JP2003505024A - Microbial, cell, and tissue storage - Google Patents

Abstract

  (57) [Summary] Compositions for preserving living microorganisms, cells or tissues comprise (a) a preservative combination of (i) a non-reducing disaccharide and (ii) a filler; and (b) a buffer. Also described is a method of preserving viable microorganisms, cells or tissues, the method comprising combining viable microorganisms, cells or tissues with a storage solution comprising a non-reducing disaccharide, drying the combination to at least 80 wt. Forming a dry preparation having a% solids content, and counting viable microorganisms, cells, or tissues in the dry preparation, wherein the dry preparation provides a predetermined count of viable microorganisms, cells, or tissue. Which can be combined with an aqueous buffer to obtain an aqueous preparation comprising

Description

Detailed Description of the Invention

The present invention relates to the storage of microorganisms, cells and tissues. The invention has particular applicability in the storage of a wide range of such biological materials, as well as in particular in the storage of those biological materials that degrade in aqueous solutions at room temperature. . The invention in its most preferred aspect relates to the storage of microorganisms, cells and tissues in a living state.

A wide variety of biological reagents are known to degrade rapidly when stored at room temperature, enhancing the adoption of a range of methods for storage and preservation of such reagents. There is. Known methods include simple refrigeration as well as storage at ambient temperature after lyophilization. In some cases, effective storage methods are not available. Instead, an assessment of sample degradation can be made between the preparation and the analyte, thereby estimating the original composition of the sample.

Methods for preserving microorganisms, including bacteria, yeast, and fungi, using storage beads are known. A suspension of microorganisms is made in a cryopreservative and then mixed with storage beads. The cryopreservative is removed and the beads are frozen and stored, typically at a temperature of -70 ° C. To recover the microorganisms, the beads are removed from the frozen storage and either placed in broth or rolled on growth medium. This method has various drawbacks. A storage temperature of -70 ° C requires specialized refrigeration equipment. Freezing necessarily results in significant physical damage to microorganisms. Special cryopreservatives are required, which can be expensive. Finally, the beads are typically 2-4 mm in diameter and are awkward to handle.

In some known examples, it is necessary to obtain viability counts from a water sample that probably contains bacteria. Currently, this is done routinely by transporting the test sample in solution from where it is obtained to where it is counted. It is a known phenomenon that during transport, the number of viable bacteria in a sample decreases over a period of time. To address this, bacterial counts are performed very accurately and at predetermined time intervals after the sample is taken. The count is then adjusted according to the number of reductions calculated since the sample was taken. The inherent disadvantage of this existing method is that it is accurate in view of the risk of breakage of the container containing the bacterial sample and the known rapid deterioration of the viability of the bacterial sample over time. The inconvenience is that the operation is forced on different time scales.

EP-A-0229810 describes the protection of macromolecules such as proteins using a solution of trehalose. However, it does not mention that there are special difficulties with the storage of microorganisms.

It is widely recognized that food safety is highly dependent on bacteriological methods capable of detecting small numbers of pathogenic bacteria in both raw and processed materials. Sub-infectious dose contamination can quickly become dangerous given suitable growth conditions such as rehydration of dry products or exposure of wet products to elevated ambient temperatures.

[0007] In food products, one of the problems controlling the performance of routine microbiological tests to test for the presence or absence of marker organisms is to provide appropriate standardized controls. Many laboratories rely on wet suspensions cryopreserved on beads at -20 ° C for several weeks or broth refrigerated at 4 ° C and refreshed weekly. Inoculum is prepared by making constant dilutions from stored suspensions, but the actual counts obtained can vary widely depending on the density and viability of the organism. Fresh grown bacteria are usually less damaged than cells that have been quiescent for some time and, as a result, have a higher plate efficiency. But,
Marker organisms of interest to the public health and food industries are often non-replicating and damaged. Furthermore, they are subject to thermal stress during the manufacturing process.

US-A-5733774 describes a method and a composition for producing a stable bacterial formulation, which comprises a step of drying bacteria, which is combined with a powder or granular non-aqueous carrier. And the step of packaging the bacteria in a sealed container that is impermeable to gases and water vapor, and the method also includes removing substantially all oxygen from the interior of the container. However, this method is impractical in that it requires laborious efforts to remove gases and water vapor and oxygen from the stored material. Moreover, prolonged survival of stored bacteria has not been demonstrated.

WO-A-90 / 04329 describes storing mammalian cells in a process which comprises the steps of freeze-drying mammalian cells and storing them at about 4 ° C. The freeze-drying process poses a significant risk of loss of stored material viability.

[0010] WO-A-99 / 00485 discloses a composition for preserving microorganisms using starch optionally mixed with albumin.

The present invention aims to overcome or at least alleviate the problems associated with the art and to provide improved storage of biological materials such as microorganisms, cells and tissues.

