GB2361709A

GB2361709A - Vessel for transporting micro organisms

Info

Publication number: GB2361709A
Application number: GB0009993A
Authority: GB
Inventors: Stephen Minter
Original assignee: Moorlodge Biotech Ventures Ltd
Current assignee: Moorlodge Biotech Ventures Ltd
Priority date: 2000-04-26
Filing date: 2000-04-26
Publication date: 2001-10-31
Also published as: GB0009993D0

Abstract

The present invention relates to a vessel (2) for transporting micro organisms, the vessel comprising a vial (4) defining a first chamber (6) and a cap assembly (8) releasably attachable to the vial by vial engaging means (10, 12), the cap assembly (8) comprising an outlet (14) which may be connected to a vacuum pump to expel air from the first chamber (6) to lyophilise material therein, which outlet (14) is sealable. Also provided is a method of preserving micro organisms for transport using said vessel.

Description

2361709 VESSEL FOR TRANSPORTING MICRO ORGANISMS The present invention

relates to vessels for transporting micro organisms and the use of such vessels for shipping micro organisms, such as bacteria.

Micro organisms may require transportation from one laboratory to another, or for example to a culture collection centre. For such transport it is important that the micro organisms are preserved in a dry suspended state and that when they reach their destination they may be resurrected into a viable culture. Over the years, microbiologists have developed various methods for the storage or preservation of micro organisms, including subculturing, drying, freezing drying (1yophilisation) and freezing. Other methods, such as storage under liquid paraffin, in distilled water, and liquid drying (i.e., L-drying), have also been used with mixed success.

For preservation during transport, it is generally preferred that micro organisms are freeze dried or Iyophilised to suspend the micro organisms.

Freeze drying involves the removal of water by sublimation from a frozen culture. Micro organisms are grown on a suitable growth medium, aliquots are suspended in an appropriate freeze drying liquid in glass ampules or vials, and placed in the freeze drying apparatus, where they are ftozen, and exposed to a vacuum. The water vapour from the culture is either trapped in a refrigerated condenser unit, or on phosphorous pentoxide. After freeze drying, the cultures are sealed in their vials, often under vacuum or in an inert gas, and are stored at room temperature, refrigerated, or frozen.

Two methods of freeze drying are commonly used in industry, namely centrifugal and shelf freeze-drying (See, R. H. Rudge, "Maintenance of Bacteria by Freeze-Drying," in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, [ 199 1 pp. 31-44).

When dealing with micro organisms it is desirable to avoid unnecessary handling of the micro organisms and exposure to air, which are deleterious to sterile conditions 2 and also may place a worker at risk of infection. It is also desirable to avoid contamination of the sample, including cross contamination, which may occur during transfer of the sample from one vessel to another. Also it is important that each vessel containing the sample is clearly labelled and therefore it is desirable to use as few vessels as possible in the transport of one sample to reduce the opportunity for mislabelling of a sample vessel. Further, any vessel that has come into contact with micro organisms must be either sterilised or disposed in accordance with the appropriate risk level procedures.

Although freeze drying has been widely used to preserve various organisms, there are problems associated with this method. The glass vials/ampules currently used for transporting micro organisms do not provide safe or efficient means for transporting micro organisms. For example, glass ampules are generally sealed closed with a flame (e.g., a torch), requiring some care in order to avoid injury to the operator. Glass ampules may be broken during the freeze drying process or during transport.

When the glass vials reach their destination and it is desired to resurrect the contents, it is necessary to cut open the glass vial, transfer the freeze dried contents to a vessel of resuscitating liquid and then transfer the resurrected micro organisms to culture medium. Some arnpules are very difficult to open, requiring filing in order to sufficiently weaken the glass so that the ampule can be broken. This presents risks of contamination of the culture through the introduction of contaminants through the filed area of the ampule, as well as risk of injury to the operator, should the ampule unexpectedly break. In addition, there is the risk of injury and inoculation from the broken glass (i.e., the operator may be cut on the edge of the broken glass and be inoculated with the organisms present in the ampule). This is of particular consideration if the organisms are pathogenic. Thus, there are major safety considerations associated with the use of current freeze drying methods and apparatus.

Further, as glass vials need to be cut, this means that they are not reusable, even with sterilisation, and need to be carefully disposed of according to the appropriate risk level procedures, adding further expense.

3 Further, for resuscitation of the micro organisms the freeze dried sample may need to be transferred to a further vessel containing resuscitating liquid, which further vessel requires labelling. Alternatively the resuscitating liquid may be added to the glass vial but this is undesirable as addition of liquid may wash glass shards present after cutting the ampule into the sample, and if for example a pipette is used to add liquid to the ampule, there is a risk that the pipette may become contaminated with the sample and therefore a separate pipette should be used for each sample, further adding to the expense.

