Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

• This invention relates to the storage of microorganisms, cells and tissue.
• the invention may be applied to the storage of a wide range of such biological materials but in particular has a special applicability to storing those biological materials that deteriorate at room temperature in aqueous solution.
• the invention in its most preferred aspect relates to the storage of microorganisms, cells and tissue in a viable state.
• a method of preserving microorganisms (including bacteria, yeast and fungi) is known that uses storage beads.
• a suspension of the microorganism is made in a cryopreservative and then mixed with storage beads.
• the cryopreservative is removed and the beads frozen and stored at a temperature of typically -70°C.
• a bead is removed from frozen storage and placed in broth or rolled onto growth medium. This method has various drawbacks. Storage temperatures of
• EP-A-022981 0 describes protection of proteins and like macromoiecules using a solution of trehalose. However, the particular difficulties associated with storage of microorganisms is not addressed.
• US-A-5733774 describes methods and compositions for producing stable bacterial formulations, and involves drying bacteria, combining them with a powder or granular non-aqueous carrier and packaging the bacteria in sealed packaging impermeable to gas and water vapour, the method also including removing substantially all oxygen from inside the package.
• the methods described are impractical in that strenuous efforts must be taken to remove gas and water vapour and oxygen from the stored material. In addition, prolonged viability of stored bacteria has not been demonstrated.
• WO-A-90/04329 describes storing mammalian cells in a process that includes lyophiiizing mammalian cells and storage at about 4°C.
• the lyophilizing step introduces significant risk of loss of viability of the stored material.
• WO-A-99/00485 discloses a composition for preserving microorganisms, using starch optionally mixed with albumin.
• the present invention seeks to overcome or at least mitigate problems associated with the art and to provide improved storage of biological material such as microorganisms, cells and tissue.
• a further object is to provide a stored biological material which is suitable for transport, including via the postal system, without excessive risk of damage to the biological material during transport.
• a more specific object of the invention is to provide a method of storing microorganisms, cells and tissue in a viable state for extended periods of time.
• the invention provides a method of preserving viable microorganisms, comprising drying an aqueous preparation of viable microorganisms, cells or tissue in the presence of non-reducing disaccharide to form a dried preparation, counting the viable microorganisms, cells or tissue in the dried preparation to obtain a counted dried preparation, and storing the counted dried preparation, wherein the counted dried preparation when reconstituted with water or aqueous medium forms an aqueous preparation containing a predetermined number of viable microorganisms, ceils or tissue.
• the invention provides a composition for preserving viable microorganisms, cells or tissue, the composition comprising:
• the invention provides a process for preserving viable microorganisms, cells or tissue, comprising the steps of:
• biological material such as microorganisms, cells or tissue can be stored for extended periods of time in a stable form and whilst retaining significant viability.
• Specific embodiments of the invention provide individual dried preparations of bacteria suitable for storage and which are readily reconstituted with water or other aqueous buffer to yield viable bacteria.
• a 25 l drop of bacterial suspension forms a lenticule with a dry weight of 8mg, containing from 10-10 8 CFU's of single organisms or mixtures. It can be reconstituted for use in 10 minutes.
• a further advantage of the invention over other methods of preserving viability in microorganisms is that samples of biological material can be prepared having a predetermined, known number of viable microorganisms, ceils or tissue, due to the extended viability of stored material in compositions of the inventions. Samples can thus have a defined numbers of viable organisms per stored sample. Hence, they can be used for quantitative experiments, and in situations where presence of absence of very low-numbers of marker organisms is a stringent test of the processes being controlled.
• count preparations containing bacterial numbers ranging from 10 cfu to 10 8 cfu have been prepared in accordance with the invention, stored and resuscitated with no significant loss of viability.
• the confidence limits of the cfu counts have been established by performing viable counts on multiple lenticules. The counts are usually within 95% confidence interval, even for the very low count preparations.
• the invention provides a versatile alternative to the maintenance of bacterial cultures either on slopes, wet suspensions at 4°C, cryo-preservation at low temperature, freeze-drying or spray-drying.