Accordingly, it is an object of the present invention to provide a method of storing biological material that does not require refrigeration below 0 ° C. and does not require freezing of the stored material.

Another object is a solid state stored biological material which is acceptably stable and suitable for long-term storage at 4 ° C., wherein the solution of biological material is easily prepared. To provide a biological material that can be reconstituted into.

A further object is to without excessive damage to biological material in transit,
Providing stored biological material suitable for transport, including via the mail system.

A more specific object of the present invention is to provide a method of storing microorganisms, cells and tissues in a long-term viable state.

Accordingly, in a first aspect, the present invention provides a method of preserving viable microorganisms, which method comprises drying an aqueous preparation of viable microorganisms, cells, or tissue in the presence of a non-reducing disaccharide. Forming a dry preparation, counting viable microorganisms, cells, or tissues in the dry preparation to obtain a counted dry preparation, and storing the counted dry preparation. The dried preparation, when reconstituted with water or an aqueous medium, forms an aqueous preparation containing a predetermined number of viable microorganisms, cells, or tissues.

In a second aspect, the invention provides a composition for preserving viable microorganisms, cells or tissues, the composition comprising: (a) (i) a non-reducing disaccharide and (ii) A combination of a preservative with a filler; and (b) a buffer.

In a third aspect, the invention provides a method of preserving viable microorganisms, cells or tissues, which method comprises: (a) treating the microorganism, cell or tissue with a composition of the invention. In combination to form a stock preparation; (b) drying the stock preparation at or above the triple point of water; and (c) storing the resulting dry preparation.

[0019]   The advantage of the present invention is that biological materials such as microorganisms, cells or tissues are stable
It can be stored in the form for a long time, but retains significant viability. Features of the invention
Certain embodiments provide individual dry preparations of bacteria suitable for storage, which
It is easily reconstituted with water or other aqueous buffer to obtain viable bacteria. Therefore, 25
A μl drop of bacterial suspension formed a dry weight of 8 mg of lenticule, which was 10 to 10 ⁸ Includes a single organism or mixture of CFUs. It is reconfigured and used in 10 minutes
obtain.

A further advantage of the present invention over other methods of preserving microbial viability is that viable microorganisms, cells, or tissues are preserved due to the extended viability of the stored material in the compositions of the present invention. A sample of biological material having a predetermined, known number of can be prepared. Thus, the sample may have a defined number of viable organisms per sample stored. Thus, they can be used in quantitative experiments and in situations where the presence or absence of very few marker organisms is a rigorous test of the method to be controlled.

A defined number of preparations containing bacterial numbers in the range of 10 cfu to 10 ⁸ cfu have been prepared according to the invention, stored and reconstituted without significant loss of viability.
Confidence limits for cfu counts have been established by performing survival counts on multiple lenticles. Counts are usually within 95% confidence intervals, even for very low count preparations. The present invention provides a versatile alternative to maintaining bacterial cultures on either slope, 4 ° C wet suspension, cryopreservation at low temperature, freeze-drying, or spray-drying.

In use of the present invention, a sample of biological material comprises (a) (i) non-reducing disaccharide and (ii)
A combination of a preservative with a filler; and (b) a buffer. The non-reducing disaccharide: filler weight ratio is suitably 5: 1 to 0.5: 1, preferably
It is about 3.5: 1 to 1.5: 1 and the solids content is generally at least 20% by weight.

The non-reducing disaccharide can be selected from the group consisting of trehalose, sucrose, maltose, lactose, cellobiose, isomers thereof, and mixtures thereof.
Preferably trehalose or a combination of trehalose with other non-reducing disaccharides is used.

In the compositions of the present invention, the bulking agent is typically or comprises a high molecular weight protein. Preferably, the bulking agent is a matrix protein having a molecular weight of 100 kD or less and preferably in the range of 50-100 kD.
Most preferably, the matrix protein is a relatively inactive globular protein such as albumin. Gelatin (normally having a molecular weight in the range 170-200 kD) is not suitable. Suitable albumins include ovalbumin, fetal bovine albumin, and lactalbumin. In an embodiment of the invention the matrix protein is serum albumin, more particularly bovine serum albumin. Matrix protein is conveniently 5 to 2 per 100 ml.
0 g, preferably 8-12 g, and more preferably about 10 g per 100 ml.
Is present in the solution.

The buffer should include components that are selected to maintain the pH of the solution at a value at which the microorganism, cell, or tissue is stable, and that will not selectively salt out as its concentration increases in the drying process. Is. One suitable buffer is phosphate buffered saline (PBS) at a concentration of 0.1-1M, preferably 0.15-0.5M.

The above components together form a solution which, when dried, yields a solid pellet, typically a flexible pellet containing the stored biological material.

