DE2215551A1 - Combined device for the sampling and cultivation of microorganisms - Google Patents

Description

The generally recognized clinical procedure for the quantitative determination of the microorganism content of a liquid sample is the pour-plate technique. After this In the procedure, a series (approximately 6) of dilutions of the liquid sample is made. A known volume of each dilution is filled into separate Petri dishes containing agar that has not solidified (not set), whereby nutrients have been added to the agar and the whole is kept at a temperature of about 45 ° C. Purified agar is the preferred suspension medium for the nutrient because it

209841/1209

is inert, non-toxic and has no nutritional value for most microorganisms. In any case, this will be the appropriate one Dilution of the sample volume withdrawn in .. the mixture of nutrients and agar are stirred in and then the whole thing is allowed to gel. A quantitative value is obtained after the incubation Count the number of colonies per unit area in an or several dishes that have a suitable colony density. This procedure is too tedious and tedious for routine use time consuming. In addition, the pouring plate technique requires a relatively large amount of volume during the incubation.

A semi-quantitative breeding process that is more economical Mass screening of the bacteria Developed in the urine, uses a dropper pipette to dispense a single drop of urine when the tip of the pipette is held on a plate covered with agar, which agar in turn contains nutrients. This procedure is done with various urine samples from the patient repeated until a drop in each of 10 discrete bleaches is placed on the plate. Then you bring them Place plate in the incubator for 12 to 18 hours. According to the in-. Cubation will change the appearance of the colonies within each drop as well as the appearance of the edge of the drop with a series of standard images of known bacterial counts (bacterial counts). ..- .. · -:

Another attempt to simplify this process is that Plate immersion (dip slide technique), in which a surface covered with nutrients (e.g. a glass slide or a spoon covered with a nutrient / agar mixture - ;.) taken from a sterile container and immersed in the liquid sample in such a way that all of the nutrients are submerged, and finally the surface is allowed to drain and rearranges the surface for incubation, in the sterile container. B-ei another. Are revelations

209841/1209:

221 ^ 551

clear, sterile polystyrene containers with a nutrient / agar medium inside overdrawn. The liquid sample is poured into the container, poured out again and then the container sealed in order to isolate the culture medium and the whole incubated. The latter development is in the article by Mackay-ScoHay "A simple quantitative and qualitative microbiological screening test for bacteria in urine "in the journal elin. path. (1969) 22, 651-653.

An instrumental method for counting viable bacteria and to determine their antibiotic susceptibility, see the article by Bowman et al "Capillary Tube Inspection Device for mechanized microbiology "(Science, Vol. 15 8, pages 7 8-83). The suspension of bacteria (and antibiotics, if used) is added to a molten mixture of nutrients and agar. These materials are blended and the resulting mixture is used to fill up the desired number of capillary tubes. The filled tubes are then closed at the ends with red sealing wax or plastic stoppers. The prepared ones Capillaries are examined both before and after incubation. In a variant of the method, the capillaries, which are later to be filled with the mixture of sample and nutrients, with a uniformly standardized amount Antibiotic pre-coated, The antibiotic is dispersed in a medium, such as purified agar, which is inert and as Thread is filled into the capillaries and then dried.

The devices of the present invention simplify sampling a lot and improve the effectiveness of the whole procedure by combining the functions of sampling and cultivation. The preferred device is an internally sterile, transparent disposable tube (e.g. a pre-plugged pipette of the type used in bacteriology or serology or a dropper pipette) that was previously over

209841/1209

a known area of the inner surface with a culture medium has been coated for colony-forming microorganisms (such as bacteria, yeast or mold). In addition, will a combination sample container cultivation tube described, in which the cultivation tube, the inner surface of which with a Nutrient medium is lined, is releasably attached to a wall of the sample container, the interior of the cultivation tube is in flow communication with the interior of the sample container. In this way, the inside of the cultivation tube if desired, the liquid sample can be exposed without a liquid sample being removed from the sample container itself must become.

The term "solid" used in the present application for Description of the condition of the nutrient agar deposits used means that this material is present as a stiff gel that spreads out as a layer along a wall can carry yourself.