• a sample of biological material is combined with (a) a preservative combination of (i) a non-reducing disaccharide and (ii) a bulking agent; and (b) a buffer.
• the weight ratio of the non-reducing disaccharide:bulking agent is suitably 5: 1 to 0.5: 1 , preferably about 3.5: 1 to 1 .5: 1 , and the solids content is generally at least 20% by weight.
• the non-reducing disaccharide may be selected from the group consisting of trehaiose, sucrose, maltose, lactose, cellobiose, isomers thereof and mixtures thereof.
• trehaiose or a combination of trehaiose and other non-reducing disaccharide is used.
• the bulking agent typically is, or comprises, a high molecular weight protein.
• the bulking agent is a matrix protein which has a molecular weight which is less than 100kD and preferably is within the range 50-1 OOkD.
• the matrix protein is a relatively inert globular protein such as albumin.
• Gelatin (which normally has a molecular weight in the range 1 70-200kD) is not suitable.
• Suitable albumins include ovalbumin, foetal calf albumin and lactalbumi ⁇ .
• the matrix protein is serum albumin, or more specifically bovine serum albumin.
• the matrix protein is conveniently present in solution in an amount between 5 and 20g per 100ml, preferably 8-1 2g and more preferable around 10g per 100ml.
• the buffer is selected so as to maintain the pH of the solution at a value at which the microorganisms, cells or tissues are stable and should contain components that do not selectably salt out as their concentration increases during the drying step.
• One suitable buffer is phosphate buffered saline (PBS) at a concentration between 0.1 and 1 M, preferably 0.1 5-0.5M.
• a particular embodiment of the invention comprises: (i) 5 to 30 wt% non-reducing disaccharide; (ii) 1 to 1 0 wt% bulking agent; and (iii) 60 to 94 wt% aqueous buffer.
• the composition may further comprise a monosaccharide, for example a reducing sugar such as glucose.
• a monosaccharide for example a reducing sugar such as glucose.
• the monosaccharide can be present at 0.1 to 3 wt%.
• compositions can be or comprise a water-soluble polymeric carbohydrate.
• a structural additive can be or comprise a water-soluble polymeric carbohydrate.
• carboxymethylcelluiose is used, though other additives are suitable, such as hydroxyalkylcellulose.
• the structural additive be water soluble and that all other components be water soluble so that when a dried composition of the invention is reconstituted in water it dissolves substantially completely leaving a clear solution. Insoluble or partially soluble sugars and bulking are preferably avoided. Thus, starch which does not fully dissolve or dissolves leaving a cloudy preparation is preferably avoided.
• a colouring agent is further optional.
• compositions of the invention are particularly suited to storage of microorganisms, selected from the group consisting of bacteria, viruses, protozoa and fungi.
• compositions of the invention is initially generally in the range of from 10-50%.
• Drying may also be carried out by passing a draught of air, inert gas or a mixture thereof over the preserving preparation in the presence of a drying agent.
• Silica gel can be used, as can other drying agents. It is also optional to use a substantially oxygen-free environment.
• the process comprises:
• the process comprises depositing the preserving preparation onto a hydrophobic surface prior to drying.
• a small volume of liquid is deposited and dried, forming a small solid composition, referred to as a pellet or lenticule. Once dried this pellet is highly convenient to handle.
• the volume of preserving preparation that constitutes a small volume can be in the range of from 5-100 ⁇ .
• the process of the invention as applied to bacteria is to heavily seed a supportive medium with a culture and incubate it optimally until early lag phase.
• a supportive medium with a culture and incubate it optimally until early lag phase.
• the typical yield is 10 11 colony forming units, CFU's. This growth is harvested into a small volume of low strength saline and mixed with 2.5ml of solution. 25 I spots are dispensed onto a hydrophobic surface and dried above the triple point.
• the 100 or so resulting primary so-called lenticuies each contain around 1 0 8 CFU's and are stored at about -20°C, above the eutectic, under desiccation.
• Strains that have been preserved by the invention include Staph. aureus, Staph. epidermidis, Bacillus cereus. Bacillus subtilis, Listeria monocytogenes, Str. faeca/is, Str. pyrogenes, Str. agalactia, Str. pneumonia, Enterococcus faecium, Cl.
