GB2062005A

GB2062005A - Bacterial culturing aids

Info

Publication number: GB2062005A
Application number: GB7933162A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1979-09-25
Filing date: 1979-09-25
Publication date: 1981-05-20
Also published as: GB2062005B

Abstract

A pre-sterilised bacterial culturing package (10) comprises a first container (12) with a communication (16) to a second container (14). The first container (12) will be supplied with a measured volume of culturing medium therein and has an entry position (18) via with a sample to be tested. That inoculation will be via resealing means such as a resilient bung (24) originally protected by sealed ears of the container. After desired incubation part of the contents of the first container (12) are passed over to the second container (14) for inspection by a nephelometer to detect even low- level bacterial activity.

Description

SPECIFICATION Bacterial culturing aids The invention relates to so-called "culturing", as usually carried out in pathology laboratories.

Hitherto, so-called "blood-culturing" has usually involved procedures known as "subculturing" that are tedious, time-consuming and capable of introducing errors. Thus, conventionally, a culture medium is inoculated with a blood sample in a partitioned container also containing an agar screen with which the inoculated medium, or some of it, can be brought in contact after a desired incubation time. That agar screen is intended to collect growing bacteria colonies and permit their further growth. However, samples may have to be picked-off and plated-out for further incubation using Petri dishes, possibly even with subsequent replating before visually significant bacterial activity has taken place.These plating and replating steps constitute what we have called "subculturing" and the possibility of erroneous results must be manifest as stray bacteria may actually be introduced or picked up at least during plating-out.

It is an object of this invention to overcome or at least substantially mitigate such problems.

To this end, one aspect of this invention provides as a pre-sterilised package a container loaded with a desired culture medium, an entry position to that container for inoculation with a sample that may contain bacteria, that entry position including means affording a re-sealing of the entry position after inoculation, and a connection from that container to a second container for passage to the latter of contents of the former for inspection as to bacterial content, preferably by nephelometry.

To those skilled in the art it will be obvious that, for blood-culturing, only plasma will normally be transferred from the first mentioned container to the further container, either after settling of red blood cells naturally or by mild centrifuging. A flexible connecting tube with a captive selective closure clip is thus a useful and advantageous provision for the proposed package.

It will be appreciated that turbidity sensing using a nephelometer is much more sensitive than relying upon the development on agar of visible bacteria colonies, so that plating-out is unlikely to be required using a package as proposed herein.

A nephelometer is capable of detecting a bacterial density of 105 which is far below what is observable by the human eye.

The type of package proposed herein is very well suited to coping with the consequences of sampling in the further container before sufficient bacterial growth has occurred, say by reason of the inoculation containing a very small number of bacteria that will thus require a substantial time to multiply and make a detectable population. Thus, return of the contents of the further container to the first-mentioned container may be effected over the said connection. Alternatively, as the first-mentioned container will normally be much larger than the further container it may be satisfactory simply to make successive transfers to the latter at spaced intervals of time. There could, of course, then be an inevitable dilution of the contents of the further container, but that may be acceptable.

However, it is also proposed herein that there be two or more of said further containers each with its own connection, preferably selectively closable, to the first-mentioned container and intended to serve for successive separate sampling of the contents of the latter.

It is particularly preferred, and advantageous, for at least the first-mentioned container to be of flexible material so that loading of a further said container is by squeezing the first-mentioned container in a manner analagous to known ways of emptying blood-donor bags. The or each further container must be transparent and is preferably form-sustaining at least when loaded so as to fit snugly in a nephelometer testing holder. However, deformability of the or each further container is feasible and could assist in return of its contents to the first-mentioned container between sampling intervals, if that is desired, say for establishing a base level reading for the nephelometer.

The analogy just drawn to blood-donor bags is of particular use in explaining that the entry position may be of an essentially similar type, i.e.

sealed, prior to use. One known type of such sealed entry position has a short tube welded between seams of a bacterially inert and non-toxic container wall material, the tube being surrounded by welded-together ears of that same wall material. Then, it may well be satisfactory, after ripping apart these ears to gain access to the tube for inoculation purposes, simply to heat-crimp or otherwise seal the tube, even using a clip or plug.

However, at least in some circumstances and applications, substantial advantage is envisaged for the incorporation into the end of such a tube, or directly into the top seam, of a plug that can be pierced by an inoculating needle but will self-seal on removal of the latter. Such a plug may be weld compatable with the tube or wall material, or otherwise secured therein, say by an adhesive. A yet further alternative would be to have a thinned tube end portion or a pierceable or frangible closure thereto, so as to retain the culture medium without access to the ears yet permit piercing by an inoculating needle, the tube after inoculation, then being closed off by heat-crimping, clamping or clipping as desired.

The packages hereof are especially well suited to the implementation of blood-culturing in an automatic or partially automatic manner as will be indicated in greater detail with respect to the drawings.

