the synthetic resin suspension is then reduced under reduced pressure Pressure relieved, leaving a moist resin-packed column inside the separator. The remaining formalin ensures the security of the column. The separator is then placed in the 16mm test tube. which contains the acetic acid, brought by the edge of the Elkay tube is pressed into the 16 mm test tube and the plasma tube into the acetic acid leads into it. These tubes can be prepared and stored before use. At the time of use 3.0ml urine will be poured into the Elkay tube (which is a storage volume above the artificial heart column of about 4.5 ml) given. The Elkay tube is then grasped and slowly removed from the test tube. This has the effect in the test tube of the Elkay rim. which presses against the side wall of the test tube, a negative pressure. In this way the urine sample is sucked through the synthetic resin and introduced into the acetic acid. As a final result it gets take a 5 ml sample of urine and acetic acid, which is then taken can be filtered, stained and washed by the method according to the invention.

Although acetic acid and the exchange resin used as a diluent increase the efficiency of the method for detecting bacteriuria, there are occasional urine specimens which, due to the presence of pigments, are not removed by the anionic exchange resin. Difficulties arise. Blood hemoglobin, certain basic drugs and basic pigments in the urine of patients with certain pathological disorders cause a coating of the filter and prevent the bacteria from staining. For example, when processing a urine sample that contains blood, the erythrocytes pass through the synthetic resin, as the cells cannot enter into an exchange process with it. When mixed with acetic acid, the blood cells are lysed and the basic hemoglobin is concentrated on the filter in the form of a greenish pigment, which interferes with the staining of bacteria. This problem can be solved if such a filter is treated with hydrogen peroxide. Treatment of a filter containing hemoglobin with 0.2 ml of 30 percent H ₂ O _{2 for} 30 seconds causes the filter color to be completely cleared. A staining of the filter with Safranin-EDTA and a subsequent washing show that the peroxide has no effect on the colorability of the bacteria. In fact, the peroxide treatment often increases the color of the bacteria. Therefore, in all cases in which excess pigment appears on the filters (after treatment with synthetic resin and acetic acid), the above-described treatment with H ₂ O ₂ can be carried out for 30 seconds. It is then dyed and washed as described above.

In the case of very cloudy, bloody or dark yellow-brown urines, a precipitate is occasionally deposited or a pigmented compound on the membrane. Coloring such material would be false lead to positive results. In such cases where the urine specimens are so severe with precipitations, like phosphates, carbonates. Urals or blood, contaminated, it is possible to use the invention Procedure to use when the sample is centrifuged at low speeds, with a Sedimentation of the mentioned materials occurs without sedimentation of bacteria. A centrifugation at Revolutions on the order of 500 rpm are generally sufficient for this purpose. After a such centrifugation happens to contain the bacteria The filter protruded more easily. This procedure reduces the possibility of false positives and leads to the discovery of positive results that would otherwise be due to excess pigment build-up would have been masked.

The methods of the invention can be used in a similar manner for staining. Detect and analyze gram-negative and gram-positive bacteria in other liquids and fluid media, such as culture media, Blood. Spinal cord liquid wedge and water, as well as for marking bacteria from such liquid wedges or fluid media have been deposited on membranes, can be used.

Example I (comparative example)

E. coli (gram-negative bacteria) are added to a 100 ml sample of normal, bacteria-free urine, the final concentration being 10 ' ¹ colony-forming units (CFU) / ml. A second urine sample is mixed with Staphylococcus aureus (gram-positive bacteria) in the same way. Then the urine samples are mixed with Safranin-O (red, basic dye) in a dilution of 1: 200,000. As a control, a urine sample that has not been contaminated with bacteria is also treated with dye.

The samples are left at room temperature (RT) for 30 minutes. The 100 ml urine samples are then transferred to Centrifuge tubes centrifuged for 30 minutes at 3000 rpm. Then the tubes are checked for the presence examined by colored bacteria. The tube containing E. coli shows a pellet (precipitate) at the bottom of the tube. However, no red coloration can be seen, it is only that during pelletization gray matter typical of uncolored bacteria. In a tube containing Staphylococcus aureus, the pellet is red in color. The control tube does not contain any pelletized mass.

