FI70045B - METHOD ANORDNING OCH NAERINGSMEDIUM FOER ATT PAOVISA BAKTEREMI - Google Patents

Description

I 1 - NOTICE n n n a r [B3 (11) UTLÄGGNINGSSKRIFT 700 4 5 C (jr \ Patent uiyoiU'CLty

Patent, cMcl-it 12 CO 108G

^ ^ ^ (51) Kv.lk.4 / lnt.CI.4 C 12 Q 1/00, C 12 Μ 1/18 FINLAND - FINLAND (21) Patent application - Patentansftkning 801 1 66 (22) Application date— Ansöknlngsdag 1 1.04 .80 (FI) (23) Starting date - Giltighetsdag 1 1. θ4,8θ (41) Has become public - Bllvlt offentlig 29.1 2.80

National Board of Patents and Registration of Finland Date of publication | 31 .01 .86

Patent- och registerstyrelsen '' Ansökan utlagd och utl.skrlften publicerad (32) (33) (31) Pyydetty etuoikeus - Begärd prlorltet 28.06.79

German Democratic Republic-Democratic Republic of Germany (DD) WP 213960 Proven-Styrkt (71) VEB Pharmaglaswerk Neuhaus, Sonneberger Strasse 73, 6420 Neuhaus am Rennweg, German Democratic Republic-Democratic Republic of Germany (DD) (72) Manf Kun Schambach, Neuhaus am Rennweg,

Werner Köhler, Jena, Klaus Muller, Neuhaus am Rennweg,

Axel Stelzner, Jena, Hannelore Schleef, Jena,

German Democratic Republic-Demokratiska republiken Tyskland (DD) (74) Keijo Heinonen Ky (54) Method, apparatus and nutrient medium for the detection of bacterial hemorrhage - ------------ Method, method and culture for the detection of bacterial __________

The invention relates to a method, an apparatus and a medium for detecting bacterial blood by taking a blood sample and culturing it so that febrile conditions of unknown origin can be identified and diseases caused by known bacteria can be monitored and treated,

The method, apparatus and medium according to the invention are suitable both for taking a blood sample from a human being and for culturing the microorganisms contained in the blood by microbial log is clinical experimental methods.

Several methods and devices are available for the detection of bacterial haemorrhage, which, however, differ more or less in terms of the individual stages of operation or combinations thereof and the corresponding different components.

On the basis of the brochure material, a method for culturing blood samples is known, which essentially consists of the following steps in the examination of bacterial blood:

Blood sampling

Using a blood sampling device, a blood sample is taken from a patient's vein via a connecting tube (DE-OS 2332133), a 2 - 700 A 5 sterile syringe or a blood sampling kit. The blood sample is immediately introduced into a sterile container filled with a liquid solution, which may be a glass tube, bottle or container of varying volume, closed with a rubber stopper or a metal lid. These known vials are sealed with sterile stoppers, and unless the stoppers are sterile, they should be thoroughly cleaned with disinfectant prior to sampling. The culture vessel is inserted into the connecting tube holder by pushing it into the blood sampling section. After intravenous injection, the holder is pressed through the stopper of the culture vessel so that the vacuum draws blood from the vein. Through the blood, microbes are implanted in the liquid medium immediately. If more blood samples are needed, several culture vessels containing different media are successively filled.

- Cultivation:

The culture vessel is detached from the connecting tube retainer, the blood and liquid medium are mixed well, and then the culture vessel is incubated at 35-37 ° C for inoculation. If aerobic conditions are desired, aeration is switched on, and growth is monitored at certain intervals and possible growth as the solution coincides, followed by inoculation on solid medium. When a sample of the culture medium is taken with a sterile syringe through a sterile cannula, the stopper must be punctured. Inoculation takes place on one or more solid media in special plates or petri dishes containing agar and various nutrients. The inoculated agar cultures are further incubated. Inoculation from the culture vessel is carried out on the 3rd, 6th and 10th day of blood sampling. More detailed microbiological examination of different organisms is possible only when colony formation is observed in inoculum cultures.

The disadvantage of this known method is that the measures from blood sampling to inoculation of the inoculum cultures require several means, which makes it difficult to guarantee the avoidance of contamination.

This method of culturing npicrobes in blood samples uses known glass culture vessels filled with a liquid medium and fitted with rubber or metal stoppers.

70045 These culture vessels must be punctured or opened each time a blood sample is to be introduced into the vessel for culture or a liquid culture is to be sampled for inoculation or a gaseous nutrient is introduced into the culture space.

There is a risk of contamination at all these stages of processing, and especially during the preparation of inoculum cultures, when the culture vessel has to be opened or punctured and operated with syringes and cannulas as aids.

