DD267157A3 - METHOD AND DEVICE FOR DETECTING MICROORGANISMS IN KOERPER FLUIDS - Google Patents

Abstract

The invention relates to a method and a device for the detection of microorganisms in human and veterinary medicine, in particular blood, urine and CSF. The object is to develop a method in which minimizes the time and path of a single withdrawal of a body fluid up to their action on liquid or solid nutrient media and different microorganism cultures are generated simultaneously. The device is to create a closed extraction culture system whose constructive structure according to the component principle without preparation guarantees a universal use. According to the invention, the method is characterized in that, after a cannula puncture, any body fluid is sucked into a closed removal culture system in which a gas required for the cultivation of certain germs is aspirated by breaking one or more glass tips successively or simultaneously into two or more several vases einroemt, mixed with different liquid nutrient media and inoculated with these solid nutrient media and that subsequently cultures of different microorganisms are produced simultaneously and sequentially and visualized. Two or more collection culture systems with rupturable glass tip are connected via a Verteilerstueck with a common Injektionskanuele. Fig. 2 shows a multiple-shoot culture system. Fig. 2

Description

For this 2 pages drawings

Field of application of the invention

The invention relates to a method and a device for the detection of microorganisms in body fluids in human and veterinary medicine, preferably for the detection of germs in the urine by bladder puncture, in CSF by lumbar puncture or in the pathology in thoracic or Herzpunktionen, wherein by means of the device according to the invention the different body fluids are separately taken by puncture and their germs are cultured for microbiological-clinical test investigation.

-2- 26/157 Characteristic of the known state of the art

For the diagnosis of germs in different body fluids, various method and testing devices are known, which differ considerably from one another in terms of their individual procedures or their amount of goraid and thus the quality of the test results.

The bladder puncture in humans is made of diagnostic green tone exclusively for the detection of bacterial pathogens in infectious progenies of the kidneys and urinary tract.

It is carried out after the detection of spontaneous urine in the clinical laboratory and evidence of bacterial inflammation and Zwononstrahlharnkulturon in likrobiologischen laboratory keino definitive Diagnoso is possible.

These ambiguous findings are:

In mixed cultures, i. simultaneous detection of several types of pathogens, in particular of pathogens that are not clearly pathogenic,

- discrepancy between the nuf indicating a disease clinical picture and dom inappropriate finding of urine culture,

- relatively low numbers of pathogenic germs in the urine culture.

In the case of women under wraps, bladder puncture for targeted therapy is always required if don Lochlienfluß and the prohibition of Urnkatheterisiorung always suspected of inflammatory Urnwegprozoß.

As a rule, bladder puncture is performed for diagnostic purposes according to the following procedure:

- detection and disinfection of the puncture area,

- puncture or long cannula (syringe),

- transfer of the punctate into sterile transport containers for examination in the microbiological laboratory,

- transport to the microbiological facility as quickly and safely as possible in order to avoid temperature effects,

- Microscopic examination and culture on plate cultures.

One known method is to immerse a plastic carrier with a nutrient medium in a cup of urine.

The Plastoträger is inserted into a Piasto tube (as a shell) and incubated. The possibilities of contamination and other disadvantages are given in several points.

- The free urine from the urinary tract becomes infected the open bechor.

- It is schlochto sealing possibility of the carrier in the plaster ear and poor storability as a system (only S months). According to DE-WP 136622 a microbiological Nährbodonträner, in particular for quantitative and qualitative Urindiagnostik known, wherein the culture medium is dipped with a certain surface in a liquid sample and then incubated in a closed cylindrical vessel with stopper, Further systems for Nachwei3 of microorganisms are as Open or semi-open systems for the study of urine or cerebrospinal fluid (spinal fluid) are known, but work in two phases. By means of syringes or ampoules with cannula, any body fluid is aspirated and cultured in different vessels or containers on different nutrient media.

Another, according to DE-WP 149843 known combined blood culture collection system, consisting of a blood collection · and a blood culture system, characterized in that the vessel of the blood culture system as a vial with a ring-shaped narrowing dor length of the ampoule the required ratio of a solid to a liquid Nährmodium, preferably the ratio 1: 3, adapted.

The annular constriction has a crushing ring and fixes the position of the solid Nährn odiums, blood culture and blood collection system are arranged movable by an elastic connector to each other. This combined system also has significant procedural advantages and high contamination safety due to its design advantages.