The experiment is with crystal violet and toluidine blue repeated. In both cases this is in the E. coli tube deposited pellet not colored, while the tube containing Staphylococcus aureus contains a pellet with the The color of the dye used has: dark blue when using toluidine blue. and purple when using crystal violet.

Example 2

100 ml samples of normal, collected urine are ^{treated with 10 h} CFU / ml E. coli and Safranin-O at a dilution of 1: 200,000. The control samples are treated with dye but not with bacteria. All samples are tested with the tetrasodium

treated with the salt of EDTA in the concentrations given below. The samples are 30 minutes leave at room temperature and then put in 100 ml centrifuge tubes Centrifuged at 3000 rpm for 30 minutes to deposit the bacteria.

The bacteria pellet that forms at the bottom of the tube is determined by the amount of bacteria present in it Safranin rated. 0 = no staining of the pelletize

Bacteria; + = Traces of red coloring; +, ++. + + + and + + + + mean increasing amounts of dye. that sticks to the bacteria. Furthermore, after the Centrifugation of the obtained supernatant liquid for its absorption at the wavelength of the dye (520 nm) examined to determine the percentage of the dye removed by the bacteria. The results are summarized below.

Table I.

Final EDTA Urine containing E. coli concentration Pellet adsorption in the urine without 0 0.423 0.01 + 0.390 0.02 + 0.320 0.03 + + 0.280 0.04 + + + + 0.190 0.05 + + + + 0.185 0.06 + + + + 0.196 0.07 + + + + 0.199

removed

Konlrollunn adsorption egg Pellet 0.420 0 0 0.415 0 0 0.424 0 0 0.409 0 0 0.416 0 0 0.421 0 0 0.409 0 0 0.424 0 0

removed

0 6

23 33 54 56 53 52

The results in Table I show that in the presence EDTA binds considerable amounts of dye to the bacteria and occurs in the pellet.

When the above experiment is repeated with the following basic dyes, the same results are obtained obtained: crystal violet, toluidine blue, methylene blue and neutral red.

Example 3

According to Example 2 with various microorganisms using Safranin-O as Model dye studies carried out. The results are summarized below.

Table II

Test organism

Gram dye adhesion dye no 0.05 m

exercise EDTA EDTA

E. coli - 0

S. aureus + + + ■ + - +

Proteus vulgans - 0

Pseudomonas - 0

Group A Strep. + + + + +

Group D Strep. + + + + +

Klebsiella pn. - 0

colored pellets from urine are more effective than the diluted dye used above (i.e. 1: 200000) is the case. Even at the higher dye concentrations, EDTA is responsible for the adhesion of dye required on gram-negative microorganisms.

It turns out that in the case of gram-negative microorganisms, the basic dye binds to the bacteria in the urine the presence of a chelating agent is required while gram-positive bacteria colored by the dye both in the presence and in the absence of a chelating agent.

Further experiments show that basic dyes in a dilution of 1: 5000 in the formation of

Example 4

1: 500 suspensions of Safranin-O are prepared in the diluents listed below. The samples are then 30 minutes at 1.06kg / cirr (15 psi) autoclaved. After cooling, the autoclaved samples are filtered through a 0.22μιη-Μί11ίροΓ0-Filtcr filtered. The filtrate is then mixed with the same volume of 0.1 M EDTA, creating a final Safranin dilution of 1: 1000 and an EDTA concentration of 0.05 m result.

10 ml samples of normal urine are then passed through glass fiber epoxy finite filters 13 mm in diameter and a porosity of 0.65 µm. The membranes through which the urine passes are connected to the Dye-EDTA stock solutions given below have been treated by adding the stock solutions Bring in contact with the membranes for 1 minute. Then the dye preparation is through the Membrane sucked. The membrane is then decolorized twice with 5 ml of acetic acid at pH 3 each time washed.