Meanwhile, however, mention is made of a method aspect of this invention wherein a package hereof has its entry position opened, a blood sample is inoculated into the culture medium of the first container with resealing of said entry position, the package is incubated, after a desired incubation period the first container is deformed to transfer plasma onto the second container, and the second container is scanned for turbidity.

Preferably, a captive clip is operated to close the connection between the containers at inoculation time and to open that connection at transfer time. Obviously, that connection may be closed again after transfer.

Practical implementation of the invention will now be described, by way of example, with reference to the accompanying drawings, in which: Figure 1 shows a preferred package thereof; Figures 2 and 3 show details of the package of Figure 1; Figures 4, 5 and 6 show possible modifications; Figure 7 shows, schematically, turbidity sensing using a nephelometer; and Figures 8 and 9 show, schematically, automation applied to blood-culturing using preferred packages hereof.

Referring first to Figures 1 to 3 a package 10 comprises a first or main container 12 and a second or sampling container 14 with an interconnection 1 6 between them. The first or main container 12 also has an initially sealed entry position 1 8.

The first or main container 12 is of flexible plastics material, such as approved for use in making blood-bags but checked as non-toxic to bacteria, and comprises two opposed walls, 21, 22 that are welded together (23) at or near edges thereof, usually by high frequency welding. The connection 1 6 is a flexible plastics tube sealed in position between weld seaming 23.

Sealed in the upper weld seam 23, is a plug 24 at said entry position 18 and between-sideseaming making a neck 19. Access to the plug 24 is via ears 25 and 26 that are edge-welded together at seam 27, but can be ripped apart. The plug 24 is of a self-sealing type, say of a suitably resilient synthetic rubber or plastics material, that is pierceable by an inoculating needle and will automatically reseal itself on removal of that needle. The plug 24 is secured by welding or adhesive as desired, suitable or convenient.

The first or main container 12 is shown loaded with culture medium 30, which will be a measured volume as desired for culturing, for example 40 ml or 80 ml, for which there may be two sizes of package to be made-up to 50 ml or 100 ml by inoculation in use.

The connection 1 6 is secured, say via a bung 28, to the second container by welding or adhesive or, perhaps, only an interference fit if that is acceptable for culturing. The connection 1 6 has a captive clip 32 thereon with a keyhole slot having clearance and closure parts 34 and 36, the clip 32 being operated by sliding the closure slot part 36 over the connection tube 16.

Figure 1 also shows a second connection tube 40 between the containers to serve as a vent for displaced air or other gas during transfer. That tube 40 extends from a position of the main container 12 above, and spaced from, that of the tube 1 6 to facilitate transfers from container 12 to container 14, either by pouring or by expression on squeezing the container 12. The venting tube 40 could be of much smaller bore than that 16, if desired, and may be embodied as a double-tube, see Figure 4, that is separated only adjacent to the main container 12. Then, a single clip could be used for both of the tubes 16 and 40, though Figure 1 actually shows a separate such clip 42.

The purpose of the venting tube 40 is, of course, to prevent air-locking on transfer between the containers, and its end 44 within the sampling container 14 is shown extending thereinto to avoid blocking if the contents thereof are to be poured back into the container 12.

The provision of a venting connection 40 can be avoided by alternative arrangements such as shown in Figures 5 and 6. In Figure 5, bung 28 carries the tube 1 6 and a further short tube 44 that is shown initially sealed by heat-crimping at 46. Venting can be achieved by severing the tube below the heat-crimping 46, and even resealing by heat-crimping after transfer to the second container 14. Figure 6 shows a short tube 48 that carries a porous bacteria barrier plug 50, for example a cotton-wool plug that may be fused therein.

If the volume of the container 14 is sufficient compared with the volume of the connection tube and/or the container 14 is flexible at least in part, and initially at minimum size, transfers can be made by squeezing the container 12 without any need for venting arrangements, thus simplifying the package 10.

The entire package 10 as thus described will be made complete with culture medium and then subjected to sterilisation for supply as a sterile unit in suitable packaging.

In use, the unit will be broken-out of its packaging, the ears 25, 26 separated as in Figure 2 and torn apart to break the weld 27 and give access to the plug 24, the plug 24 pierced by the needle of a hypodermic syringe for addition of the desired volume of patients blood, and the needle removed whereupon the plug 24 will seal. The thus-inoculated container 12 will then be incubated for a prescribed or desired period after which the clip 32 will be released and a small quantity of plasma and culture medium passed over tube 16 to the coupling container 14 for testing, with any appropriate steps taken for venting if required.

Preferably, prior to transfer to container 14, the contents of the container 12 are checked to see that red blood cells and stroma have settled out. It is advantageous if the container 12 is sufficiently transparent for that to be checked visually. If not, or if settlement is not satisfactory, the container 12 will be centrifuged to ensure that what is passed to container 14 will not have unwanted inclusions.