Another disadvantage of this known method is that weakly growing bacterial colonies are difficult to detect from liquid medium or solid agar.

Based on the brochures, a two-step method of culturing microbes in the blood, the so-called The Gibco-Bio-Cult system with several media, a special safety guard and a wall separating the liquid and solid medium. It is not necessary to inoculate these culture vessels at certain intervals and to open the vessel for this purpose. The liquid medium into which the blood has been mixed can be drained into the second chamber by tilting the vessel while rinsing the solid agar on the septum so that any microbes contained in the solution adhere to it, and then the culture medium can be poured back into the space provided.

The disadvantage of this two-stage culture system is that, due to the relatively large volume due to the structure of the vessel, it is not possible to establish a direct connection between the vein and the blood sampling device and the culture vessel. Blood sampling from a vein and delivery to a culture vessel is mainly by means of syringes, and only in the case of small volumes by means of tubing or the like connected to cannulas.

Although this method of culturing microbes in biphasic blood allows colonies to grow faster on agar, there is a high risk of contamination due to separate blood sampling. In addition, also in this culture method, the optical detection of colony formation during incubation is difficult, and for this reason, the microbiological examination of weak-growing cultures is also difficult. Thus, in the case of certain microbes contained in the blood, the solid agar phase must be rinsed again at certain intervals with the medium blood mixture.

DE-OS 2221096 and DE-DS 2332133 describe blood sampling devices and cannulas.

The blood sampling device according to said blood sampling system includes a cannula with its fastener, wherein the end inside the cannula is surrounded by a vent valve. The cannula valve can be moved from a position that allows air to enter through the narrow orifice, allowing sampling to a position that prevents air or blood from passing through the orifice.

The fastener includes a tubular main portion and a portion with a conical surface inside. The tubular part is inserted into an evacuated container with a pierceable stopper. The conical closure closes the opening after the cannula head has punctured the culture stopper.

The blood sampling system and the culture system for microbes in the blood are connected to each other in a known manner by means of this fastener.

The cannula unit according to OE-OS 2332133 likewise comprises a front part which is inserted into a vein and a back part which is inserted into the vein.

This cannula unit is equipped with a valve 1 which prevents the flow of fluid from the collection vessel into the patient's bloodstream, as well as a device from which the blood flow can be seen.

The cannula also preferably has a tubular fastener suitable for insertion into a culture vessel. After intravenous injection, the blood sampling system and the culture vessel are connected to each other using a fastener. As mentioned above, there is also a risk of contamination of the entire system in this loose connection, and in addition to this, complex supplies must be constantly on hand.

DE-PS 1110357 discloses vacuum ampoules for sterile blood and fluid sampling, with a cannula containing a cannula inserted from below at the closed top, and 70045 which have been sealed at the lower end under high vacuum.

These vacuum ampoules are for blood sampling only, and the aspirated blood must be separately transferred to the culture medium. Cultivation requires nutrients that provide favorable growth conditions. Corpuscular and non-corpuscular antibodies in the blood can kill or inactivate a large number of bacteria. When it is desired to perform blood tests by culturing the bacteria contained in the blood, several agents are available that inhibit the action of these antibodies. Thus, a polyanethol sulfonate is known which is very good for this purpose. At the same time, it has anticoagulant properties. The known substances are not suitable for biphasic culture systems because their antibacterial properties are too weak and they cannot make the bacterial colonies formed during incubation visible.

It is an object of the present invention to provide a method, apparatus and medium suitable for testing bacterial hematopoies in which the disadvantages of previously known methods, apparatus and media can be avoided, in which the risk of contamination is substantially lower, in which blood sampling and inoculation of bacterial cultures can be carried out more easily. , and avoiding the use of hitherto complex blood sampling and culture equipment.

It is an object of the present invention to provide a method for testing bacterial hematopoies in which the time and distance required for blood flow from a vein to a liquid and solid medium is substantially reduced, and a device for maximizing blood sampling and culturing the bacteria in the sample. and to provide a culture medium that develops anticoagulant and antibacterial properties during blood sampling and testing, and an embodiment of the present invention includes a method of detecting bacterial blood in which blood is drawn through a blood sampling device into a culture vessel with a vacuum and / or the medium into which the blood sample is mixed, and it is essential in the invention that the sucked blood simultaneously infects the liquid medium and, when mixed therewith, also the solid medium. When the culture vessel is then kept at a certain temperature in a certain oblique position, a number of colonies appear in each medium, which become visible -: - :, -, έ '; ·,:,. : 70045 e thanks to the indicator dye contained in the latuS3ine id.

The bacterial hematopoietic test device according to the invention consists of a blood sampling and blood bacterial medium, in which the culture vessel is an ampoule with an annular constriction which divides the ampoule into two parts in the required volume ratio of solid and liquid medium, preferably 1: 3.