The membranes are rated as follows: 0 = complete discoloration of the membrane, which looks white: + = slightly reddish: + + = colored red to purple. All results with the exception of "0" represent false positive results. The turbidity of the stock solutions is also assessed: 0 = no turbidity and +, + +, + + + and + + + + = increasing turbidity. The dyes are then stored at ambient temperature. The quality of the suspension is assessed in a similar manner after 24 hours.
The results are shown in Table III.

Table III

Thinning agent for the dye

Membrane turbidity of the dye-EDTA storage

bevvertur.2 Iösune nacl;

0 hrs. 24 hrs.

Distilled water

Saline solution

Trypticase Soy Broth (undiluted) I: 10 broth in water

1: 100 broth in water

Tryptose phosphate broth

1:10 broth in water

1: 100 broth in water

Trypticase soy broth 1:10 in saline Tryptose phosphate broth 1:10 in saline solution 5% glucose in water

10% calf serum in water

1 % sodium acetate

-ι-Ο + + -ΙΟ O O O O O O O O O O

(not done)

Added to urine
Teslbactericn

The results show that only using Table V undiluted tryptose phosphate broth. Trypticase soy broth and glucose solution result in a colored product, which can be completely removed from the membrane by washing with acid. All other diluents including dilutions of the broths in water or saline, give some Extent one coloration of the membrane. Furthermore, if stored overnight with all dye-ED-TA mixtures, with the exception of those in undiluted broths, at least a reflective fall.

E. coli
S. aureus
Pseudomonas
Group A Strep.

Urine + bacteria

Staining before staining after

to washing to washing

Example 5

10 ml urine samples are mixed with 0.05 m EDTA and Safranin-0 treated at a dilution of 1: 10000 in trypticase soy broth and treated for 30 minutes at room temperature left. The samples are then passed through 13 mm - O ^ pm-bacteriological membranes (epoxy glass fibers) given. The membranes are then treated with 5 ml of acid as described below washed. The membranes are then rated, where 0 means no color on the membrane and the signs +. + +. + + + and + + + + mean an increasing membrane color. The results are summarized below.

The results of these experiments show that the dye adhering to the bacteria can be removed by washing with a organic acid is not removed, while the free, dye adsorbed on the membrane is removed by such washing.

50

Table IV

Washing agent

normal urine (no bacteria) staining before staining after washing after washing

Acetic acid. pH value 3 + + 0

Citric acid. pH value 3 ++ 0

HCl. pH value 3 + + + +

H ₂ SO ₄ , pH 3 ++ +

Nitric acid. pH value 3 ++ + +

60

Similar experiments are carried out with samples that contain bacteria are added, carried out using acetic acid with a pH of 3. The results will be accordingly as rated in Table IV and are summarized in Table V.

Example 6

The examination of patient urine for bacteriuria to a pathognomic extent (ie the bacterial concentration in the urine is 10 ⁵ CFU, ml or more) is explained below. A 9 ml urine sample is mixed with 1 ml of a 10-fold Safranin-O EDTA concentrate, mixed and left to stand at 25 ° C. for 30 minutes. The sample is then placed in a vessel which is sequentially equipped with a clarifying 5 nm polypropylene felt filter of 25 mm diameter and a bacterial filter of 13 mm diameter (0.65 μm white glass fiber epoxy filter. Value 3 can be decolorized) is connected. The urine is passed through the polypropylene filter and then through the bacterial filter by means of negative pressure. After the entire sample has passed the filters, the filters are treated with 5 ml of acetic acid with a pH value of 3, which contains formalin in a dilution of 1: 500, this liquid mixture being sucked through both filters by means of negative pressure. The 13 mm fiberglass epoxy liner is then examined for color. The color is compared with a nomogrum, the result of which is the expected number of bacteria based on the color intensity of the membranes.

When the above method is used in an actual clinical trial, the membrane surface turns orange-gold, indicating approximately 10 ⁷ CFU ml of bacteria in the patient's urine.