Despite the number of methods available for preservation of microorganisms for transport, it is clear that improved methods are needed. Improved methods and devices should be economical, easy and safe to use and provide for long-term viability of preserved cultures.

It is an aim of a preferred embodiment of the present invention to provide a vessel for transporting micro organisms which obviates or mitigates at least one problem associated with the prior art, whether referred to herein or otherwise.

Accordingly, the present invention provides a vessel for transporting micro organisms, the vessel comprising a vial defining a first chamber and a cap assembly releasably attachable to the vial by vial engaging means, the cap assembly comprising an outlet which may be connected to a vacuum pump to expel air from the first chamber to lyophilise material therein, which outlet is sealable.

By providing the vessel as a two piece assembly of a vial and cap which are releasably attachable the requirement of prior art glass ampules to cut the vessel to gain access to material contained therein is removed. Instead the cap may simply be removed from the vial for access to material contained therein. This avoids the risk of injury to a worker from shards of glass produced on cutting the glass ampule and risk of contamination of the sample with such glass shards. Further, as the cap and vial are releasable attachable it would be possible to re-use the vessel, after suitable

4 sterilisation. This avoids the need for disposal of potentially biohazardous material and also reduces cost.

Preferably the vial comprises unbreakable material, for example plastics material. The plastics material is preferably sufficiently resiliently deformable to undergo vacuum treatment without cracking. Further the plastics material is preferably sufficiently rigid to prevent collapse of the chamber containing the sample prior to lyophilisation.

Making the vial out of plastics material reduces the risk of the vessel breaking during freeze drying or transport, as compared to glass ampules.

Preferably the cap comprises plastics material. The plastics material is preferably sufficiently resiliently deformable to undergo vacuum treatment without cracking. Further the plastics material is preferably sufficiently rigid to prevent collapse of the cap during vacuum treatment.

Preferably, and for safety reasons, the vial and cap assembly are constructed from conventional, preferably hard, plastic. However, in alternative, less preferred embodiments, any appropriate vial and cap assembly material, such as metal, or other materials may be employed. In the preferred embodiment, the vial and cap assembly are comprised of plastics material such as polypropylene, polyethylene, or another polymeric material. Preferably the vessel and particularly the first chamber of the vial is sterile.

It is contemplated that various vial formats will be used in the present invention. However, in one preferred embodiment, the vial is designed so that it will stand upright on a flat surface. In this embodiment, the bottom (i.e., closed end of the vial is flat. Nonetheless, it is contemplated that round-bottomed vials will also find use in alternative embodiments of the present invention.

Whilst it is not intended to limit the type of vial engaging means, it is preferred that the vial engaging means comprises threads. Alternatively, the cap may be of dimensions in relation to the vial so as to fit in sealing engagement in the vial, forming a "stopper".

One preferred embodiment of the vial engaging means includes an o-ring or other sealing device, either in the cap or on the vial to ensure sealing engagement between the vial and the cap.

Preferably the cap is tapered towards the outlet to aid in attachment of a vacuum pump to the cap. The outlet is preferably sealable by heating, although an outlet cap may be provided for sealing the outlet. The outlet cap may be threaded or of suitable dimensions to form a "stopper" in the outlet.

The cap assembly outlet is preferably is arranged to connect indirectly to a vacuum pump.

According to a preferred embodiment the vessel further comprises a sealable vent. The vent may be provided in the vial or the cap or may be provided in an insert which may fit in sealing engagement between the cap and vial of the vessel. The vent is provided to allow resuscitating liquid to be added to the vessel without the need to remove the cap from the vial, thus reducing the risk of aerosol contamination of the area surrounding the vial and also reducing the risk of contamination of the sample.

It is envisaged that the vent may take many forms. A preferred form of vent comprises a sealable outlet which may be opened by the user to allow liquid to be added to the vial without the need to remove the cap from the vial. According to a preferred embodiment, the vent may allow attachment to the vessel of a reagent dispenser. A suitable reagent dispenser may comprise a reagent chamber and a nib for hermatically sealing the reagent chamber, a portion of the dispenser being deformable to break or otherwise displace the nib to allow reagent contained in the dispenser to be delivered into the vial chamber.

6 According to a further preferred embodiment the vessel comprises immobilised desiccant within the cap. The term "immobilised desiccanC refers to the placement of desiccant within the cap of the vessel of the present invention such that the desiccant is retained in the cap. Thus, the desiccant does not come into contact with any micro organisms present in the vial.