[0027]   A particular embodiment of the invention is   (I) 5 to 30% by weight of non-reducing disaccharides;   (Ii) 1 to 10% by weight of filler; and   (Iii) 60 to 94% by weight of aqueous buffer.

The composition may further comprise a monosaccharide, for example a reducing sugar such as glucose.
Monosaccharides may be present at 0.1-3% by weight.

Further optionally included within the composition is a structural additive, which can be or can be a water soluble carbohydrate polymer. Carboxymethylcellulose is used in embodiments of the invention, but other additives such as hydroxyalkyl cellulose are also suitable.

It is preferred that the structural additive be water soluble and all other components be water soluble so that the dry composition of the present invention will dissolve substantially completely when reconstituted with water. A clear solution. Insoluble or partially soluble sugars and fillers are preferably avoided. Thus, starch that does not dissolve completely or dissolves into a hazy preparation is preferably avoided.

[0031]   The colorant is also optional.

The methods and compositions of the present invention are particularly suitable for the storage of microorganisms selected from the group consisting of bacteria, viruses, protozoa, and fungi.

The solids content of the composition according to the invention is initially generally in the range 10-50%.

When the biological material is combined with the composition of the invention, it is dried above the triple point of water and preserved by storing the resulting dry preparation. The drying step can easily be carried out at atmospheric pressure and good results have been obtained in the examples of the invention described in more detail below. Vacuum may also be used in the step of drying the composition.

Drying can also be carried out by passing air, an inert gas, or a mixture thereof over the storage preparation in the presence of a desiccant. Silica gel may be used and other desiccants may be used. The use of a substantially oxygen free environment is also optional.

In a further embodiment of the present invention, the method comprises: (i) passing air, an inert gas, or a mixture thereof over said preservation preparation in the presence of a desiccant, Partially drying the stock preparation; and (ii) drying the partially dried stock preparation at a further reduced temperature.

In a particular embodiment of the invention, the method comprises the step of placing the storage preparation on a hydrophobic surface before drying. Preferably, a small amount of liquid is placed and dried,
It forms a small solid composition, called a pellet or lenticule. Once dried, the pellets are very convenient to handle. The volume of stock preparation that makes up the small volume can range from 5 to 100 μl.

In a particular embodiment, the method of the invention applied to bacteria is to inoculate a culture in support medium and incubate optimally until the early induction phase. On a standard 90 mm plate, the typical yield is 10 ¹¹ colony forming units (CFU). This growth is taken up in a small volume of hypotonic saline and mixed with 2.5 ml of solution. 25 μl spots are distributed on a hydrophobic surface and dried above the triple point. 100 or the primary so-called lenticules thus obtained each contain about 10 ⁸ CFU and are stored under dry at about −20 ° C. above the eutectic point.

Strains conserved according to the present invention include Staph. Aureus, Staph. Epidermi
dis, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Str. fa
ecalis, Str. pyrogenes, Str. agalactia, Str. pneumonia, Enterococcus fae
cium, Cl. perfingens, Sacchromyces, E coli (including O157), Salmonella sp
, Ps aeruginosa, Klebsiella sp, Proteus sp, Campylobacter sp, Helicobact
er pylori, Vibrio parahaemolyticus, Acenitobacter sp, Serratia marcesans
, Haemophilus influenzae, Bordetella pertussis, N. menigitidis, N. gonor
rhea, Bacteroides sp, and Salmonella gold coast. Mycoplasma, viruses, and fungi can also be preserved by the present invention.

Compositions of the invention may be, for example, (1) on a parafilm or similar hydrophobic surface; (2) free; and (3) directly dried in microtiter strips, many For all of these, storage at −20 ° C. in the dry aids in extending the shelf life.

In a further aspect of the invention there is provided a dry composition of the invention further comprising live microorganisms and having a solids content of at least 80%. The viable microorganisms are preferably bacteria, or a mixture of different strains of bacteria, or a combination of bacteria and mammalian cells.

This aspect of the invention also provides a dry composition having a defined microbial count, which microbial count remains substantially stable upon storage at ambient temperature. A defined count means that the content of the dry composition is known or defined, either directly or indirectly, and so reconstitution with a buffer gives an aqueous preparation with a defined count of viable microorganisms. Means that.

When reconstituted with water or an aqueous buffer solution, it is preferred that the composition is sufficiently dispersible—that is, all components are water soluble and readily dispersible.

The method for preserving live microorganisms, cells or tissues of the present invention comprises: (a) combining live microorganisms, cells or tissues with a preservative solution containing a non-reducing disaccharide; (b) said (a). Drying the combination of to form a dry preparation having a solids content of at least 80% by weight; and (c) counting the viable microorganisms, cells, or tissues in the dry preparation. Thereby, the dry preparation can be combined with an aqueous buffer to obtain an aqueous preparation containing a predetermined count of viable microorganisms, cells, or tissues.