308 142/446

Example 7

To determine whether or not a patient is responding well to an antibiotic, the procedure of Example 6 repeated at intervals. An antibiotic effect is shown when after the start of antibiotic Therapy does not reveal any bacteria or the color intensity is reduced. on the other hand shows a constant or increasing color intensity a bacterial resistance to the administered Antibiotic.

The patient of Example 6 is receiving therapy with keflin (penicillin-type antibiotic). The urine is examined again according to Example 6 on the following day. The results are again positive. The result is an orange-red color, which ^{indicates a bacterial concentration of about 10 7} CFU / ml. This results in a resistance of the microorganisms to keflin. Gentamicin is then given. After examining the urine the following day, the filter surface shows a not entirely white color, which indicates an absence of bacteria or a very low concentration of bacteria. The latter antibiotic thus proves to be effective.

25th

Example 8

As an alternative to the method of Example 6, an examination for bacteriuria in pathogenic concentrations can be carried out can also be carried out as follows: A 10 ml urine sample is carried out by a bacteriological 0.65 μm glass fiber epoxy filter of 13 mm diameter (see. Example 6) given in order to deposit the bacteria contained in the urine. The membrane is then 30 Treated seconds to 1 minute with 0.5 ml of a mixture of 1: 1000 Safranin-O / 0.05 m EDTA. The dye is then sucked through the membrane. The membrane is made according to Example 6 with acetic acid in portions washed from 3 to 5 ml. A pe; tion can be removed through a lateral drainage to remove excess Remove dye. The other servings are removed through the filter. The membrane staining is then compared with a nomogram to determine the degree of bacteriuria.

Example 9

Plicemia or bacteremia can be determined as follows: A blood culture is prepared by diluting a blood sample 10-fold with culture broth. The sample is incubated at 36 ° C. Samples of 3 ml volume are taken from the blood culture every hour (for the first time 3 hours after the first incubation period at 36 ° C.). The samples are passed through a 2 μm clear membrane to remove blood cells and cell fragments. The filtrate is then treated with dye and EDTA as in Example 6, with the exception that the 3 ml sample is only mixed with 0.3 nil dye-EDTA test solution. The sample is then allowed to stand for 30 minutes at 25 ⁰ C and, then according to Example 6 one by one by the Klärfiltei and the glass fiber-epoxy-filter. After washing with 5 ml of acetic acid / formalin, the color of the filter is determined and compared with a nomogram. The results obtained when examining a patient's urine are shown below.

Table VI

Sld. after
Start of breeding

Coloring the
membrane

Evaluation of the
Bacteria growth *

3 White 4th pale pink 5 orange 6th Orange red 7th dark red

Evaluation as in Example 2, based on the color of the filler.

The results show that, according to the invention, a bacterial concentration of at least 10 ⁵ CFU / ml can be detected in the culture after an incubation period of only 4 to 5 hours. This incubation time is shorter compared to the minimum time required for the formation of turbidity, even with clear (blood-free) liquids. Such opacities generally only appear after 24 hours.

Example 10

5 ml of spinal fluid from a patient with sepia Meningitis are added to 50 ml of culture broth and incubated at 36 ° C. In the following specified 3 ml samples are obtained from the culture at intervals and processed according to Example 9. To the same At times, the culture is checked for the appearance of severe turbidity, which is indicative of bacterial growth would be observed. The results are summarized below.

Table VII visible cloudiness
the culture Coloring the
membrane Evaluation of the
Bacterial growth * Hours after
Start of breeding O O O O ++ White
pinkish orange
orange
Orange red
Red
dark rol 0 3
4th
5
6th
7th
8th

Evaluation as in example 9

According to the invention, the presence of bacteria can be detected 3 hours before turbidity occurs in the Determine cultivation media. This means a considerable benefit for patients with serious illnesses, like meningitis.