It is contemplated that various desiccants may be used in the present invention. In one embodiment, the desiccant is selected from the group consisting of CaC12. CaO, NaOH, MgO, CaS04 (e.g., Drierite TM), H2S04, silica gel, Mg(C104)2, and P205. In an alternative preferred embodiment, the desiccant provides an atmosphere within the vial (i.e., when the vial and cap are engaged) that contains approximately 1-3% moisture. In a particularly preferred embodiment, the desiccant is silica gel.

The use of the desiccant within the vessel helps to maintain low moisture levels within the vial. The interior of the vessel is preferably provided with a dry biologically inert atmosphere. In some embodiments, the atmosphere is oxygen-free. This atmosphere aids in preservation of viable, dried organisms. Those skilled in the art are aware of many methods for producing such an atmosphere, including the use of vacuum. The use of desiccant in the cap also assists in maintaining a suitable atmosphere within the interior of the vial.

For added safety (i.e.., to prevent leakage), and to further prevent deterioration of the preserved organisms, a sheath or film may be used to seal the vessel. It is intended that the sheath or film be comprised of aluminium laminate or any suitable plastics material that is capable of tightly scaling the device and maintaining the vacuum. It is also contemplated that the sheath may either completely surround the vial and cap, it may surround only a portion of the vial and cap device, or it may surround only the area where the cap and vial attach.

According to a further embodiment the first chamber of the vial may comprise Iyophilisation media. The Iyophilisation media may comprise one or more cryoprotectants or may comprise bovine serum albumin, sucrose, fraction V, casein 7 peptone, soy peptone, NaCI, K2HP04, dextrose, trehalose, thioglycollate or ascorbic acid.

As used herein, the term "cryoprotectant" refers to compounds or substances which prevent damage to cells during the Iyophilisation or freezing process. It is intended that the cryoprotectants of the present invention include any compounds or substances that protect the cells from shearing or other mechanical damage that is possible during the preservation process. It is intended to encompass compounds and substances that prevent damage due to the formation of ice crystals during the freezing process. It is further intended that the term encompass compounds and substances that prevent damage that occurs during the rehydration (i.e., revival) of organisms.

As used herein, the term '1yophilisation medium" refers to the medium in which organisms are suspended prior to lyophilisation. In one embodiment, it is contemplated that cryoprotectants be included in the lyophilisation medium. It is not intended that the lyophilisation medium be limited to any particular formulation. Indeed, it is contemplated that various formulations will be successfully used in the present invention. For example, it is contemplated that the medium may contain compounds or enzymes such as oxygen-scavenging or other enzymes, anti-oxidants, and other substances that are suitable for use in lyophilisation media. As used herein, the term 9yophilisation solution" refers to lyophilisation medium within which microorganisms are suspended or contained.

According to a preferred embodiment the exterior of the vial is equipped with a labelling area for providing information about the dried microorganisms located within the vial. For example, this area allows for the inscription of the name (i.e., genus and species) of the organisms, their accession number (e.g., the number assigned to the particular culture by a culture collection), the date of preservation, etc. This labelling area is intended to provide convenience to both the operator preserving the culture.. as well as identify the culture for the operator who revives the culture. It is not intended that the labelling area comprise any particular size. The labelling area may be large or small, depending upon the amount of information needed to be 8 included on the vial. In addition, the labelling area may be an etched region, especially if on plastics material, or it may be an area to which a label may be taped or adhered.

According to a further preferred embodiment, for convenience and safety during the manipulation of the vessel the exterior of the vial is striated, such that it is easy to grip.

According to a second aspect of the invention there is provided a method of preserving micro organisms for transport using the vessel according to the first aspect of the invention, the method comprising:

(a) placing micro organisms in the vial; (b) attaching the cap assembly to the vial to provide a vial and cap combination; (c) attaching a vacuum pump to the cap outlet and expelling air from the first chamber to lyophilise the micro organisms; and (d) sealing the cap assembly outlet.

Preferably, the method according to the second aspect of the invention comprises the additional step of placing lyophilisation medium comprising one or more cryoprotectants in the vial either before, after or simultaneously with the micro organisms.

The method according to the second aspect of the invention may further comprise reviving the preserved micro organisms. In one embodiment, the reviving comprises adding rehydration medium to the preserved culture within the vial and cap combination.

As used herein, the terms "revival" and "rehydration" refer to the process of reviving the preserved micro organisms. While it is not intended that the present invention be limited to this method, rehydration is intended to encompass the addition of liquid or fluid to the preserved micro organisms within the vessel. The suspension of organisms so produced may then be used to inoculate suitable microbiological media.