To calculate the viability count, at least a first and a second dry preparation are formed, viable microorganisms, cells, or tissues in the first dry preparation are counted, and thereby the first dry preparation. It is preferred to estimate the number of viable microorganisms, cells or tissues in the dry preparation of 2. Therefore, counting is done indirectly.

When a large number of such dry preparations are made, the method counts selected samples of the dry preparation to determine the viable microorganisms, cells, cells, etc. in the remaining dry preparations.
Alternatively, it may include the step of estimating a tissue count.

The drying step can be carried out in air or an inert gas or mixtures thereof. 1
In one embodiment, the drying step is carried out in an atmosphere of reduced oxygen concentration (compared to air), the advantage of which is that the oxidation of biological material during the drying step is reduced. In another embodiment, the drying step is performed in a reduced humidity atmosphere. In a further embodiment, the pressure is reduced during drying.

The drying step may conveniently be carried out at room temperature and may also be carried out at temperatures down to about 4 ° C. Higher drying temperatures may also be used, but these must take into account the increased degradation of biological material caused by the elevated temperatures.

The drying step is intended to obtain a solid composition comprising biological material and preferably having a water content in the range of 10% by weight or less. The water content is preferably in the range of 4-8% by weight, and most preferably about 6% by weight ("about" is ± 2).
Give a tolerance of%). As will be appreciated, the ideal moisture content of a solid stored composition depends on the nature of the material being stored.

After this method has been used to produce a solid composition containing biological material,
It can be stored either at room temperature, or conveniently at refrigeration temperature, typically about 4 ° C.

Dissolving a stored solid in water or a buffer solution to reconstitute the active biological material from the stored solid is a fairly straightforward procedure.

In an embodiment of the invention the storage method is carried out under sterile conditions. Similarly, in an embodiment of the invention, reconstitution of an aqueous solution of biological material is performed with sterile water or sterile buffer.

The present invention is applied to biological materials that cannot be stored in aqueous solution at room temperature indefinitely. Biological material should be stored at ambient temperature (20 ° C) for up to 5 days,
It can be considered to have no acceptable stability, especially if it has a half-life of 2 days or less. Particularly unstable biological materials are those with a half-life of 24 hours or less.
Examples of biological materials suitable for storage according to the present invention include microorganisms, including bacteria and viruses, cells, and samples of biological solutions such as serum samples.

In an embodiment of the invention this storage method is used to store a biological sample. For storage of bacteria, the storage solution optionally contains a membrane stabilizer prior to drying. Suitable membrane stabilizers include glycerol, egg yolk, and mixtures thereof. Storage of microorganisms such as bacteria using the method of the invention is particularly advantageous when the bacteria are in solid storage form, as they are easy to handle and to transport, including by mail service. Bacteria in the storage form of the present invention are relatively stable, and this allows viability counts to be obtained over a more flexible time scale than previously possible.

In a preferred embodiment of the invention, the method for storing bacteria comprises the step of providing a nutrient medium for the stored bacteria.

A first solution of matrix protein and mono- or disaccharide in 100 ml of buffer can be prepared according to the present invention. If necessary, a small amount of peptone in the range of 0.01 to 1 g is added.

A second solution can be prepared that acts as a nutrient medium for the bacteria. This nutrient medium routinely contains nutrients essential for bacterial growth / survival. In addition, a bacterial membrane stabilizing component may be included, if desired. Bacteria are grown / cultured, then pelleted and mixed with a small amount of nutrient medium. The nutrient medium containing a small amount of (1) viable bacteria is mixed with a larger amount of stock solution than (2) and dried to obtain a residual solid having a water content of about 4-8% by weight.

To reconstitute a viable bacterial sample, the solid is dissolved in water or a buffer or nutrient solution.

In another embodiment of the invention, the preparation of biological material (B) to be stored is mixed with the solution (S) of the invention in a B / S ratio of 1: 5 to 1:10. To be done. 10-50 μl
The volume of the mixed solution in the amount of
Alternatively, they are placed in microtiter strips. This results in a small volume of separated liquid. At this stage, the biological material is in the stabilizing solution in a predetermined volume of droplets containing the desired volume of biological sample. The separated volume is then dried to obtain the remaining solid lenticules or pellets having a water content in the range of 4-8% by weight, preferably about 6% by weight.

This process differs from freeze-drying in that the drying temperature is not below 0 ° C. or the pressure is not significantly below atmospheric pressure. The stabilizing solution typically contains an initial level of dissolved solids of about 30%. As the drying progresses, the individual volumes become very viscous and, on hydrophobic films, take the form of plano-convex discs, ie lens-like. As the drying progresses further, the disc becomes solid and glassy. Although the size and weight of the solid pellets can vary within wide limits, the present invention is particularly applicable to the production of these relatively small solid pellets.