The invention, as well as the objects and advantages, are described below explained in more detail with reference to the drawing.

Fig. 1 shows an elevation of an internally sterile pipette which is partially is shown in section to show the lining of the nutrient medium as well as those present at both ends Plugs that maintain sterility;

FIG. 2 shows a modification of the pipette according to FIG. 1, in which pipe segments which can be connected are connected to one another in order to the diagnostic use and / or the antibiotic sensitivity tests with the device according to the invention simplify and with the inner surfaces of the Segmeri * te are covered with different culture media;

Fig. 3 shows a dropper pipette according to. the present Invention is coated on the inside and sealed at the lower end piece and in a sterile container is arranged and

209841/1209

Figure 4 shows an elevation, partly in section, of the combination Sample container / cultivation tube.

In Fig. 1, the pipette 10 is made of a transparent material, preferably a plastic. The pipette 10 has a stopper 11 made of cotton or similar fibrous material, which is arranged in the passage 12 at the mouthpiece end of the barrel 13. The inner surface of the passage 12 is at least partially covered with a layer I ¹ + of a suitable solid nutrient medium. The same material forms the tip plug 15 at the lower end piece. After the stopper 15 has been removed in a manner still to be described, the liquid to be examined is drawn into the mouth by sucking on the mouthpiece with the mouth or generating a negative pressure in some other way, for example by means of a flexible bellows, not shown.

The presence of the sterile barrier, the stopper 11, provides protection against the passage of microorganisms in both directions. At the same time, the stopper 11 does so in the presence of a suitable cultivation environment (ie oxygen-containing for alrobe bacteria; no or only a low oxygen content for analrobe bacteria ) It is not necessary to hermetically seal the mouthpiece of the pipette 10. This is important, since thereby the undesired fogging of the pipette wall, which is often encountered in completely closed breeding devices is ^s, automatically vermiedenbder minimized.

The layer 14 and the plug 15 of nutrient are inserted, by drawing the nutrient into the sterile pipette in a warm, free-flowing state to the desired height and then drain the nutrient. A thin layer of the nutrient remains on the inner surface of the barrel 13 and covers them evenly. The nutrient material gels and the last part of the dripping nutrient that comes through

2 0 9 8 41/12 0 9

the gelling is more viscous, collects in the drip tip 16 and forms the stopper 15. The presence of the stopper 15 is a sure sign that the pipette has been coated and has not yet been used. That way, both the Nutrient layer 14 as well as the plug 15, which together with the plug 11. the inside of the. Pipette is kept sterile, manufactured in one and the same operation »It is only necessary to applying pressure to the bore immediately before use (e.g. by blowing through the mouthpiece), which is sufficient to remove the plug 15. The removal of this plug is used to clean the tip for sampling and growing and likewise to the plastic surface of the To clear the passage 12 at the tip 16 for connection to a clay plug, not shown, to be used later, as will be described later. In addition, the removal of the plug leads to the original dimensions and surface conditions the tip, which the flow and dropping properties determine.

In use, the liquid is up to a predetermined level below the upper limit of the layer 14 in the The pipette is pulled in and then it is allowed to drain. In the case of pre-plugged pipettes, the Liquid-nutrient contact established. With unplugged 'Pipes, those without the plug 11, only becomes a single one Liquid-nutrient contact established ^ if preferred, by lifting the liquid into the tube and then quickly turning the tube around and allowing it to drain.

In the case of the pipette 10, the open end of the dropper tip is then reclosed, e.g. by turning the tip 16 into a thin plate made of clay. The outer surface of the pipette 10 is disinfected and the entire unit can then be in vertical position at room temperature or in a thermostatically controlled oven, not shown, are incubated. These

Incubation is usually 18 to 24 hours at a time Temperature of about 37 C. A very large number of these culture pipettes can be stored or incubated in a small volume will.

After incubation, the pipette can be easily checked for microorganism growth be examined, e.g. for bacterial colonies by using the reflected light in the background, to which The purpose is to arrange the pipette with its longitudinal axis at a small angle to the rays emitted by a light source. Easy visual readings for bacterial

3 9
concentrations of 10 to 10 bacteria / ml can be obtained.