9 The inoculated media are then incubated under conditions suitable for the growth of the organisms, and colonies of organisms present in the preserved culture observed.

In a preferred embodiment of the second aspect of the invention, the micro organisms are bacteria.

According to a third aspect of the invention there is provided a vessel according to the first aspect of the invention in which the vial and cap assembly are in sealing engagement, the vessel containing lyophilised micro organisms as produced according to the second aspect of the invention.

The vessel according to the third aspect of the invention is suitable for transport over long distances. The invention further provides for longer viability (i.e., longer shelf life of the cultures) than other methods commonly used for transport of dried cultures (e.g., cultures dried onto inoculating loops, such as Cultiloops TM [available from Carr Scarborough Microbiologicals, Inc., Stone Mountain, Ga.] or cultures dried onto swabs).

As used herein, the term "viability" refers to the ability of a culture to grow. For example, a "viable culture" is comprised of live organisms that are capable of metabolism and growth, while a "non-viable culture" is comprised of cells that are either dead or sufficiently damaged that they are not capable of good growth (i.e., no or abnormally small visible colonies are present), even under optimal conditions for their growth. It is recognised that in some circumstances, a viable culture is not capable of growing well or at all, on a given medium (e.g. microbiological media) under certain conditions, such that growth is visible to the eye. However, it is contemplated that placing such a culture in or on an appropriate medium (including cell cultures or living organisms, such as chick embryos), and under appropriate conditions, the organisms will be capable of growth.

As used herein, the term "shelf-life" refers to the time period during which a culture remains viable. For example, the present invention provides cultures that have long shelf lives. The preserved cultures maintain their viability for long periods of time, as compared to cultures preserved using other methods (e. g., weeks, months, or even years). The viability of the culture is measured following revival of the preserved organisms.

The term "sample" in the present specification is used in its broadest sense. On the one hand, it is meant to include a specimen or culture (i.e. , a "sample of microorganisms"). On the other hand, it is meant to include both biological (e.g., organisms isolated from clini cal specimens) and environmental samples (i.e., organisms isolated from the environment, rather than a living host). These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms.

Biological samples may be animal, including human, fluid or tissue, food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing instruments. apparatus, equipment, disposable, and non- disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.

Whether biological or environmental, a sample suspected of containing microorganisms may (or may not) first be subjected to an enrichment means to create a "pure culture" of microorganisms. By "enrichment means" or "enrichment treatment," the present invention contemplates (i) conventional techniques for isolating a particular micro organism of interest away from other microorganisms by means of liquid, solid, semisolid or any other culture medium and/or technique, and (ii) novel techniques for isolating particular micro organisms away from other microorganisms. It is not intended that the present invention be limited only to one enrichment step or type of enrichment means. For example, it is within the scope of the present invention, following subjecting a sample to a conventional enrichment means, to subject the resultant preparation to further purification such that a pure culture of a strain of a species of interest is produced. This pure culture may then be preserved using the vessel and methods of the present invention.

As used herein, the term "culture" refers to any sample or specimen which is suspected of containing one or more microorganisms. "Pure cultures" are cultures in which the organisms present are only of one strain of a particular genus and species. This is in contrast to "mixed cultures," which are cultures in which more than one genus and/or species of microorganism are present.

As used herein, the term "organism" is used to refer to any species or type of microorganism, including but not limited to bacteria, and fungi. As used herein, the term fungi, is used in reference to eukaryotic organisms such as the moulds and yeasts, including dimorphic fungi.

An embodiment of the present invention will now be described, by way of example only, with reference to the following drawings, in which:

Figure I shows a side cross-sectional illustration of a vial and cap combination according to an embodiment of the first aspect of the invention; Figure 2 shows a plan view of the vial and cap combination shown in Figure 1; Figure 3 shows a side cross-sectional illustration of a vial and cap combination according to a further embodiment of the first aspect of the invention; and Figure 4 shows a plan view of th e vial and cap combination shown in Figure 3.

Figures I and 2 show a vessel 2 according to one embodiment of the first aspect of the invention. The vessel 2 is made up of a vial 4 defining a first chamber 6 and a cap assembly 8 releasable attachable to the vial by exterior threads 10 on the vial engaging with interior threads 12 on the cap assembly 8. The cap tapers towards an outlet 14 which may be connected to a vacuum pump (not shown) to expel air from 12 the first chamber 6 to lyophilise material therein. The outlet 14 is adapted so as to be scalable, preferably by heat.

From Figure 2 it can be seen that the vial 4 and cap 8 are circular cylindrical and that C1 the outlet 14 is open.