Example 11

The antimicrobial susceptibility of bacteria can be determined as follows: A bacterial suspension in a broth rich in organic components is diluted and divided into aliquots (one aliquot each for the antibiotic to be examined and one control) and placed in wells in a cuvette in contact with an antimicrobial elution disc brought. A control corresponding to the point in time ü is produced by adding formalin to the original inoculum and incubating and reading it under the test conditions. The cuvettes are moved by briefly rotating them at 200 rpm in an incubator at 36 ° C. and then incubated until several generations of growth occur, that is to say 1½ ° ^s 3 hours. The cultures are then stained according to Example 6 and filtered. The color intensity of the cultures containing the antimicrobial agents is compared with the control value. Resistant cultures show the same color intensity as the control sample, while sensitive cultures show less color. The staining of partially resistant cultures reads in between.

Example 12

The antibiotic concentrations in blood can be determined as follows: Serum is obtained from a patient's blood and subjected to a 2-fold serial dilution in culture media. 1 ml samples of the various serum dilutions are placed in tubes and each treated with 0.1 ml of a suspension of the original bacteria isolated from the patient (approx. 10 ⁵ CFU / ml). The tubes are then incubated at 36 ° C for 2 to 3 hours.

At the same time there will be an attempt with a second series Tubes carried out. The tubes of this second series contain 1 ml samples that contain the blood of the patient any antibiotic present in known concentrations is given. The serially diluted antibiotic samples are then treated with 0.1 ml of bacterial suspension as described above and incubated.

A control sample is prepared by placing a 1 ml sample without antibiotic in a tube with Treated 0.1 ml bacterial suspension and incubated.

After the 2 to 3 hour incubation period, the samples are stained, filtered and washed according to Example 8. The membrane stains are scored for the most dilute serum sample and the least concentrated antibiotic sample (minimum inhibitory concentration = MIC), which has an inhibitory effect on bacterial growth show to determine. This means that the least concentrated serum sample containing an im Compared to the control sample has a lower staining intensity or the lowest antibiotic concentration, in which a less intense color occurs compared to the control sample, is determined. By multiplication of the degree of dilution with the minimum inhibitory concentration one obtains the concentration of the Antibiotic in the patient's blood.

Example 1 3

Multiple urine samples with a positive reaction to bacteriuria (determined by streaking and counting colonies), which due to suspended solids difficult in the device to detect bacteriuria to be handled, are diluted with an equal volume of acetic acid diluent from the pH value 2.5 mixed with a content of 0.05 m glycine. Corresponding urine samples are used as controls mixed with an equal volume of sterile water. The entire samples (2.5 ml urine + 2.5 ml acetic acid or 2.5 ml of sterile water) are then filtered through a 0.65 μm filter of 10 mm diameter. The flow rate is determined. The filters are then stained and according to the above Versions washed with acetic acid of pH 3. The color intensities of the membranes are determined. The results are summarized below.

I:
W. Table VIII infection CFU, ml Staining intensity of the Filters, Fj, '. Piilient by Closing speed * No. Urine + water Urine + acetic acid P. Proteus 10 " clogged + / 1.7 F. 10 E. coli 10 " + /1.9 + + '0.9 ψ- 11th E. coli 3x10 ' clogged + / 1.4 I. 12th Pseudomonas 5x10 ' clogged + /1.6 1'. ¹ X-
I ¹ I- " 13th S. aureus i χ 10 " 0 / 1.8 + / 0.7 14th Enterococci 6x10 ' clogged 0 / 1.7 K 15th Enterococci 2x10 ' 0 1.9 + /0.9 F. 16 S. epidermidis 8x10 ' 0 / 1.9 0 / 1.4 ι? 17th K. pneumoniae 10 " + '2.0 + + / 0.9 $ 18th S. marcescens 10 " + /1.8 + /0.8 t:
». ' 19th

The counter indicates the color intensity of the l'iller surface. while the denominator is that required to filter the entire sample Indicates Zeil in minutes.