For example, if urine samples contain more than 10 viable bacteria / ml, it is generally assumed that the appropriate Patients have a severe urinary tract infection.

At lower concentrations, i.e. up to and including

3
10 bacteria / ml the pipette remains essentially free of colonies. Similarly, the pipe wall of the pipette can be very

7 9 high concentrations, namely 10 to 10 bacteria / ml, the to the naked eye appear free of colonies and only at the border between the inoculated and the uninoculated nutrient area a clearly defined ring occurs. When this ring is found, the examining person knows that the individual Colonies are not visible because the colonies are microscopic or because the nutrient is completely overgrown and thus indicates a very high concentration of bacteria. In the case of

4 6

Trations in the range of 10 to 10 bacteria / ml can be proportional Colony densities per unit length of pipette can be observed.

In addition to purified agar, various others can also be used Suspension media can be used, e.g., agarose, cellulose derivatives, resins, pectins and gelatin. The to the selected Nutrients added to the suspension medium can be, for example: a broth of trypticase soybeans, veal infusion or Brain-Heart Infusion Medium.

209841/1209

Cultures of samples, e.g. urine, taken from specific patients can be easily identified by having a Color coding used, e.g. layers of different colored clays, which are introduced into the drip tip or colored Markings on the mouthpiece of the pipette or by using two numbered stickers, one of which has been removed and attached to the dog piece.

After use, the plastic pipettes can be easily disposed of by bringing them to sterilization temperatures heated above the softening point, thereby reducing the amount of lumen they occupy.

The pipette configuration per se, in contrast to a simple tube with a small diameter and coated as described, is particularly suitable, since the reduced nozzle-like opening on the pointed end piece gives the device a significant flow property. This reduction enables the user to control the uptake of the liquid sample in a cohesive mass and the dripping rate in order to achieve a uniform distribution of the liquid sample over the area provided with nutrients. The first option allows the uptake and inoculation of an amount of less than 0.2 ml volume with a nominal 1 ml pipette. This is a feature of considerable importance in the creation of urine specimens from babies, old people, or people who have been catheterized. This low required Probenvolumeh allows the use of a larger sample portion for microscopic and chemical examinations. The culture tube , as described in the article by Mackay-Scollay , into which the liquid sample can be easily poured , requires at least 2 to 3 ml of the liquid sample for effective inoculation. \

Considering a section transverse to the central axis through the <tube according to the invention, the open area should be associated with)

2 09841/1209!

Nutrient-lined bore preferably has a circumference between a maximum of about 13 mm to accommodate the required sample volume reduce to an acceptable amount, and a minimum of

■ about 4 mm to avoid a confusion between diametrically arranged To decrease colonies when observed. It can each

: but also bore circumferences between approx. 1.5 mm and approx. 2 3 mm be used.

■ Although the tubes used, with the exception of the tip, are usually manufactured as straight circular cylinders, the structure (and the passage 12) need not have this shape, but can

Other geometric cross-sections, such as triangles and Square, can be used. The pipette configuration is also

'as a tool for mixing a liquid sample, e.g. of urine, useful to homogenize them before culturing.

; In the case of transparent tubes, the inside evenly with

Nutrient are covered and have neither the stopper 11 nor the tip 16 containing a> stopper 15, can after the! Manufacture can be sterilized by sealing them in a plastic container and irradiating them. After inoculation you can

they are sealed at both ends, ί

} If desired, passage 12 can be made with a number

successive layers of different nutrient materials be lined, which partially overlap the previous layers and so a hole with a large number j successive different nutrient media for loading; vaccination and so a useful diagnostic device represent. There was a nominal 1 ml pipette on top of her Inside with trypticase soy agar (trypticase soy agar) (TSA), which in turn is partially overlaid (approximately in the middle of the barrel) with MacConkey agar became. A gram-positive organism was grown in it. The Gram-positive bacteria grew on the TSA, but not on MacConkey agar.