A relatively small amount of blood to be examined allows a reliable identification of different bacteria.

The disadvantage of this system is that it has so far been applicable only to blood as body fluid, d. H. in seinor application is relatively narrowly limited.

According to DE-OS 2850147 a device for the detection of microorganisms is known, which consists of a provided on both sides with a shutter transparent tube, wherein at one of the two closures coated with one or with variously solid nutrient media, in the tube hineinragondei carrier is fixed.

The carrier is preferably in the form of a slide. The device according to the invention is part of the entire container combination. The method for detecting the microorganisms is to introduce the sample to be examined into a first container containing a liquid nutrient medium, then contact it with the surface of the solid medium of a second container, incubate and examine it.

In addition, a system for the investigation of microorganisms according to dor DE-OS 3136251 as a device for the detection of microorganisms in liquids and on Oherflächen known.

The device consists of a provided at one end with a tight-fitting closure transparent tubular housing, wherein the closure is fixed with a solid nutrient medium, in the closed state into the housing protruding culture medium is fixed and is characterized in that the culture medium carrier from oiner to one Polygonal body folded deep-drawn Folio consists and don donieften outer surfaces of the polygonal body is filled with solid nutrient medium. The Mehrkantkörpor is preferably designed as a triangular body and the support surfaces have different nutrient media. Dor culture carrier is sealed germ-tight in the housing. Through this system, it is possible to simultaneously detect different germs or fungi in a liquid sample to be examined.

Nevertheless, this system also has the considerable amount of uncontrollable contamination as a so-called "open multi-ling system".

In addition to the aerobes and anaerobes as bacteria, the fungi are known as pathogens. Mushroom-induced diseases are growing. This development of fungal diseases is' u, a. due to skin-damaging effects caused by detergents, cosmetics and environmental influences.

Since the possibilities of sepsis with respect to the types of germs are very large and can definitely not be determined, whether it is a bacteremia caused by aerobic or anaerobic microorganisms or whether it is a fungaemia, it is necessary in case of suspicion respective special systems with the special nutrient media. This is impractical in the sense that at least two venipunctures must be performed. Special difficulties are to be expected if bad venous conditions, as in patients with diabetes mollilus, are present. Multiple puncturing of the veins of the patients is no longer required if the culture systems are provided with a U-shaped or star-shaped preassembly with the cannula attached to one end and the culture vessel at the other end.

The well-known collection culture systems set forth work with a very small sample of blood, urine or cerebrospinal fluid. This is achieved by passing the sample directly from the cannula into the culture medium vessel. Other systems with a hose al? Transmission work, require a much larger amount of sample. The application is therefore not suitable for pediatrics.

- Systems for urine and CSF are not yet manufactured industrially, a comparison is therefore poorly possible.

- The useful life is 18 months for the Kulturentnahmesystome, other systems for blood and urine examination can be used only 6 or 9 months.

Object of the invention

The aim of the invention is to provide methods and apparatus for the detection of different microorganisms in Kötper / Iüssigkeiten in which a high contamination safety gogebon, disadvantages of the previous high equipment expense are avoided and any boliebige body fluid to different microorganisms, especially bacteria, germs or Mushrooms, being examined.

Explanation of the essence of the invention

The invention is therefore based on the object to develop methods for the detection of different microorganisms to any body fluid, preferably in blood, in cerebrospinal fluid or in urine, in which the time and way a single withdrawal of a body fluid until its action on several liquid and / or solid nutrient media minimized and created different microorganism cultures simultaneously and to create devices as a closed extraction culture systems whose constructive structure according to the component principle without preparation ensures universal use.

According to the invention, the method for the detection of microorganisms in body fluids, boi sucked the blood in a closed blood collection and culture system, mixed with a liquid nutrient medium inoculated a solid nutrient medium and on this produces a bacterial culture visible, dad ' gekennzeichnet \ characterized in that after a Cannula puncture is aspirated with any body fluid, preferably blood, urine or cerebrospinal fluid, into a closed total collection culture system, sequentially or simultaneously metered into two or more vessels of the system, inoculated with different liquid nutrient media and with these solid nutrient media, and thereafter in the nutrient media different cultures of microorganisms are simultaneously and sequentially created and visualized.