Further, in the culture vessel of the device according to the invention, the liquid and solid media are located successively and are in contact with each other when the culture vessel is at an angle of Q to 135 °, and the volume and position of the solid medium are determined by the annular narrowing.

The outer surface of the annular taper has a fixed rupture ring, which makes it possible to separate the parts of the container neatly from each other without cracks and chips. The diameter of the through-hole formed by the annular taper is preferably about 6-10 mm, since the amount and position of the solid agar phase are determined by the beginning of the radius of the taper cross-section.

The culture vessel of the device according to the invention is further connected to the blood sampling device by an elastic connector, preferably made of transparent soft plastic, which allows the axes of the blood sampling device and the culture vessel to be angled and freely movable relative to each other, forming a closed unit.

The lower end of the injection cannula tube of the blood sampling device according to the invention extends around the sharp-tipped top end of the culture vessel so that the glass tip can be broken before the blood sampling.

In addition, there is a protective cover around the rupture tip, the elastic fitting and the blood sampling device.

The bacterial hemorrhage medium consists of a liquid and a solid part containing antibacterial agents, anticoagulants and a pH-dependent indicator system, and the liquid part of the medium should preferably contain indomethacin and carrageenan (Indomethazine), Carrageenan.

The advantages of the method and device according to the invention are that the combined .7 '> f': ·.

...... 70045 by hitherto separate measures for a single blood sampling and culture combination, the qualitative level of research has improved with the reduction of the risk of contamination.

In addition, an even smaller amount of blood can be handled, from which subcultures can also be inoculated, since the method according to the invention uses effective media containing antibacterial additives and solid media containing indicator dyes. The composition of the medium with its additives thus makes it possible to cultivate a very small amount of bacteria, i.e. to use a small blood sample, and thus to keep both parts of the culture age at an optimal size compared to known culture vessels. Because both liquid and solid media are infected simultaneously and bacterial growth can be detected optically, considerable simplification of the method and omission of bacteriological steps have been achieved.

In addition, the direct export of a blood sample to a two-part culture dish, the rapid infection of both media after blood sampling, and the incubation of bacterial cultures in a two-part culture dish without repeated filling steps and the risk of contamination ensure a rapid and reliable diagnosis.

The combination of liquid and solid medium in the culture medium according to the invention makes it possible to keep the vessel at an angle and sometimes to tilt it in order to accelerate the growth of certain bacterial colonies so that the solid agar surface is washed with liquid.

According to the invention, it is also possible to make further cultures from the contents of the culture vessel. Once bacterial growth has taken place, the parts of the culture vessel can be separated by cutting at the annular taper and then examining each medium separately. Thanks to the annular tapered rupture ring, it is possible to make the cut smoothly and as safely as possible.

The method and device according to the invention are illustrated in the following by way of an embodiment. The example is illustrated by the accompanying drawing.

Figures 1 and 2 show an embodiment according to the invention in a schematic longitudinal section.

.-- · e ;; - ::: '; Ί .. ..

70045

In the bacterial hemorrhage test method, a blood sample is aspirated from a patient's vein with a blood sampling device consisting of a cannula or syringe system of a certain structure and the sample is passed into a glass culture vessel with a vacuum or a specific gas and mixed with the liquid medium. The ingested blood thus infects the liquid medium containing antibacterial additives and at the same time the solid medium in the culture medium becomes infected with the medium-water mixture. Bacterial culture is then performed in both media at a certain temperature and at a certain oblique position of the vessel, depending on the bacterial quality to be examined. Bacteria that proliferate in either medium can be detected with an added indicator dye, preferably added to the solid medium.

The device according to the invention consists of a blood sampling device and a culture vessel for bacteria contained in the blood. The culture vessel is a two-part glass body, culture vessel 1.2, which according to the invention is an ampoule with part 1 and part 2. Culture vessel 1.2 contains an annular constriction 3 which divides the length and volume of the vessel in a certain ratio, preferably in a ratio of 1: 3.

Around the surface of the annular constriction 3 there is a fixed rupture ring 9 which, due to a certain tension exerted on the glass, makes possible the culture vessel 1; 2 separating the two parts neatly and as safely as possible. The diameter of the annular taper depends on the passage of the solid medium and is about 6-10 mm.

The liquid and solid media are in succession and in contact with each other in the culture vessel 1, 2 so that the solid agar 10 in part 2 and liquid medium 11 in part 1 of age 1, 2 are determined by the location and volume of the solid agar according to the retaining annular taper 3.

The volume and thus the size of the culture age 1, 2 can be adjusted to match the size of the blood sample. Because the media contain certain anti-bacterial additives that inhibit the body's own antibodies, it is possible according to the invention to take a small blood sample and keep the volume and proportions of vessel 1, 2 small and still increase the small initial bacterial count and show a visible reaction. Preferably, the culture vessel 1, 2 is an ampoule.