• perfingens Sacchromyces, E coli (including 0157), Salmonella sp, Ps aeruginosa, Klebsiella sp, Proteus sp, Campylobactersp, Helicobacter pylori, Vibrio parahaemolyticus, Acenitobacter sp, Serratia marcesans, Haemophilus influenzae, Bordetella pertussis, N. menigitidis, N. gonorrhea, Bacteroides sp and Salmonella gold coast. Mycoplasma, viruses and fungi can also be preserved by the present invention.
• compositions of the invention may be supplied in a number of formats, e.g.
• a dried composition according to the invention further comprising a viable microorganism and having a solids content of at least 80%.
• the viable microorganisms are preferably bacteria, or a mixture of bacteria of different strains, or a combination of bacteria and mammalian cells.
• This aspect of the invention also provides a dried composition having a defined microorganism count wherein said microorganism count remains substantially stable in storage at ambient temperature.
• defined count is meant that the content of the dried composition is known or predetermined, directly or indirectly, and that therefore once reconstituted with buffer an aqueous preparation is obtained having a defined count of viable microorganisms.
• composition is fully dispersible when reconstituted with water or aqueous buffer solution - ie that all components are water soiuble and readily dispersible.
• a method of preserving viable microorganisms, cells or tissue of the invention comprises:-
• the dried preparation can be combined with aqueous buffer to yield an aqueous preparation comprising a predetermined count of viable microorganisms, cells or tissue.
• the viable count it is preferred to form at least first and second dried preparations, counting the viable microorganisms, cells or tissue in the first dried preparation and thereby estimate the number of viable microorganisms, cells or tissue in the second dried preparation. Thus the count is made indirectly.
• the method can comprise counting a selected sample of those dried preparations so as to estimate the count of viable microorganisms, cells or tissue in the remaining dried preparations.
• the drying step can be carried out in air or inert gas or mixtures thereof.
• the drying step is carried out in an atmosphere of reduced oxygen concentration (compared to air) , an advantage of this being that oxidation of the biological material during the drying step is reduced.
• the drying step is carried out in an atmosphere of reduced humidity.
• the pressure is reduced during drying.
• the drying can conveniently be carried out at room temperature and can also be carried out at temperatures down to around 4°C. Higher drying temperatures could also be employed, though these would have to be weighed against the increased deterioration of the biological material caused by the increased temperature.
• the drying step is intended to result in a solid composition containing the biological material and preferably having a water content in the region of less than 10 percent by weight.
• the water content is preferably in the range of 4-8 percent by weight and most preferably around 6 percent by weight (the term "around" allowing a tolerance of + /- 2%).
• the ideal water content of the solid stored composition will depend upon the nature of the material to be stored.
• the storage method is carried out under steriie conditions.
• the re- constitution of aqueous solution of biological material is carried out using steriie water or sterile buffer.
• the invention is of application to biological material that cannot be stored indefinitely at room temperature in aqueous solution.
• a biological material may be regarded as having an unacceptable stability if it has a half life of less than 5 days, especially less than 2 days when stored at ambient temperature (20°C).
• Especially unstable biological material are ones having a half life of less than 24 hours.
• Examples of biological material that are suitable for storage according to the invention include microorganisms including bacteria and viruses, cells and samples of biological solutions such as serum samples.
• the storage method is used to store a bacterial sample.
• a membrane stabilizing agent include glycerol, egg yolk and mixtures thereof.
• glycerol glycerol, egg yolk and mixtures thereof.
• the bacterial in the stored form of the invention are relatively stable and this enables the viable count to be obtained over a more flexible time scale than previously possible.
• the bacterial storage method includes providing a nutrient medium for the stored bacteria.
• a first solution may be prepared according to the invention containing a matrix protein and a mono-or di-saccharide in 100ml of buffer. It is optional to add a small amount, in the range of 0.01 -1 g of peptone.
• a second solution may be prepared acting as a nutrient medium for the bacteria.