A typical pellet produced according to the present invention can weigh up to 1 mg, and its weight can be 1-100 mg or less. It is understood that the final weight and size are determined by the volume and concentration of the solution before drying and the water content of the solids. Thus, for example, the volume of solution being dried is 1 to 1
The final dry weight is about 0.33-33 mg, preferably 3.3-16 m, in the range of 00 μl, preferably 10-50 μl, and when the solids content is about 33% by weight.
It is g. The final dry solid disc is typically 1-5 mm in diameter. They are then suitable for storage below 4 ° C., and preferably under dryness, and do not deteriorate significantly at ambient temperature, eg during shipping. They can be stored in the presence of dehydrating agents such as silica gel at temperatures down to -20 ° C or lower.

The exact composition of the stabilizing solution will depend on the material being stored. For example, many buffers may be suitable, but it is essential that the components of the buffer do not selectively salt out as the concentration of the buffer increases as drying progresses.

It is understood that there is some experience in selecting the materials used in the stabilizing solution. The base components are designed to ensure that the solid material dries to a glass-like solid that is hard and free of crystal formation. Other additional ingredients such as glycerol (membrane stabilizer), ascorbic acid (reducing agent), peptone, amino acids, calcium, magnesium and phosphate ions, charcoal, and soluble starch can be added as needed. These additional materials are typically present in amounts of 0.01 to 1% w / v. Additional ingredients are added to act as plasticizers, reducing agents, nutrients, membrane protectants, and antioxidants.

[0064]   Preferably, the composition comprises water in an amount of 4-8% by weight of the total composition.

The marker dye is optional and it functions only for convenience in identifying the stored material. The same solutions and methods can be used for storage of bound antibody.

Occasionally, during desiccation, the viable counts of some organisms such as Aeromonas are significantly reduced. This must be tolerated, but once dehydrated no further increase in viability loss occurs.

[0067] Example 1 Stock solution formulation   Prepare a solution with the following composition:     Trehalose 240g     Bovine serum albumin (filter sterilization) 80g     Glucose 10g     CMC (BDH, low viscosity) 20g     Pages physiological saline solution (Oxoid) 1 liter

BSA was analyzed and contains only 5 milliequivalents of sodium. Page
s Saline is used because it stabilizes biological entities. It is a dilute saline solution containing low concentrations of sodium, magnesium, potassium, and phosphorus. Other buffers may also be used. Add EC approved food coloring from time to time. All were tested at high concentrations against various bacteria and had no deleterious effect on viability.

Once prepared, the solution is autoclaved before use. Note that the concentration of ingredients increases during dehydration. 25 μl drop is about 7 mg, ie about 7 μl
When dehydrated to, everything is concentrated 3 times.

Example 2 Dehydration of Stock Solution The stock solution from Example 1 is force dried under aeration of sterile air in a drying cabinet containing silica gel for several hours (3 to 3 for individual lenticules on parafilm strips). 4 hours; overnight if dispensed into wells of plastic pieces). After that, they are transferred to a sealed plastic box containing silica gel and placed in a refrigerator at 4 ° C. to finish the dehydration process. This can take 7 days. If they become "hard" by visual inspection, transfer to a plastic jar with a screw cap containing silica gel and store at -20 ° C. Samples are removed after 7 days and at 3-6 month intervals thereafter for viability count testing.

Drying the lenticules under reduced pressure is optional. The silica gel at the bottom of the cabinet is regenerated by heating in a dry heat oven until it becomes a light blue color. Other desiccants may also be used.

Example 3 Preparation of Bacteria for Solution Typically, stored bacterial strains were subcultured on solid medium to produce heavy growth,
That is, about 10 ¹¹ colony forming units (cfu) are obtained. After proper incubation (usually overnight at 37 ° C, as bacteria should be in the early lag phase of the growth cycle)
, Collect colony growth in a sterile loop holding approximately 100 mg of material. The collected material is suspended in about 0.5 ml Pages saline and then added to 2.5 ml lenticulating solution and mixed very thoroughly to avoid clamping. Satisfactory mixing can be achieved by aspirating small volumes up and down in a pipette with a fine tip.

Once a uniform suspension is formed, the inoculated liquid is dispensed on a hydrophobic surface such as Parafilm into droplets weighing approximately 1 mg, onto a flat sheet material such as Perspex. Stretch to. 100 such spots, each containing approximately 10 ⁸ cfu, are sufficient.

The inoculum was given these high counts to create a defined count of lenticules.
Can be subdivided from "stock" lenticules. Miles and Misra viability counts were performed, and lenticules were rehydrated with maximal recovery dilution (MRD)
It is then added to the lenticulation solution at the required concentration, ie low count lenticules are prepared from stock lenticules without a culture step.