Testing of the sample, say 2 or 3 ml, but as much as 10 ml, passed over to the container 14 is indicated in Figure 7 using nephelometry. An opaque container holder 60, say of aluminium, has a cavity closely fitting the container 14 at least at aperture 62 in the bottom of the holder for illumination by light 64 from source 66. At least one, and preferably several light detectors, such as semiconductor photo cells are located in one or more apertures 68 in the side or sides of the holder 60 for producing a signal on line 70 indicative of received light, which will obviously depend on the extent to which bacteria in the sample causes light to be scattered. Line 70 is an input to a low-noise semiconductor amplifier 72 that provides an output on line 74 to an indicator or recording device 76.

It will be appreciated that, if desired or required, the apparatus 10 is capable of use in taking a base reading by immediate testing of a sample after inoculation of the main container 12. To do so may require immediate centrifuging of the container 12, followed by sampling and nephelometer testing in container 14. The sample may be returned to the container 12 if dilution of a subsequent sample is unacceptable or not desired, and the subsequent growth of bacteria may be improved if the container 12 has its contents remixed by shaking or the like. Alternatively if the main container wall material is sufficiently transparent, a base reading may be taken directly therefrom.

Figures 8 and 9 show possible at least partial automation wherein multi-station incubator 80 has a pivotted plate type container squeezer 82, or one for each station, say using initially centrifuged container 12 or taking them out for centrifuging before transfer. Alongside the incubator/squeezer apparatus is a multi-station nephelometer testing apparatus 84. The apparatus of Figures 8 and 9 is clearly very readily susceptible to automatic control using purpose-designed electronics, etc., or computer apparatus, in order to set inoculation times, transfers and nephelometer operation. Clips or clamps for the tubes 1 6 and 40, if present, may also be automatically controlled if desired, and the apparatus is particularly suitable for the use of a container 12 having two or more sample containers 14 separately connected thereto for successive sampling purposes without return of contents of a sample container 14.

Claims

1. A pre-sterilised bacterial culturing package comprising a first container loaded with a desired culture medium, that first container having means at an entry position that enables both inoculation of its contents with a sample that may contain bacteria and re-sealing of that entry position after inoculation, a second container adapted for inspection of its contents as to bacterial content, and a connection from the first container to the second container for passage to the latter of contents of the former after a desired incubation period following inoculation.

2. A pre-sterilised package according to claim 1 , further comprising a further connection between said second container and the first container for air displacement purposes during transfer over the first-mentioned connection.

3. A pre-sterilised package according to claim 2, wherein said first-mentioned and further connections start at different locations on the first container to assist contents transfer.

4. A pre-sterilised package according to claim 1, wherein the second container has a vent for air release during transfer over said connection.

5. A pre-sterilised package according to claim 4, wherein the vent has a porous bacterial barrier plug.

6. A pre-sterilised package according to any preceding claim, wherein the or each connection has a selective closure clip or clamp associated therewith.

7. A pre-sterilised package according to claim 6, wherein the clip or clamp is captive on a tube of a said connection.

8. A pre-sterilised package according to any preceding claim, wherein the second container is transparent to electromagnetic waves used for inspection by nephelometry.

9. A pre-sterilised package according to any preceding claim, wherein the second container is much smaller than the first container.

10. A pre-sterilised package according to any preceding claim, comprising a plurality of said second containers each with the separate connection or connections to the first container.

11. A pre-sterilised package according to any preceding claim, wherein the first container is of flexible material.

12. A pre-sterilised package according to claim 11 , wherein the or each connection is a or said tube fused into a seam of the first container.

13. A pre-sterilised package according to claim 11 or claim 12, wherein the entry position means is, prior to use, protected by releasably fusedtogether ears of said flexible material.

14. A pre-sterilised package according to any preceding claim, wherein the re-sealing means comprises a resilient plug anchored in the first container.

1 5. A pre-sterilised package according to claim 14 with claim 11, wherein the plug was fused into a seam of the first container.

16. A pre-sterilised package according to claim 14 with claim 11 , wherein the plug was secured into a tube itself fused into a seam of the first container.

17. A pre-sterilised package according to claim 1 5 or claim 16, wherein seams are fused into said first container to each side of said plug or plug tube to form a neck thereat.

18. A pre-sterilised package according to claims 12 to 17, wherein said fusing is by way of ultrasonic handing.

1 9. A pre-sterilised package substantially as herein described with reference to, and as shown in, the accompanying drawings.

20. A method of bacterial culturing wherein a package according to any preceding claim wherein the first container has its contents inoculated with a sample, at least the first container is inoculated for a desired period of time, some of the contents of the first container are transferred to the second container, and the second container is inspected for bacterial content using a nephelometer.

21. A method according to claim 20, wherein the whole package is incubated prior to contents transfer.

22. A method according to claim 20 or claim 21, using a package according to claim 11, wherein contents transfer is by deforming the first container.

23. A method of bacterial culturing substantially as herein described with reference to the drawings.

24. Bacterial culturing apparatus for carrying out a method of claim 23 and substantially as herein described with reference to and as shown in Figures 8 and 9 of the drawings.