209841/1209

In addition, one of these nutrients in the overlap may contain antibiotics that have been mixed with it to simultaneously the antibiotic susceptibility of the cultured organisms to determine. It can make thin layers of nutrient agar can be applied inside these pipettes so that even expensive nutrient agar layers can be used economically,

Another construction for such a multiple media arrangement is shown in Fig. 2, in which the tubular construction by assembling a number of interconnectable segments 21 is achieved. These segments can be produced in this way be that each has a lining 2 2 with a different nutrient and thereby the possibility of choosing the nutrient medium for inoculation with the same sample creates. One of the segments, segment 23, can be used as a mouthpiece contains a plug 24 made of cotton or a suitable one cellular material. The segment 26 can be used as a drip tip. Form in compound form several of these sections 21 a longitudinally extending, tubular, pre-plugged bore, lined with nutrients, essentially of the type already described Way can be used.

The construction of FIG. 2 from individual segments creates the possibility of not only having antibiotic sensitivity to study a number of antibiotics, but it also simplifies the use of different concentrations of the Antibiotics, to determine both the appropriate antibiotic for use and its optimal concentration. In addition, this examination device is inexpensive since it only requires thin layers of nutrient suspension medium. Also, as shown in Fig. 3, a dropper pipette 31, e.g., a nasal dropper, which is removable in a sterile Container 32 is arranged and designed according to the present invention, can be used. The barrel 3 3 is inside evenly with a layer 34 of nutrients and

2098A1 / 1209

Suspending medium covered, as already described above for the pipette construction and this leads in the same way to Formation of the plug 36 in the tip. As in the aforementioned embodiments, air pressure is used to transport to effect the liquid sample from the sample container to the nutrient layer. This is done with the dropper pipette 31 by applying a negative pressure by first compressing the flexible bellows 37, which can for example consist of rubber and then let go.

The culture is produced by removing the pipette 31 (together with the attached bellows 37 and the cap 38) from the Removes container 32, pushes out the stopper 36 by forcefully squeezing the bellows 37, the tip of the pipette. 31 immersed in the sample liquid and then releases the bellows 37, causing it to return to its normal shape. The one created in this way Negative pressure draws the sample liquid into the pipette 31 and this covers a certain part of the layer 34 there. The liquid is then expelled and the pipette 31 is returned to the container 32 and the cap 38 is again with it connected to the container 32. The entire unit 39 (the inoculated pipette 31 and the container 32) is then incubated.

Another device for combining sampling and Cultivation is shown in FIG. This unit 40 is of particular value in those cases in which the donor has to the liquid to be examined, such as urine, in the M-I cup, which is provided with the lid 42 which is tightly connected therewith can be, e.g. by a snap fit or with a screw cap, after the sample has been filled. In the illustrated Construction is a growing tube 43 removable with the Cover connected, namely by a tight Übagang on the collar 44, whereby the volume 46 in flow communication with the Volume 47, the interior of the cup 41 is.

209041/1209

The inner wall of the cultivation tube 43 is uniform over a known area with a suitable, solid culture medium, covered. It is therefore only necessary to use the unit 40 to turn around from the position shown in order to remove the liquid sample from the cup 41 in a simple manner and without contamination to bring into contact with the culture medium layer 4 8. After inoculation, the unit 40 is used for storage or for the transport is returned to the upright position shown in FIG.

The inoculation can be carried out very shortly after the sample has been taken and afterwards, e.g. during the time of transfer the sample from the clinic to the laboratory, the cultivation tube 43 can drain. After draining, it is only necessary to separate the tube 43 from the cover 42, to attach a closure (not shown) and the tube 43 in the vertical Able to incubate. The remainder of the liquid sample in the beaker 41 can be used until further tests are carried out to be covered. The combined structure created in unit 40 thus obviates the need for a liquid sample can be taken from the sample container, for example by pouring, in order to obtain a culture.

The devices of the present invention are particularly useful for determining the microorganism content of urine samples, e.g. for screening purposes. Additionally for clinical applications, each can have a microorganism content in the concentration range suitable for the device containing liquid can be examined in the same way.