Example 4 Preparation of cells from urine for storage by lenticularization Leukocytes and epithelial cells from human urine are stored and used to create a simulated clinical sample for QA purposes.

One liter of urine is pooled from clinical samples known to have high cell counts. Erythrocytes are removed by adding 20 ml glacial acetic acid dropwise with stirring. It also dissolves in phosphate. The urine is left to slowly sediment the remaining cells. It is not possible to centrifuge at this stage because the cells stick together.

After 2-3 hours, most of the supernatant is decanted and about 100 ml of 0.1 M disodium dihydrogen phosphate buffer at neutral pH is added to neutralize the acetic acid. The suspension is left again and most of the supernatant decanted. Phosphate buffered saline (PB
S) is added and the volume is returned to about 100 ml. It is currently a suspension of white blood cells, epithelial cells, and a few bacteria. The cells are fixed by the addition of 1 part in 800 of glutaraldehyde and the morphology is preserved. The cells are pelleted by centrifugation at a slow speed, leaving the bacteria in the supernatant. Cells can be rinsed once with PBS and then stored at -4 ° C or cryopreserved by addition of 30% glycerol and storage at -20 ° C. These may be added to the stock solution of Example 1 at the desired concentration, with or without bacteria or other biological material, and dehydrated in the usual manner.

Example 5 Preparation of Red Blood Cells for Lenticleization Sheep red blood cells are prepared for storage by fixation in glutaraldehyde and precipitation as described in Example 4.

Example 6 Preparation of Simulated Feces Biodegradable plant material was dried, ground and heat treated with enteric pathogens to mimic fecal material for QA testing and using the solution of Example 1. And store.

Example 7 Preparation of Background Flora for Simulated Pharyngeal Swabs A blood agar plate culture of pharyngeal swabs containing no pathogenic bacteria but containing a wide range of representative non-pathogenic oral flora is selected. To do. Biomass is pooled from several plates and stored as in Examples 1-3. These can then be rehydrated and added to the lenticulated solution at the appropriate dilution to provide a high, medium, or low background count. The target pathogen suspension can be added again at a known concentration and the complex suspension is dispensed and dried as before. These can be used in National External Quality Assessment Schemes such as UKNEQAS.

Alternatively, the stock solution containing background flora and pathogens is dispensed into plastic cups and dehydrated. The product is rehydrated with a defined volume of MRD. Soak standard cotton swabs in suspension and each will reach the standard volume, so some swabs should be treated according to the standard operating procedure (SOP) for culturing pharyngeal swabs.
Test in each laboratory that receives C samples.

Example 8 Other Microorganisms Preserved by Lenticulization The materials and methods of Examples 1-3 are used to store Cryptosporidium vesicles, certain fungi such as Candida, and mycoplasma. Mycoplasma are cultured in broth for several days before storage. The cells are then microcentrifuged and resuspended to half their original volume in stock solution. The stored material is rehydrated on solid medium and carefully spread into a circular area with a radius of approximately 1 cm. After several days of incubation, the surface of the plate is examined under a low power microscope to see mycoplasma colonies.

The virus has been successfully stored, preserving its morphology for electron microscopy QA samples, and preserving influenza A and adenovirus viability for over a year.

Example 9 Lenticle Rehydration Viable microorganisms are recovered from stored samples by rehydration. Typically, the stored sample may be placed on the surface of a suitable supporting solid medium such as blood agar and rehydrated for 10 minutes. It can then be spread over the surface of the plate in the usual way with a sterile plastic rope.

Alternatively, the stored sample is treated with a volume of liquid, typically the maximum recovery dilution (MRD
) And completely dissolved. The liquid is then swirled to ensure even mixing and sampled as desired.

Example 10 Use of a Preparation of the Invention in a "Spiking" Dry Food for a Food EQA Scheme Food EQA schemes typically have very low counts (10 cf in 25 g of dry food).
It is desired to assess the ability of u) to detect the presence or absence of a particular pathogen. Low count preparations are particularly suitable for this. Two sets of preparations, each with or without 10 cfu of target organism, are supplied. One preparation is rehydrated directly on solid medium and colony counted. The other is added to 25 g of dry food, followed by a controlled laboratory process. 100% recovery is not expected due to the presence of free radicals, toxins, or competing organisms in the food. A detection failure rate of 5% is acceptable to the food industry, but ideally the reference material should be supplied near the detection limit to provide a sufficiently rigorous test.

Thus, results of 5% or more from processed foods warrant further study of processing if they do not correspond to results from direct plating of paired lenticules. Salmonel is a persistent Salmonella, monophasic and relatively rare
La gold coast has been used in this study to date.

Introducing such present / absent lenticules into a food EQA scheme is straightforward. Lenticle content can be dictated by the particular process being controlled. The ability to produce up to 10,000 identical preparations at a time has great advantages in that duplicate samples are available to some test laboratory empirical difficulties in their food processing.