• This nutrient medium will routinely include essential nutrients for growth/survival of the bacteria.
• it may optionally also include bacterial membrane stabilizing components.
• the bacteria are grown/cultured and then pelleted and mixed with a small volume of nutrient medium.
• a small volume of (1 ) the nutrient medium containing the viable bacteria is mixed with (2) a larger volume of the storage solution and subjected to drying as before to give a residual solid with water content approximately 4- 8 percent by weight.
• the solid is dissolved in water or buffer or nutrient solution.
• a preparation of the biological material to be stored (B) is mixed with a solution of the invention (S) in a ratio ⁇ of B/S ⁇ of between 1 :5 and 1 : 10.
• Volumes of the mixed solution in an amount of between 10 and 50 /I are dispensed onto a hydrophobic film, for example parafilm, or into microtitre strips. This results in small separated liquid volumes.
• the biological material is in a stabilizing solution in a droplet of given volume which contains a desired quantity of biological material.
• the separated volumes are then dried to give residual solid lenticules or pellets with a water content in the range 4-8 percent by weight, preferably around 6 percent by weight.
• the process differs from freeze drying in that the drying temperature is not below 0°C nor the pressure significantly below atmospheric.
• the stabilizing solution typically contains an initial level of around 30 percent dissolved solids.
• the individual volumes becomes highly viscous and, when on the hydrophobic film, assume the shape of a plano-convex disk, i.e. lens like.
• the discs become solid and glass-like.
• the size and weight of the solid pellets can vary over a wide range, the invention is particularly applicable to the production of these relatively small solid pellets.
• Typical pellets produced according to the invention may weigh less than 1 mg and their weights can be as 1 - 1 00mg or lower. It will be appreciated that the final weight and size is determined by the volume and concentration of the solution before drying commences and the water content of the solid. Thus, for example, when the volume of the solution being dried is in the range 1 -100 ⁇ l, preferably 10-50 vl and the solid content is around 33 wt%, the final dried weight will be around 0.33-33mg, preferably 3.3-16mg.
• the final dried solid discs are typically between 1 -5mm in diameter. They are suitable thereafter for storage at 4°C or less, and preferable under desiccation, and will not deteriorate significantly at ambient temperature, e.g. if in transit. They can be stored at reduced temperature, down to - 20°C or lower, in the presence of a dehydrating substance, e.g. silica gel.
• composition of the stabilizing solution will vary according to the materials being preserved. For example, many buffers may be appropriate, though it is essential that the components of the buffer do not selectively salt out as the concentration of the buffer increases as drying proceeds.
• the basic ingredients are designed to ensure that the solid material dries out into a glass like solid which is firm and free of crystal formations.
• Other additional components may optionally be added such as glycerol (membrane stabilizing), ascorbic acid (reducing agent), peptone, amino acids, calcium, magnesium and phosphate ions, charcoal and soluble starch. These additional substances are typically present in amounts between 0.01 and 1 percent weight by volume.
• the additional components are added to act as plasticizers, reducing agents, nutrients, membrane protectors and anti-oxidants.
• the composition contains water in an amount of between 4-8% by weight of the total composition.
• a marker dye is optional, functioning only for convenience in identifying the stored material.
• the same solution and method can be used for the storage of conjugated antibody.
• a solution is prepared having the following composition:-
• Bovine serum albumin 80 g
• the BSA is analyzed and contains only 5 milli-equivalents of sodium. Pages saline is used because it stabilises biological entities. It is a dilute saline containing low concentrations of sodium, magnesium, potassium and phosphorus. Other buffers may also be used. EC-approved food colourings are added on occasion. All have been tested at high concentrations against various bacteria and had no deleterious effect on viability.
• the solution is autoclaved prior to use. Note that the concentrations of the ingredients will increase during dehydration. If a 25 /I drop dehydrates to c. 7mg i.e. c. 7 ⁇ , everything is concentrated by a factor of 3.