Figures 3) and 4 show a vessel 2 according to a further embodiment of the first aspect of the invention. As shown in Figures 3 and 4, in one embodiment of the cap, the underside of the cap 8 includes about its periphery a rim16 of such dimensions in relation to the vial 4 to sealingly engage the mouth of the vial 4. The underside of the cap 8 further comprises a protruding circular lip 18. Lip 18 together with rim 16 define a groove 20 for receiving an elastomeric or plastics material oring, washer or other sealing device for co-operation with the mouth of the vial to ensure sealing engagement between the vial 4 and cap 8. Lip 22 also defines on the interior thereof an area in which desiccant material may be immobilised.

Figures _33 and 4 also show a vent 24 which is provided on the cap 8. The vent 24 provides access to the chamber 6 without having to separate the vial 4 from the cap 8. As can be clearly seen from Figure 4 a removable stopper 26 seals the vent 24.

In Figures J3) and 4 the vessel 2 is shown containing lyophilised micro organisms 28 within the chamber 6 and with the cap outlet 14 sealed. The vessel 2 is Figure 3) also shows a labelling area 30 on which information concerning the micro organisms contained in the vial may be written.

EXAMPLE

Preparation of Mist Dessicans METHOD - USING GLASS FILTRATION APPARATUS 1. Wherever possible, sterile filtration apparatus must be prepared at least the day before preparation of mist dessicans. Assemble apparatus as shown in Figure 1 - 13 Plug side vent with cotton wool. Wrap apparatus in autoclave bags so that upper and lower sections can be unwrapped separately. Secure bags in place with autoclave tape.

2, Autoclave at 12CC for 15 minutes, then place in oven to dry.

3. Take 10OmI bottle (or more if required) frozen horse serum from the cold store. Thaw by standing bottle(s) in warm water.

4. Add 333mI sterile nutrient broth to every 10OmI horse serum (thawed) used, mix thoroughly.

5. Weigh out 10g glucose per 10OmI horse serum. Add glucose in small amounts, Zn shaking until all glucose has been dissolved. (NB. shaking is required to prevent formation of solid Iumps"). The resulting solution is "mist dessicans".

6. Take filtration apparatus and remove wrapping from upper section only. (NB. Filter tap must be kept covered until used). Carefully clamp it vertically in a retort stand.

7. Pour mist-dessicans from 5 into top of filtration apparatus using a small funnel.

8. Connect pressure tubing to compressed air tap with the other end connected to top of filtration apparatus.

9. Switch on pump. Open air tap.

10. As mist dessicans is forced through filter into reservoir, remove tubing and add more to upper section. Care must be taken not to let all the mist dessicans pass through the filter. A layer approximately 2cm deep must always be kept above filter.

14 11. When all mist dessicans has been added and only approximately 2cm remains switch off tap and remove tubing from filter apparatus.

12. Carefully transfer filtration apparatus (still in stand) to a safety cabinet.

13. Remove wrapping from lower section of filter apparatus.

14. Dispense approximately Iral through tap into sterile nutrient broth. Label "First ml-. Dispense remainder of mist dessicans into sterile vessels (in approximate volumes) except for final ml. Dispense this into nutrient broth as before.

15. Dismantle filter apparatus. Discard membrane filter, filter paper and cotton wool plug. Wash remainder in water and allow to dry in oven.

16. Plate out a drop from each of the broths in 14 onto nutrient agar plates.

17. Incubate broths plates and mist dessicans at 3TC for 3 days. If turbidity occurs in the broths or the mist dessicans (in these, slight precipitation is not uncommon) the batch of mist dessicans should be discarded.

18. If no contamination in broths, on plates or in mist dessicans is detected, transfer mist dessicans to 25'C incubator until required.

19. Cheek broths and plates at 3TC after 7 days.

METHOD - USING DISPOSABLE CLYDE FILTER 1. Take 1 0OmI bottle (or more if required) frozen horse serum from the cold store. Thaw by standing bottle(s) in wann water.

2 Add 3333mI sterile nutrient broth to every 10OmI horse serum (thawed) used, mix thoroughly.

3. Weigh out 1 Og glucose per 1 0OmI horse serum. Add glucose in small amounts, shaking until all glucose has been dissolved. (NB. shaking is required to prevent formation of solid "lumps").

4. In safety cabinet remove filter ftom bag.

5. Tighten (twist) the syringe into "T" connector.

6. Insert the hose from the filter into mist dessicans.

7. Pull the syringe plunger gently until the barrel fills with mist dessicans.

8. Remove the blue sterility sheath from the tip of syringe (NB Do not touch the outlet of the filter).

9. Dispense approximately ImI into sterile nutrient broth by pushing plunger gently down, forcing mist dessicans through filter outlet. Label 1 st ml". With plunger, repeat filling and discharging the mist dessicans to dispense the remainder (except final I ml) into sterile vessels in appropriate volumes. Dispense the final I mi into nutrient broth as before.