Example 14

A urine sample that has an excessively high precipitate content and is heavily pigmented is according to Example 13 using acetic acid as a diluent processed. Although the urine sample is free of any visible precipitate, a clog occurs in the yoke the membrane so that the test cannot be terminated. The test is repeated by taking 3 ml of urine over a column packed with 5 g of anionic exchange resin (A109-D, Diamond Shamrock, CL "form) and 2.5 ml of eluate are taken up in 2.5 ml of acetic acid as a diluent. The 5 ml sample is then passed through a 0.65 µm filter for what only 0.2 minutes are required. After staining and washing with acetic acid from pH 3, like explained above, the result is a red-colored filter surface, what indicates bacteriuria.

When processing another urine sample that gives a negative bacteriuria result when streaked out, the filter becomes clogged simply using acetic acid as a diluent. If the above explained Treatment with the exchange resin and the inclusion of acetic acid as a diluent carried out, after staining and washing the membrane is not completely white, which is a indicates negative result.

Thus, with the simultaneous use of acetic acid with a pH of 2.5 as a diluent and the exchange resin solve clogging problems. Bacteriuria examinations can thus be carried out immediately. This eliminates the 24- to 48-hour waiting times that arise when streaking out and examining the growth of colonies result.

Example 15

Blood-containing urine is passed through the anion exchange resin of Example 14. 2.5 ml of eluate are taken up in 2.5 ml of acetic acid with a pH of 2.5 as a diluent. The entire sample with a volume of 5 ml is then passed through e ^; n 0.65μπι-Ρί1ΐεΓ given by 10 mm in diameter. A greenish pigment coating results on the membrane. Coloring the filter with Safranin / EDTA and washing it at pH 3 results in a light green filter surface. A urine sample is then processed again according to the procedure outlined above, with the exception that the filter is treated with 0.2 ml of 30 percent hydrogen peroxide for 30 seconds prior to staining. After this treatment, the peroxide is sucked off through the filter. It is then stained and the filter is washed with acetic acid at pH 3. The results show a strongly positive bacteriuria, since the membrane surface is not colored red. A smear test shows that the urine sample contains E. coli in an amount of 10 ⁶ CFU / ml.

Another blood-containing urine is processed according to the method explained above, but the peroxide treatment is omitted. Due to the pigment content, it is impossible to tell if bacteria are present. After the above-described treatment of the greenish filter surface with H ₂ O _{2 has been carried out} , there is no pigment on the membrane. After staining and washing with acetic acid, the result is a typical, not entirely white color, which indicates a negative test result. No bacteria can be detected when smearing.

Another urine sample that contains a fluorescent yellow pigment gives a yellow filter surface. The resin treatment cannot remove the pigment. After staining, there is a strong yellow color on the filter surface. The pigment can be removed by treatment with H _{2 O.} The usual treatment according to the invention gives a red color. The sample thus contains bacteria, which can then be confirmed by streaking out.

Claims (9)

Patent claims: 1. A method for detecting bacteria in liquid samples by staining bacteria at a pH above ⁷ , 0, the dye preparation additionally containing a salt of ethylenediaminetetraacetic acid or citric acid, according to Patent 2938511, characterized in that the bacteria according to the Coloring concentrated. 2. The method according to claim 1, characterized in that the tetrasodium salt of Ethylenediaminetetraacetic acid used. 3. The method according to claim 1, characterized in that the concentration of the bacteria by depositing the bacteria on a semi-permeable membrane that has an average Has pore diameter of 0.2 to 1.0 μm and no significant amounts of free Dye absorbed. 4. The method according to claim 3, characterized in that the membrane is an epoxy-glass fiber filter used with a net positive surface charge. 5. The method according to claim 3, characterized in that the dye in organic media is resolved. 6. The method according to claim 5, characterized in that the membrane after deposition the stained bacteria with an organic acid washes with a pH of 2.7 to 4.0. 7. The method according to claim 1, characterized in that the intensity of the coloration of the Comparing bacteria to a known standard. 8. The method according to claim 1, characterized in that one aliquot part of the bacteria treated with an antimicrobial agent prior to staining and the relative intensity of the staining of the each stained concentrated bacteria determined. 9. The method according to claim 3, characterized in that that the membrane with an organic acid with a pH of 2.5 to 2.6 washes and determines whether the membrane is discolored by this washing process.