• a storage solution from Example 1 is subjected to forced drying under a draught of filtered air in a drying cabinet containing silica gel for a few hours (3-4 hours for individual lenticules on Parafiim strips; overnight when dispensed into the wells of plastic strips). Thereafter, they are transferred to sealed plastic boxes containing silica gel and left in the fridge at 4°C to complete the dehydration process, which may take 7 days. When they have become "hard”, which seems to be judged by eye, they are transferred to screw-capped plastic jars containing silica gel and stored at -20°C. Samples are removed for viable count testing after 7 days and at 3-6 monthly intervals thereafter.
• Drying the lenticules under reduced pressure is an option.
• the silica gel, in the base of the cabinet, is regenerated by heating it in a hot air oven until it is bright blue.
• Other desiccants may also be used.
• the bacterial strain to be preserved is subcultured onto solid media to obtain heavy growth i.e. c. 10 11 colony forming units (cfu). After appropriate incubation (usually overnight at 37° C, such that the bacteria should be in the early lag phase of their growth cycle) the colonial growth is collected in a sterile loop, which holds c. 100 mg of material. This collected material is suspended in c. 0.5ml Pages saline, which is then added to 2.5 ml of lenticulating solution and mixed very thoroughly to avoid clumping. Satisfactory mixing can be achieved by sucking a small volume up and down in a fine-tipped pipette.
• the inoculated fluid is dispensed in drops weighing c. 1 mg onto a hydrophobic surface such as
• White blood cells and epithelial cells from human urine are stored to be used in making simulated clinical samples for QA purposes.
• the cells are rinsed once in PBS and can then be stored at -4°C or cryopreserved by the addition of 30% glycerol and storing at -20°C. They can be added to the storage solution of Example 1 at the desired concentration, with or without bacteria or other biological material, and dehydrated in the usual manner.
• Sheep red blood cells are prepared for storage by fixing them in glutaraldehyde as in Example 4 and sedimenting them.
• Biodegradable plant material is dried, crushed and heat-treated with enteric pathogens to simulate faecal material for QA tests and stored using the solution of Example 1 .
• Blood agar plate cultures of throat swabs are selected containing no pathogenic bacteria, but a wide variety of typical, non-pathogenic, mouth flora.
• the biomass is pooled from several plates and stored as in Examples 1 -3. Thereafter, these could be rehydrated and added to fresh lenticulating solution at appropriate dilution to provide high, medium or low background counts.
• Suspensions of the target pathogen can be added, again at known concentrations, and the complex suspensions dispensed and dried as before. These can be used in National External Quality Assessment Schemes such as UKNEQAS.
• the storage solution containing the background flora and pathogens is distributed and dehydrated in plastic cupules.
• the product is rehydrated with a defined volume of MRD.
• Standard cotton swabs are dipped into the suspension, as each will take up a standard volume, several swabs being tested in each laboratory receiving the QC sample, according to their standard operating procedure (SOP) for culturing throat swabs.
• SOP standard operating procedure
• Examples 1 -3 Cryptosporidium oocysts, certain fungi such as Candida, and Mycoplasma are stored.
• Mycoplasma are cultured in broth for several days prior to storage.
• the cells are then spun out in a microfuge and resuspended in half the original volume in storage solution.
• the stored material is rehydrated on a solid medium and carefully spread over a circular area of c. 1 cm radius. After several days' incubation, the surface of the plate is examined under a low-power microscope to view the colonies of Mycoplasma.
• Viruses have been successfully stored, preserving their morphology for electron microscopy QA samples, and preserving viability in Influenza A and Adenoviruses for over a year.
• Viable microorganisms are retrieved from the stored sample by rehydrating them.
• a stored sample can be placed on the surface of a suitably supportive solid medium such as blood agar, and allowed to rehydrate for 10 minutes. Thereafter, it can be spread with a sterile plastic loop over the surface of the plate in the usual way.
• a stored sample can be added to a volume of a liquid, typically maximum recovery diluent (MRD) and allowed to dissolve completely. The liquid is then swirled to ensure even mixing and sampled as desired.
• MRD maximum recovery diluent
• Food EQA Schemes typical want to assess the ability to detect the presence or absence of very low counts ( 1 0 cfu in 25g dried food) of specific pathogens.