10. Discard filter 11. Plate out a drop from each of the broths in 9 onto nutrient agar plates. 12. Incubate broths plates and mist dessicans at 3TC for 3 days. If

turbidity occurs in the broths or the mist dessicans (in these, slight precipitation is not uncommon) the batch of mist dessicans should be discarded.

133. If no contamination in broths, on plates or in mist dessicans is detected, transfer mist dessicans to 25'C incubator until required.

16 14. Check broths and plates at 30'C after 7 days.

Centrifugal Freeze Drying Method 1. INTRODUCTION

NCIMB cultures are normally freeze-dried in a two-stage process. The first stage (primary drying) is carried out in a centrifugal freeze dryer, where the liquid cultures are centrifuged at low speed to prevent frothing while the drying chamber is being evacuated. Centrifuging is stopped when the cultures have frozen (as a result of the drop n vapour pressure as the dryer is evacuated). Water vapour removed during drying is trapped in a refrigerated condenser. Cultures are then transferred to a secondary vacuum dryer, where further water is removed overnight with a phosphorous pentoxide water trap. Although only the primary stage involves centrifugation, the whole process is referred to for convenience as centrifugal freeze drying.

2. MATERIALS/EOUIPMENT Centrifugal freeze-dryer Mist desiccans Secondary stage vacuum dryer Cultures for drying Ampule constrictor Scissors Larninar flow cabinet Plug pushing tool Sterile Pasteur and 2ml serological pipettes Sealing torch Prepared ampules and cotton plugs HT vacuum tester 3. METHOD Primary drying I. Switch on freeze-drier.

17 2. Spin down any liquid cultures.

Assemble pipettes, vessels, mist desiccans and cultures in the laminar flow cabinet, where all vessel filling must be done.

4. Using 2ml pipettes, wash the growth from slopes with 2-4 ml mist desiccans, pooling as you go, return the suspension to the mist desiccans bottle and replace the cap.

5. Liquid cultures should be centrifuged and resuspended in mist dessicans. 6. Take up the suspension in a Pasteur pipette to within I 2cm of the cottonwood plug. Dispense culture into vessels as in 7 below, re-capping each vessel as you go.

7. a) For standard cultures, dispense 4 or 6 drops into each vessel, taking care not to touch the sides as you insert and withdraw the pipette.

b) For QC cultures, ensure the suspension is absorbed by the thick paper strip. Therefore, dispense enough to soak the strip but do not leave any excess in the vessel.

8. Load the vessels on to the centrifuge plate of the freeze-drier, balancing each vessel with another placed directly opposite.

9. a) For standard cultures, put the plate into the drying chamber, replace the lid, switch on the centrifuge and evacuate the chamber.

b) It is not necessary to centrifuge QC cultures because all liquid has been absorbed by the paper strips.

18 10. After 20 minutes, switch off the centrifuge. Leave the cultures to freeze dry for hours.

11. Remove the vessels from the freeze-drier.

Second!a dryin 1 Working at the bench, and flaming the mouth of each vessel as you remove its cap, change the lint caps on the vessels for cotton wool plugs, inserting each plug about 1.5cm into the vessel and removing the steel rod.

2. Cut off the plugs flush with the mouth of the vessel and push them down with the special tool (final cotton plug size: approx. 15 mm).

Switch on the secondary stage drier.

4. Constrict the vessels. 5. Note the pressure in the secondary dryer. Ensure that manifold nipples are adequately greased and attach the vessels, turning them gently clockwise to ensure a good seal. Blank off any spare nipples with vessel stubs.

6. When the vessel bank is full, open its valve and check that the required vacuum is achieved (it should be less than 2x10-2 mbar). If not, check the tightness of each vessel in turn.

7. Take a second pressure reading at least 1 hour after opening all valves to ensure drying is progressing satisfactorily.

8. Leave vessels on the drier overnight.

19 9. Record time and pressure, then seal off the vessels with the gas torch attached to the dryer. The flame should be 3-pointed, blue and approx. 2in high. Adjust the gas and air valves of the burner to achieve this. Keep checking the pressure in the drier while you are scaling off.

10. When all vessels have been sealed off, switch off the drier. 11. Check the vacuum in the ampoules with the HT sparker attached to the drier.

Hold the tip of the sparker close to the vessel without touching it. If the vessel glows violet the vacuum is OK. If it does not, the vacuum is unacceptable and the vessel must be rejected.