Example 11 Simulated Urine Storage Materials and Methods Strains Routine isolates from urinary tract infections were collected over a period of 1 month. These are E
E. coli, "Coliforms", Klebsiella sp., Pseudomonas, and Str. faecalis. Furthermore, the inventors have found that the NCTC strain of Acinetobacter, Ps. Fluorescens MRS.
A and C. albicans were selected to expand the range of organisms included in the panel. Select strains for appearance of representative colonies, seed on support medium, usually blood agar,
And incubated overnight for maximum yield. These cultures,
Stored by the method of Example 1 and stored at -20 ° C until further testing. Retain a portion of the lenticle at room temperature, 30 ° C, and 37 ° C for 7 days to reconstitute,
And cultured. Strains showing a substantial decrease in counts at higher temperatures were excluded, provided that accelerated alteration tests could predict long-term storage potential. Pus and epithelial cells from the ureter were collected by pooling a substantial number of fresh urine samples submitted for routine experiments. To 1 liter of this pool was added 20 ml of glacial acetic acid and mixed quickly. After standing at room temperature for 4 hours, most of the cell contents settled to the bottom in 20% volume. Decant the supernatant, and the remaining 200 ml at 1,000 rpm
The cells were pelleted by very light centrifugation at. These were washed twice with PBS and cryopreserved at -20 ° C as a 10% suspension until needed. Group 0 positive red blood cells were also cryopreserved as a 10% suspension at -20 ° C.

[0090]   Urine specimens were reconstituted in stored solution at a defined level with stored bacteria.
It was prepared from a cryopreserved cell suspension. These levels are based on the levels of fresh urine samples.
Set to resemble a bell, and either 10 single organisms or mixtures ⁵ Included counts above and below CFU. In addition, some samples have pus
It contained cells or red blood cells or both in easily observable concentrations. The mixture
Distribute into 8-well flat-bottomed microtiter strips with different samples at each position.
I put it. Four batches were prepared for a total of 32 samples. However, one sample
Replicated in 2, 3, and 4. This set also includes some completely negative samples
Also included. A batch of strips is assembled into a plate, labeled and then
Forced air in a drying cabinet at 20 ° C and then 72 ° C on silica gel at 4 ° C.
The process was completed by leaving it to dry for a period of time. Until using the plate −
Stored at 20 ° C.

Media All routine culture media was obtained from Oxoid in powder form and prepared in situ according to the manufacturer's instructions. Columbia Agar Base CM331, CLED CM with 5% horse blood from TCS for blood agar plates. Solutions were prepared from stock reagents as needed.

Procedure and Test Before use, 100 μl MRD was added to each well to rehydrate the strips,
Allowed to stand for 10 minutes and shaken to disperse the bacterial and cell suspension. These specimens were examined by standard laboratory operating procedures. The cells were observed in situ with the specimen under an inverted microscope. Care was taken to ensure that the strip was correctly positioned in the strip depending on its position 1-8 in order to identify each sample. Each specimen was spread with half of a standard 90 mm CLED plate on top of it using a standard 1 μl loop and incubated aerobically at 37 ° C. overnight. The plates were read, scored, and recorded by the same individual who processed the specimen the day before. Sensitivity testing and identification was performed on some of the isolates.

All specimens in the four batches were independently tested at various times over a year by both microscopy and culture to confirm retention characteristics. To improve the accuracy of these tests, the counts were calculated by Miles and Misra (1938, Journal of Hyg since the capacity obtained by the standard loop method varies considerably.
iene, 38, 732-749).

[0094] result   The results are shown in Table 1.

[0095]

[Table 1]

The results show no evidence of reduced counts of stored organisms over a period of 35 months except for one year or a single Klebsiella sample. Moreover, the morphology of the cellular components of the sample remained unchanged.

Example 12 Storage of E Coli Lenticles containing E Coli were prepared according to Example 3 and viability counts on blood agar or stock solutions were taken on days 1, 90 and 220. It was
The results are shown in Table 2.

Accordingly, the present invention provides an effective method of storing biological material in solid form. The material is acceptably stable in solid form and is readily reconstituted into a working solution. Many practical applications of the invention, such as storage of bacterial samples for transport, are obvious.