Growing anal robes requires a low or no environment during incubation in the pipette Oxygen content can be maintained or the pipette is provided with such an environment and sealed after inoculation.

209841/1209

For those nutrient materials that change color as bacteria grow, it may be necessary to do a relative one to place a thick layer of nutrient material inside the barrel of the pipette or the cultivation tube. Based on the given Teaching, it should be easy for the technical staff to produce the required structure. It will be a Pipette selected with a sufficiently large diameter so that the nutrient-lined passageway that after the introduction of the nutrient remains, has a sufficiently large cross-sectional area within the specified limits.

Many bacteriological studies are based on identification specific properties of colony growth, e.g. size and shape of the colonies, surface properties, Color and gloss as well as color changes in the specific agar used. Therefore, the tube or the pipette should be made of one material be made to allow easy cutting of the walls in the area of any 'colony of interest, so that the colony can be removed for examination by standard methods.

209841/1209

Claims (15)

Claims 1. Disposable device for the combined sampling of liquids and the cultivation of microorganisms contained therein, marked by a) a hollow, longitudinally extending, transparent wall having part (10) with a first and a second Tail and b) at least one layer (IU) of sterile nutrient material for the growth of microorganisms that uniformly cover the inner surface area of the hollow part covered and so forms a longitudinally extending passage (12) which is lined with the nutrient material and has a circumference in the range from about 2 3 mm to about 1.5 mm, bringing a liquid sample into the hollow part and in contact can be drawn with the nutrient lining. 2. Apparatus according to claim 1, characterized through a porous barrier (11) in the first end piece the hollow part (10), which prevents the passage of microorganisms and allows the free exchange of air. 3. Apparatus according to claim 2, characterized in that the barrier is a plug (11) is made of a fibrous material. 4. Apparatus according to claim 3, characterized in that the barrier is a cotton plug (11) is. 5. Apparatus according to claim 2, characterized in that the second end piece of the hollow part (10) is in the form of a drip tip (16) which forms a narrowed channel for the passage of liquid, wherein the narrowed channel has a removable plug (15) of nutrient material contains. 209841/1209 6. Apparatus according to claim 1, characterized z ei c h η e t that the second end piece of the hollow part (10) has the shape of a drip tip (16) that constricts one Forms channel for the passage of liquid, the narrowed channel having a removable plug (15) of nutrient material contains. 7. Apparatus according to claim 1, characterized in that the circumference of the nutrient-lined Passage (12) is in the range from about 13 to about t mm. 8. Apparatus according to claim 2, characterized that on the first end piece of the hollow part (31) an elastic, compressible device (37) is attached and this end piece closes, with which air can be pushed through the hollow part. 9. Apparatus according to claim 1, characterized that the hollow part consists of a plurality of tubular segments (23, 21, 26) connected to one another exists, of which at least two are lined inside with different nutrient materials. 10. Apparatus according to claim 9, characterized that the segment (23) which forms the first end piece of the hollow part, a porous barrier (24) contains. 11. Apparatus according to claim 9, characterized that the segment (26) which forms the second end piece of the hollow part, the shape of a Has a drip tip. 20984Ί / 1209 12. Apparatus according to claim 1, characterized in " that at the first end piece of the hollow part an elastic compressible device is attached and this closes the first end piece and thus air can be pressed through the hollow part. 13. Device for the combined liquid sampling and the Breeding of microorganisms contained therein, characterized by a) a walled container (41) which encloses a first volume (47) and contains a liquid as well as a closure part (42) and b) a hollow, longitudinally extending, transparent walled part (M-3) which is detachably and sealably connected at an end piece to the container and extends outside the container, the part "(43) at its distal end is closed, and _(4?) c) the hollow part (43) has at least one layer of one for the growth of microorganisms has suitable sterile nutrient material on the inner surface area and forming a second volume (46), the second volume in fluid communication with the first Volume (47) stands. 14. Apparatus according to claim 13, characterized in that the walled container (41) and the nutrient-lined part (43) through Snap lock are connected to each other. 15. Apparatus according to claim 13, characterized in that the lined with nutrient Part (43) is connected to the closure part (42). 209841/1209