Freeze drying strips INTRODUCTION

Preparation of freeze-dried QC culture strips containing defined low numbers of organisms.

METHOD A J27'C organisms: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Clostridium sporogenes 1. Inoculate from liquid nitrogen seed stocks 100 nil growth medium CM I (BH1 for 3 7'C for c. 24 h.

Clostridium sporogenes) and incubate at) 2. a) Harvest by centrifugation all broths.

b) Resuspend pellet in 10 ml MRD (Maximum recovery diluent). c) Prepare a dilution series, (initial dilutions in MRD, critical test dilutions must be conducted in mist desiccans) and store at 2-5T.

d) For each organism carefully pipette 100 Vtl of each test suspension (3 appropriate serial dilutions) into vessels (without test strips). Deposit each 100 RI aliquot at the base of the vessel without touching the sides.

e) Freeze dry, resuscitate, count and incubate plated serial dilutions of these vessels overnight a) Prepare a fresh dilution series, from the original suspension refrigerated the previous day. (From these counts, an estimate of nearest most suitable dilution. or adjustment thereof, necessary to achieve final numbers of 20-150 organisms per final freeze-dried vessel (minus strip) inoculated. b) Fill all batch vessels by carefully pipetting 100 ttl onto filter strips. C) Prepare 5 extra naked glass ampoules for post count and internal QC procedures.

4. "Resuscitate with: ml MRD, I naked glass ampoule per organism; plate and incubate as in I (e).

5. Examine post freeze-drying batch counts and pass or reject product if<20 or > 15 0; or if contaminated. B 33 O"C organisms: Bacillus subtilis, Candida albicans, Aspergillus niger I. Inoculate 100 ml growth media (CM 1 for Bacillus subtilis; Difco malt extract broth for Candida albicans) or 2 Difco malt extract agar plates (for Aspergillus niger) and incubate at 30T over weekend. 2. As 2 above protocol for 37T organisms. Incubate counts for 48 hr at 300C. I 3. As 3) above protocol for 37T organisms, except for incubation temperature. 4. As 4 above protocol for 337T organisms except for incubation temperature.

Incubate at 20T over weekend.

21 5. Carry out counts.

SuRgested test dilutions for each organism (of resuspended pellet) NCIMB Number " 4 Escherichia coli: -5,-6,-7 500) Pseudomonas aeruginos -5,-6,-7 50067 Staphylococcus aureus -5,-6,-7 50080 Clostridiunisporogenes -3, -4, -5 50099 Bacillus subtilis -3,-4,-5 50090 Candida albicans -4,-5,-6 50010 Aspergillus niger -3,-4,-5 50097 Resuscitate by resuspending contents of vessel in:!1.0 ml of appropriate liquid growth medium or MRD, allow to stand for 10 min, agitate strongly by hand and spot plate out onto a well dried appropriate agar plate.

For Clostridiunisporogenes counts need to be performed using pour plates of fastidious anaerobe agar (without added blood). Incubate in a thriceflushed anaerobic jar containing anaerobic gas mixture plus catalyst. Strict anaerobiosis for incubation is essential.

Preparation of Quantitive Freeze Dried Cultures:

The final number of cfu after freeze drying is determined by both the resuscitation method in particular the volume of resuscitation medium and the counting technique used i.e. spotting spread or pour plate.

Standard Method 22 1. Bottles etc. should be prepared and sterilised. Enough bottles, etc. should be prepared to allow adequate post drying checks and in-house R&D.

2. Grow up adequate amounts of the organisms required. Thawed pre-frozen stocks can also be used.

3. Prepare a suspension of the organism from the culture. A set number of slopes and a constant volume of suitable diluent should be used. If the organism is grown in liquid culture then it should be harvested by centrifugation and then resuspended in a known volume diluent.

4. Prepare a serial dilution using the suspension prepared in (4) above. If a thawed frozen suspension is used then the dilutions can be made directly from this.

5. Fill a few bottles etc. with the required amount of the appropriate dilutions.

This is usually lml or 10Oul per bottle and 10Oul per vessel (with or without filter paper). Dilutions to be freeze-dried should be made in mist dessicans or other suitable freeze drying base.

6. Freeze dry. 7. After drying reconstitute the dilutions in the appropriate amount of resuscitation medium. Perform counts on these dilutions using the appropriate technique i.e. spot, spread or pour plate. For spread and pour plate methods the clients procedures should be used. From the results obtained determine the dilution that gives the required number of cfu after drying.