To investigate anaorobic germs, the body fluid to be examined is sucked in according to the invention simultaneously with a certain proportion of gas, preferably 5... 10% CO 2, and flows into the nutrient medium vessel filled with a specific filling gas, preferably 90... 95% N 2 the sampling system, mixes as known with the liquid nutrient medium and inoculated with this the solid nutrient medium.

For examining aerobic germs, a body fluid to be examined is also aspirated as known, flows into the vessel of the removal system in a metered dose, simultaneously inoculates several nutrient media and simultaneously or sequentially produces several microorganism cultures.

Furthermore, the orfordungsgemäße method is characterized in that in several different collection culture systems of a self-contained total collection culture system after a Kanüleneinstich the glass tips are broken in a particular order and that then successively to be examined body fluid into the different sampling culture systems for Detection of anaerobic bacteria, aerobic germs and / or fungi is sucked in a known manner.

For urine analysis method of the invention is characterized in that after a cannula puncture in the bladder of the human body a defined amount of urine is sucked into the culture-removal system, inoculated the liquid and solid nutrient medium and then grow the germs of urine on the surface of the solid nutrient medium, be visualized and determined by indicators.

The device for carrying out the method for the detection of microorganisms. In body fluids consists of a closed-Kuluursystem as bekanm for blood testing bekanm with a designed as an ampoule with an annular constriction vascular system for liquid and solid nutrient medium and with a cannula system. The device is characterized in that two or more withdrawal culture systems are connected to the vascular and breakable glass pits via a manifold to a common glass-cannulated injection cannula.

Due to the inventive construction of the device, it is possible systems with different Nöhrmodien for different microorganisms, eg. B. for various bacteria, fungi, Hefon u. a. in the form of a so-called twin or triplet system and to fill this "battery" of several systems via a Kanüleneinstich Depending on the body fluid to be examined, the ratio of liquid to fostem nutrient medium and thus the size ratio of the two vessels changes dos withdrawal and culture system.

Another identification of the device according to the invention is that the annular constriction divides the vessels dos Kul (U (system8 preferably in the ratio 3: 1 on the length of the total body and that the solid and the liquid nutrient medium in a ratio of preferably 10: 1 in The device according to the invention for CSF examination is characterized in that the vessels and the glass tip of the removal culture system are connected via an elastic connecting piece with a cone and a conical sleeve and thereby with clyr cannula.

For sampling different body fluid wedges, different sizes of cannulas can be used for the sampling culture system, thereby making the scope of application of the device variable.

The Nachwois ancestor invention for different microorganisms in different body fluids and the device according to the invention as a twins, triplets and Mohrlingssystem have significant advantages over the known influenced by high contamination process.

With the help of the bladder puncture system, the procedure for bladder puncture is simplified and safe and fast results in the detection of germs are achieved:

- With the puncture takes place at the same time Punktatgewinnung, transport and cultivation of the germs.

- There is contamination safety (Fromdkeime) by eliminating needles and syringes at de: transmission into the transport vessel.

Dor doctor can decide by observation during the incubation whether a proof of germination succeeds. In the negative case, the transport to the microbiological facility is eliminated.

- In the positive case, typical images of the colonies provide information on the pathogen or germ species.

- The cultivation of damaged germs is improved by immediate introduction into the culture medium.

- The liquid nutrient medium and 2 different solid nutrient media allow the Ar breeding of about 98% of the expected in infectious processes of the urinary system pathogenic aerobic and anaerobic bacteria.

The orfindungsgemäßo system has similar advantages in CSF investigations compared to the previously open or semi-open systems.

It is stung by means of a special cannula in the intended place of the human body and then placed the combined extraction culture system on the cone of the cannula and sucked the cerebrospinal fluid and cultured.

By choosing the cannula size, it is possible to make the scope of the combined system according to the invention variable.

It is thus possible to inoculate by a Kanüleneinstich several culture vessels with puncture blood or puncture urine. The transfer of blood or urine takes place in a closed system. The great advantage is that in addition to the reduction of punctures in patients by exclusion of contamination results in a significant reduction of false positive cultures. When using an aerobic system and an anaerobic system, the U-shaped transfer part including the cannula is filled with CO ₂ -GaS. To create a favorable growth-promoting gas filling, the anaerobic system should first be inoculated with human blood or urine; the other systems should be used in any order.

In lumbar puncture, it is possible to extract a sample of CSF from the cannula, which is used for a microscopic examination.