• Low-count preparations are particularly suited to this.
• Duplicate preparations each containing 1 0 cfu of the target organism (or not) are supplied.
• One preparation is rehydrated directly onto solid media and a colony count performed.
• the other is added to 25g of dried food, which is then subjected to the laboratory process being controlled.
• One hundred per cent recovery is not expected, because of the presence of free radicals, toxins or competitor organisms in the food.
• a failure-to-detect rate of 5% is acceptable to the food industry, but ideally the reference material should be supplied close to the limit of detection to provide a sufficiently stringent test.
• Salmonella goldcoast has been used in this work to date because it is a durable Salmonella, is monophasic and relatively uncommon.
• lenticule content could be dictated by the specific process to be controlled.
• the ability to produce up to 10,000 identical preparations at a time has the great advantage that replicate samples are available for any test laboratory experiencing difficulty with their food processes.
• Routine isolates from urinary tract infections were collected over a period of one month. These included E coli, "coliforms, " Klebsiella sp., Pseudomonas and Str. faecalis.
• NCTC strains of Acinetobacter, Ps. fluorescens MRSA and C. albicans were chosen for their typical colonial appearances, plated onto a supportive medium, usually blood agar, and incubated overnight or longer for maximal yield. These cultures were preserved by the method of Example 1 and stored at -20 °C pending further tests.
• a proportion of the lenticules were held at room temperature, 30°C and 37 °C for 7 days reconstituted and cultured. Strains showing a substantial fall in count at the higher temperatures were rejected on the premise that an accelerated degeneration test would predict their probable long term storage.
• Pus and epithelial cells from the urinary tract were collected by pooling a substantial number of fresh urine samples submitted for routine examination. To one litre of this pool 20ml of glacial acetic acid was added and quickly mixed. After standing at room temperature for 4 hours most of the cellular content had settled into the bottom 20% volume. The supernatant was decanted and the remaining 200ml centrifuged very lightly at 1 ,000 rpm, to deposit the cells. These were washed twice in PBS and cryo-preserved at -20°C as a 1 0% suspension until required. Group 0 positive red ceils were also cryo-preserved as a 10% suspension at -20°C.
• Urine specimens were made from the stored bacteria and the cryo-preserved suspensions of cells by reconstitution in storage solution at a predetermined level. These levels were set to resemble those of fresh urine samples and included counts above and below 1 0 5 CFU's either of single organisms or mixtures. In addition some samples contained pus cells or red cells or both at an easily observable concentration. The mixtures were dispensed into 8 well flat bottomed microtitre strips each position being occupied by a different sample. Four batches were made giving a total of 32 samples. However one sample was replicated in batches 2, 3 and 4. The set also included some completely negative samples. The batches of strips were assembled into plates, labelled and dried by forced ventilation in a desiccating cabinet at 20°C followed by standing at 4°C for 72 hours over silica gel to complete the process. The plates were stored at -20°C pending use.
• MEDIA All routine culture media was supplied by Oxoid in powder form which was prepared on site in accordance with the manufacturer's instructions.
• the strips Prior to use the strips were re-hydrated by the addition of 100//m of MRD to each well, left for 10 minutes and shaken to disperse the bacterial and cellular suspensions. These specimens were examined by the laboratory's standard operating procedure. Cells were observed with an inverted microscope with the specimens in situ. Care was needed to ensure that the strips were correctly orientated as the identification of each sample depended upon its position, 1 -8, in the strip. Each specimen was spread using a standard 1 ⁇ l loop onto half of a standard 90mm CLED plate and incubated overnight aerobically at 37 °C. The plates were read, scored and recorded by the same individual that processed the specimen the previous day. Sensitivity tests and identifications were carried out on a some of the isolates.
• Lenticules containing E Coli were prepared according to Example 3 and viable counts on blood agar or on storage solution taken at day 1 , 90 and
• the invention provides an efficient method of storing biological materials in solid form.
• the materials are acceptably stable in solid form and are easily reconstituted into working solution.
• Many practical applications of the invention are evident, such as storage of bacterial samples for transit.

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• 1999
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