8. Using the dilution determined in (7) fill enough bottles etc. to satisfy the total number required (see 2) with the appropriate volume of that dilution. Freezedry. The original suspension (see (4)) may be used provided it is stored refrigerated. alternatively another suspension may be prepared from a fresh 2 '33 culture provided it has been grown under identical conditions and the same volume of diluent is used to prepare the suspension.

If the dilution factor has been already determined for the organism concerned (5) (6) and (7) may be omitted.

9. Freeze dry. 10. When freeze drying is complete reconstitute and perform counts as in (8) above. Aliquots of the reconstituted cultures should also be tested for purity; usually colony morphology is sufficient.

OD Method 11. Follow (1) to (3) above. 12. Make a suspension using the freshly grown culture (or thawed pre-frozen stocks) in 0.85% saline (18ml in BIOLOG tubes). Using the BIOLOG turbidimeter adjust the optical density of this using sterile saline (or more culture) until a transmission of 35% is obtained for the suspension.

Prepare a dilution series from the suspension obtained in (12) according 14. Freeze dry appropriate dilutions and perform counts as in (5) (6) and (7).

15. Prepare the batch as required (see (8)) and freeze dry (see (9)).

16. Perform counts check for purity and record results as in (10) (11) and (12).

17. If the appropriate dilution to give the required cfu after drying has already been determined then after preparing the suspension at 35% transmission the 24 final batch can be prepared from it directly without the need to do a pre- run i.e. omit (16).

As indicated above, cultures of the organisms were grown on suitable media, placed in the first chamber of the vessel according to the first aspect of the invention and the cap attached to the vial. Primary centrifugal freeze drying is carried out according to the method described above. The cap outlet is then attached to a vacuum pump and freeze drying carried out according to the method described above. The vessel is them placed into an aluminium laminate sleeve. The vessel is then sealed with the gas torch attached to the dryer.

Such freeze dried micro organisms may be transported in the vessels according to the first aspect of the invention. The freeze dried micro organisms may be tested for their viability at monthly intervals by adding resuscitating liquid through the vent of the vessel and plating the resuscitated micro organisms on suitable media for colony counts. It is expected that the micro organisms may be stored effectively in the vessel of the first aspect of the invention for at least one year and most probably for over two years.

Claims

Claims:

1 A vessel for transporting micro organisms, the vessel comprising a vial defining a first chamber and a cap assembly releasably attachable to the vial by vial engaging means, the cap assembly comprising an outlet which may be connected to a vacuum pump to expel air from the first chamber to lyophilise material therein, which outlet is sealable.

2. A vessel according to claim 1 in which the vial comprises plastics material.

A vessel according to claim 1 in which the cap comprises plastics material.

4. A vessel according to claim 2 or claim 3 in which the plastics material is sufficiently resiliently deformable to undergo vacuum treatment without cracking.

5. A vessel according to claim 2 or claim -3) in which the plastics material is sufficiently rigid to prevent collapse of the chamber containing the sample prior to lyophilisation.

6. A vessel according to claim 1 in which the vial engaging means comprises threads.

7. A vessel according to claim 1 in which the cap is tapered towards the outlet to aid in attachment of a vacuum pump to the cap.

8. A vessel according to claim 1 further comprising a sealable vent.

9. A vessel according to claim 1 further comprising immobilised desiccant within the cap.

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10. A vessel according to claim 9 in which the desiccant is selected from the group consisting of CaC12, CaO, NaOH, MgO, CaS04, H2S04, silica gel, 1 M.(),'(C104)2, and P205.

11. A vessel according to claim 9 in which the desiccant provides an atmosphere within the vessel that contains approximately 1-3% moisture.

12. A vessel according to claim 1 further comprising a sheath capable of sealing the vessel.

13. A vessel according to claim 1 in which the first chamber of the vial further comprises lyophilisation media.

14. A vessel according to claim 13 in which the lyophilisation media comprises one or more cryoprotectants, bovine serum albumin, sucrose, fraction V, casein peptone. soy peptone, NaCI, K2HP04, dextrose, thioglycollate or ascorbic acid.

15. A vessel according to claim 1 further comprising a labelling area on the vial.

16. A vessel according to claim 1 in which the exterior of the vial is striated.

17. A method of preserving micro organisms for transport using the vessel according claim 1, the method comprising:

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18. A method according to claim 17 further comprising the additional step of placing lyophilisation medium comprising one or more cryoprotectants in the vial either before, after or simultaneously with the micro organisms.

19. A method according to claim 18 further comprising the additional step of reviving the preserved micro organisms.

20. A method according to claim 18 in which the micro organisms are bacteria.

21. A vessel according to claim 1 in which the vial and cap assembly are in sealing engagement, the vessel containing lyophilised micro organisms as produced according to the method of claim 17.