9 '-::. ·· · 9'. ϊ · '> 70045 with a breakable glass tip 4 surrounded by an elastic connecting piece 5, which is e.g. a connecting tube made of transparent soft door (such as PVC hose type 60 S klar), thus connecting the culture vessel and the blood sampling device into one closed unit .

By means of the elastic coupling piece 5, the axes of the blood sampling device and the culture vessel are arranged obliquely and in an arbitrary direction with respect to each other. The blood sampling device has an injection cannula 6 covered by a glass dome 7, the tubular lower end of which is so long that it extends around the glass tip 4 to be broken by the culture vessel 1, 2. There is a cover Θ around the rupture tip 4, the elastic connecting piece 5 and the blood sampling device of the culture vessel 1, 2.

Space 12 has been evacuated or filled with gas at a certain pressure to cultivate certain bacteria. For intravenous injection, the cover piece 3 is removed from the device according to the invention and the glass dome 7 is sawn and broken so that the sterile cannula 6 is released for use. After the cannula 6 has been injected into a vein, the culture vessel is moved around the axis of the blood sampling device so that the glass tip 4 breaks. The release of the vacuum creates a vacuum in the cannula 6, which absorbs and transports a certain amount of blood to the tube 1, 2.

At the same time as a certain amount of blood is ingested, the infection of the liquid medium 11 takes place and with this mixture also the infection of the solid agar phase 10.

When the injection cannula 6 is removed from the vein and closed with a sterile, preferably plastic stopper, the incubation of the whole culture begins, i.e. the medium east 10 and 11 is kept in a certain oblique position at a temperature, preferably between 35 and 37 ° C.

The oblique position of the culture vessel 1, 2 depends on the volume of the vessel and thus also on the amount of solid and liquid medium 10, 11, since it is intended that the liquid medium 11 only partially covers the surface of the agar 10.

Upon incubation of the media 10, 11, the bacteria begin to multiply in each of these, and the multiplication can be observed by the color change that occurs in the indicator dye added to either medium. For a more detailed microbiological examination, parts 1 and 2 of the cereal lye are separated by breaking an annular constriction 3. Γ; ΐ-ΰ · -: -: · · - * "along the 70045 fracture ring 9. This separates the liquid substance 11 from the bacteria-growing solid 10 so that both of them can be subjected to further bacteriological examinations by sampling and inoculation.

Separation of the liquid medium 11 from the solid agar phase 10 takes place by breaking the culture vessel 1, 2 in such a position that the part 2 containing the agar 10 is at the top. In this connection, it is possible to place certain gas fillers in the vessel 1, 2, in which some bacterial strains multiply better than in an oxygen atmosphere.

The liquid and gaseous parts of the medium have the following compositions which are advantageous for the invention:

Liquid part of the medium: distilled water 100 g sucrose 20 g pancreatic peptone 20 g glucose 10 g meat extract 2.5 g sodium chloride 3.0 g indomethacin 0.0028 ... 0.0035 g carrageenan 0.01 ... 0.08 g

Culture medium: sterile nutrient agar I - 0AB7 99.5 g glucose 0.5 g bromocresol purple (indicator) 0.0076 g Depending on the application, the amounts of the individual components may vary.

Claims (8)

A method for detecting bacteria, in which method the blood through the blood sampling system is sucked into the culture vessel, in which there is a vacuum and / or special gas and liquid feeding medium in which the blood sample is mixed, characterized in that the blood is drawn up into a vial. provided with a tapered portion, the aspirated blood simultaneously infecting the liquid proximal medium, which contains the opposite additives of the bactericidal substances and also including a solid proximal medium, and then the culture vessel is incubated, the liquid being in the In the lower part of the tube the solid medium is in contact with each other in that the incubatlon occurs in an oblique position, the liquid medium (11) covering the solid surface only partially, at a certain temperature, preferably 35 - 37 ° C. that the bacterial growth that may have occurred in the immediate media becomes visible thanks to the addition of indicator dye. 2. Apparatus provided with a tapered portion intended for the examination of bacteria, consisting of a blood sampling system and culture apparatus for the bacteria contained in the blood, characterized in that the culture vessel (1, 2) is a vial 1 which has an annular tapered portion (1). 3) divides the vial into two parts in the required volume ratio to the lower part arranged by a solid and liquid upper medium (10, 11) arranged to the upper part, preferably in the ratio 1: 3 * 3. Apparatus according to claim 2, characterized in that the cut-out of the annular tapered part (3) has a rupture ring (9) and the diameter of the opening of the tapered part is about 6-10 mm.