The culture collection systems are always ready for use at any time. Special preparations are not necessary in any laboratory.

Ausführungsbelsplelo

The method according to the invention and the device are subsequently illustrated and explained with reference to several exemplary embodiments.

In the attached drawing show:

Fig. 1: a schematic representation of a twin-extraction culture system; 2 shows a schematic representation of a triplet removal culture system;

The invention according Zwillingssystem (Fig, 2) consists of two vessels of the culture system, Part 1 and Part 2, which are separated from each other by the annular constriction 3 with the tension ring 8 and the '? * E and liquid nutrient medium 9 and 10 contain ,

The breakable glass tips 4 are connected via a respective olastic connection piece 5 with a distributor piece 12 and a cone with cone counterpart 13. At this distributor piece 12, the injection cannula 6 is connected for the removal of a certain body fluid, which is enclosed by the glass jacket 7. The drilling system according to the invention (FIG. 2) consists of three vessels, part 1 and part 2 of the culture system, wherein the corresponding distributor 12 is Y-shaped.

The twin system and the trilling system have, as so-called multiple systems, several separate extraction culture systems, which are each connected internally to an injection cannula 6 via a connecting piece 12.

The bladder puncture system (FIG. 2) serves for the urine intake c ·. *, The human body and for the subsequent cultivation of the pathogenic germs.

The system consists of the cannula part with the cannula 6, the elastic connecting piece 5 and vessels 1; Second

The vessels 1; 2 divided by a constriction 3 into two equal chambers. The constriction 3 is provided with a tension ring 8.

In order to provide good growth conditions for all expected germ species, a different nutrient medium (agar) 9 and 10 is introduced into each half of the vessel. There is also the possibility of arranging two solid nutrient media 9 and one liquid near bottom 10 of different composition.

It is preferably one solid nutrient medium an endoagar and the other a sodium azidagar and the liquid nutrient medium a nutrient broth.

So that the nutrient medium can not detach from the glass wall, a holding hook is attached in each case. The body will provide with a low vacuum.

After the puncture, the capillary is destroyed by sideways movement of the system and the vacuum is released. After pouring about 1 ... 2ml body fluid into the system, the puncture is terminated.

After ', onetzen of the agricultural land, the cannula 6 is closed with a stopper, and the entire system jebrütet with the cannula *. It is tilted storage of the system in the incubator or transport into the microbiological laboratory, after the Kö perflüssigkelt in vertical attitude (Spitzo up) was several seconds on the solid medium 9.

The colony growth, the number of germs and the type of germs can be read on the respective nutrient medium. This is a semi-quantitative determination.

In order to achieve optimal growth conditions in the culture system both for the aerobic and anaerobic bacteria in certain body fluids, preferably blood, the system has a gas mixture of preferably 90-95% N ₂ and 5-10%. CO ₂ .

Such a gas mixture is produced under novel conditions by the vessel body are flushed with N ₂ and the assembly of the connector and the cannula part 6 under CO ₂ atmosphere.

Only during use of the system, both gases mix and the optimal gas mixture for anaerobic bacteria is formed.

The germination begins. Turbidity of the nutrient medium 10 and the growth of lawn-like colonion on the solid nutrient medium 9 indicate the growth of the koim.

According to the type of microorganisms to be examined, the vessels 1; 2 dos twins, triplets or multi-feeder systems different types of nutrient media with different additives.

The twin system is preferably used for the investigation of anaerobic and aerobic bacteria or germs in the triplicate system, an additional simultaneous detection of fungi is possible.

The Lipuorsystem serves to examine the germs, which are in an inflammation in the Liuor.

The sterile lumbar puncture needle is inserted into the lumbar canal. After correct position of the Lipuorsystom the elastic protective cap is removed, in order to establish at the same time a connection of cannula cone and cone of the Lipuorsystoms.

After breaking off the glass tip 4, the vacuum 11 sucks the Lipuorprobe. After separation of the cone connection 13, the cone is closed again with the elastic protective cap.

The system is shown in Figure 1 and it has a special cone coupled to the lumbar puncture needle.

For various Anwondungszwecke the Sysiems invention, z. For example, for bladder and lumbar puncture or in pathology cannulas of different sizes can be used, preferably cannulas with a diameter of 0.6 ... 1.2 mm and 60 ... 100 mm lung for bladder puncture and cannulas with 0.5 ... 0.8 mm diameter and a length of 35 ... 50 mm for paediatrics.

In the collection culture systems for body fluids, preferably for blood, urine and cerebrospinal fluid, such nutrient media are particularly suitable for the liquid phase, the pancreatic peptones as nitrogen source in a concentration of at least 600 mg / 100 ml of the culture medium, meat extract in a concentration of about 0.2% w / v and carbohydrates in a concentration of at least 0.8% w / v.

The meat extract concentration shows in the range of 0.20 to 0.25% weight / volume optimal growth in the expected germ spectrum. The carbohydrate source is preferably glucose. To the medium are added inorganic salts, preferably dipotassium hydrogen phosphate and sodium chloride in a concentration of 0.2 to 0.3% weight / volume.

This nutrient broth used in the withdrawal culture system according to the invention offers good growth conditions to the germs to be cultivated. It ensures the development of sophisticated and therapy-protected germs. The preservation and the Nachwewis fewer germs from puncture materials is thereby ensured.

The solid phase introduced in the system according to the invention contains a pancreatic peptone in a concentration of preferably 1.8 to 2.0 g per 100 ml of medium. The addition of sodium chloride in a concentration of 0.04 to 0.05 g / 100 ml of medium turns out to be imsförclumd. As gelling material agar-aoar is added in a concentration of preferably 0.1 to 0.12 g / 100 ml of medium.

By adding indicators, the microbial growth is indicated by color change ^: it.

The HNonenkonzentration of the liquid and solid Phaso is adjusted so that the ready to use Entnnhmu Kultursystom a pH of 7.2 ± 0.2 is present.

Claims (7)

1. A method for the detection of microorganisms in body fluids, wherein the liquid is sucked into a closed collection and culture system, mixed with a liquid nutrient medium inoculated a solid culture medium and on this produced a bacterial culture visibly, characterized in that after a cannula puncture in a living organism any body fluid is sucked into a closed total removal culture system, in which there is a required for the cultivation of certain Keimo gas is sucked by breaking one or more breakable glass tips of the sampling system sequentially or simultaneously metered into two or more vessels, with different mixed liquid nutrient media and inoculated with these other solid nutrient media and that then produced in the nutrient media of the vessels cultures of different microorganisms simultaneously or sequentially and made visible. 2. The method according to claim 1, characterized in that after breaking the glass tip of the extraction culture system for anaerobic bacteria, the body fluid to be examined, simultaneously with a certain proportion of gas, preferably 5% CO ₂ , is aspirated, metered into the with oinem specific filling gas , preferably 95% N ₂ , filled nutrient medium vessel of the (r ntnahmesystems flows, mixed in a known manner with the liquid nutrient medium and inoculated with this the solid nutrient medium and that thereafter after breaking the glass tip of the extraction culture system for aerobic bacteria to examining body fluid, preferably blood, is sucked in a known manner and metered into the vessel of the sampling system flows and inoculated the nutrient media. 3. The method according to claim 1, characterized in that after the Kanüleneinstich the glass tip of several different extraction culture systems of the self-contained total extraction culture system are broken in a certain order and that thereafter successively a certain amount of the body fluid to be examined in the sampling Culture system for the detection of anaerobic bacteria, for the detection of aerobic germs and / or for the detection of fungi in a known manner is sucked. 4. The method according to claim 1, characterized in that after a Kanüleneinstich into the bladder of the human body, a defined amount of urine is sucked into the culture-removal system and inoculated the liquid and solid nutrient medium and that thereafter the germs of the urine on the surface of the solid nutrient medium grow, be made visible and are preferably determinable by indicators. 5. The method according to claim 1-5, characterized in that are taken in all system variants during the cultivation process of the germs in the liquid nutrient media samples for germ detection by reflux from the cannula system. 6. Apparatus for carrying out the method? for detection of microorganisms in body fluid according to claim 1, comprising a closed collection culture system for blood with a trained as an ampoule with an annular constriction vessel system for liquid and solid nutrient medium and with a cannula system, characterized in that two or more extraction culture systems with the vessels (1,2) and the breakable glass tip (4) via a manifold (12) with a common injection cannula {6) with glass jacket (7) are connected. 7. Apparatus for carrying out the method according to claim i'-6, characterized in that different sizes of the cannulas (6) can be used for the sampling culture system for sampling of different body fluids.