[0099]

[Table 2]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (34)

[Claims] 1. A composition for preserving viable microorganisms, cells or tissues, which comprises a preservative combination of (a) (i) trehalose, (ii) matrix protein filler and (iii) monosaccharide. And (b) a buffer solution. 2. A trehalose: filler weight ratio of 5: 1 to 0.5: 1.
The composition of claim 1, which is: 3. The trehalose: filler weight ratio is from 3.5: 1 to 1.5: 1.
The composition according to claim 1 or 2, wherein 4. The composition according to claim 1, wherein the filler is or comprises a protein having a molecular weight of 50 to 100 KD. 5. The composition of claim 4, wherein the filler is albumin. 6. The composition according to any one of claims 1 to 5, wherein   (I) 5 to 30% by weight trehalose;   (Ii) 1 to 10% by weight of filler; and   (Iii) 60 to 94% by weight aqueous buffer, A composition comprising: 7. The composition according to any one of claims 1 to 6, wherein the monosaccharide is a reducing sugar. 8. The composition according to claim 7, wherein the reducing sugar is glucose. 9. The composition according to claim 7, which comprises 0.1 to 3% by weight of a monosaccharide. 10. The composition according to claim 1, further comprising a structural additive. 11. The composition of claim 10, wherein the structural additive is water soluble. 12. The composition of claim 11, wherein the structural additive is or comprises a water soluble carbohydrate polymer. 13. The composition according to claim 10, wherein the structural additive is carboxymethyl cellulose or hydroxyalkyl cellulose. 14. A composition according to any one of claims 10 to 13 wherein the structural additive and all other ingredients are water soluble so that the dried composition is water. A composition which, when reconstituted with, dissolves substantially completely into a clear solution. 15. The composition according to claim 1, further comprising a colorant. 16. The composition according to claim 1, wherein the microorganism is selected from the group consisting of bacteria, viruses, protozoa, and fungi. 17. The solid content is in the range of 10 to 50% by weight.
The composition according to any one of 1 to 16. 18. A method of preserving viable microorganisms, cells or tissues, which comprises (a) combining said microorganisms, cells or tissues with a composition according to any one of claims 1 to 17. Forming a storage preparation; (b) drying the storage preparation at a drying temperature not below 0 ° C .; and (c) storing the resulting dried preparation. 19. The method of claim 18, wherein step (b) is performed at atmospheric pressure. 20. The method according to claim 18, wherein said step (b) is carried out by passing air, an inert gas, or a mixture thereof over said preservation preparation in the presence of a desiccant. The method according to 19. 21. The method according to claim 20, wherein step (b) comprises: (i) air, an inert gas, or a mixture thereof on the preservation preparation in the presence of a desiccant. Partially drying the stock preparation by passing it through air; and (ii) drying the partially dried stock preparation at a further reduced temperature. 22. The method according to claim 20 or 21, wherein the desiccant is silica gel. 23. The method of claim 1, comprising drying the stored preparation under reduced pressure.
The method according to any one of items 8 to 22. 24. A method according to any one of claims 18 to 23, comprising the step of placing the storage preparation on a hydrophobic surface before drying. 25. The method according to claim 24, wherein the volume of the stock preparation deposited on the hydrophobic surface is in the range 5 to 100 μl. 26. The method of any of claims 18-25, wherein one or more of steps (a)-(c) is performed in a substantially oxygen-free environment. 27. The composition according to any one of claims 1 to 15, wherein the composition is a dry composition, additionally comprises live microorganisms and has a solids content of at least 80% by weight. 28. The composition of claim 27, wherein the viable microorganism is a bacterium, or a mixture of different strains of bacteria, or a combination of bacteria and mammalian cells. 29. The composition of claim 27 or 28 having a defined microbial count, wherein the microbial count remains substantially stable upon storage at ambient temperature. 30. The composition of any of claims 27-29, wherein the composition is fully dispersible when reconstituted with water or an aqueous buffer solution. 31. A method of preserving viable microorganisms, cells or tissues, comprising: (a) combining viable microorganisms, cells or tissue with a preservation solution comprising the composition of claim 1; D) drying the combination of (a) to form a dry preparation having a solids content of at least 80% by weight; and (c) counting the viable microorganisms, cells or tissues in the dry preparation. A), whereby the dry preparation can be combined with an aqueous buffer to obtain an aqueous preparation containing a predetermined count of viable microorganisms, cells, or tissue. 32. A method of preserving viable microorganisms, comprising drying an aqueous preparation of viable microorganisms in the presence of the composition of claim 1 to form a dry preparation.
Counting viable microorganisms in the dry preparation to obtain a counted dry preparation, and storing the counted dry preparation, wherein the counted dry preparation is reconstituted with water or an aqueous medium. A method of forming an aqueous preparation containing a predetermined number of viable microorganisms, when applied. 33. Forming at least a first and a second dry preparation,
Counting the viable microorganisms, cells, or tissues in the first dry preparation, and thereby estimating the number of viable microorganisms, cells, or tissues in the second dry preparation. The method according to claim 31 or 32. 34. Forming a plurality of dry preparations and counting selected samples of the dry preparations to estimate a count of viable microorganisms, cells, or tissues in the remaining dry preparations. 34. The method of claim 33, including the step of: