EP1002053A1

EP1002053A1 - Medium for recovery and growth of microorganisms

Info

Publication number: EP1002053A1
Application number: EP98932405A
Authority: EP
Inventors: Peter Jeremy Stephens; Patrick Druggan
Original assignee: Oxoid Ltd
Current assignee: Oxoid Ltd
Priority date: 1997-07-11
Filing date: 1998-07-10
Publication date: 2000-05-24
Also published as: WO1999002649A1; GB9721396D0; WO1999002650A1; EP1009803A1

Abstract

Reagents for use in the recovery and growth of microorganisms, comprise (a) growth medium generating low levels of toxic oxidising species; (b) a hydrogen donor; and (c) freeze-dried OXYRASE enzyme. The combination of use of a growth medium generating low levels of toxic oxidising species together with OXYRASE enzyme enables significantly improved recovery, i.e. better and faster recovery, of stressed microorganism cells than is possible with conventional growth media. Not only are more stressed cells recoverable, but also recovery of all cells takes place in less time than under traditional conditions.

Description

Title: MEDIUM FOR RECOVERY AND GROWTH OF MICROORGANISMS

Field of the Invention

This invention relates to growth of microorganisms, and concerns reagents for use in the recovery and growth of rmcroorganisms, for instance in techniques for detecting microorganisms, for example in food samples.

Background to the Invention

For public safety and quality control purposes the analysis of samples of consumer products, such as foodstuffs and beverages, for the presence of pathogenic microorganisms is conducted on an extremely wide scale. Because pathogenic microorganisms such as Listeria and Salmonella are capable of proliferating very rapidly under e right conditions, the presence of even a single viable cell of such organisms in a foodstuff may give rise to serious infection after the foodstuff has been ingested by a human. It is therefore necessary for such organisms to be detectable even when present at extremely low levels.

A typical conventional technique for detecting a target microorganism of interest, eg Salmonella, involves incubating a sample in a pre-enrichment broth, commonly buffered peptone water (BPW) for 18 to 24 hours under conditions which encourage microorganism recovery and growth, so that any organisms present can proliferate in the sample and attain population levels which are more readily detectable. This step is known as a pre- enrichment, recovery or resuscitation step.

Portions of the pre-enrichment culture are then subcultured into selective enrichment broths and incubated for a further 20 to 24 hours. The selective enrichment broths are designed to inhibit growth of comparatively innocuous non-target microorganisms and so favour the growth of the target microorganisms. This step is known as a selective enrichment step.

After the enrichment step, the target organisms are then identified. Conventional processing involves subculturing enrichment broths onto selective differential agar plates and incubating for 20 to 24 hours. Suspected colonies of target organism are identified by visual examination, and selected suspected colonies are removed from the plates, purified and identified, eg using triple sugar iron (TSI) agar and lysine iron (LSI) agar slopes and serological tests. Purification, identification and confirmation can take up to 48 hours.

Rapid methods of identifying microorganisms, eg using ELISA, electrical based methods, nucleic acid probes, PCR etc, have been devised and can give results in as little as 30 minutes. Despite promising results in laboratory-based experiments, when used in real food enrichments even the most sensitive commercially available systems require at least 10⁴ target cells ml"¹ of enrichment broth to generate a reliable result. This requires that pre-enrichment and selective enrichment are still used in order to achieve this number of target cells. Many of the new rapid tests have an even lower degree of sensitivity which can often be further reduced by high numbers of competing microorganisms.

Despite numerous modifications to the test protocol, the culture stage of pre-enrichment still remains essential. Direct inoculation of a naturally contaminated sample into selective enrichment media often results in a failure to recover the organisms of interest. This may occur because some healthy cells are often killed when added to selective culture media and thus if they are present in low numbers this may lead to the death of the entire population of the organism of interest. Pre-enrichment allows the number of organisms to increase, thus reducing the detrimental effects of the initial killing. Furthermore, sublethally damaged cells are more sensitive to selective agents than undamaged cells, and the concentration of selective agents used is unlikely to facilitate any recovery of injured organisms. In this case pre-enrichment allows the cells of interest to repair any lesions and thus regain their resistance to selective agents, prior to enrichment. In many cases it may be necessary to recover small numbers of damaged cells and thus both events occur during pre-enrichment, ie. damaged cells are able to repair and grow to much larger numbers before they are exposed to selective agents.

It was observed more than 85 years ago that any treatment which is more or less lethal to microbial populations leaves some of the surviving organisms damaged, to a greater or lesser extent. Such cells are still viable, though their recovery by normal procedures is negatively affected in two ways. The lag times are increased considerably, even when optimal recovery media are used and the growth of debilitated cells is prevented by the majority of selective liquid media, even though they support growth of the same organisms in an unstressed state. Methods of processing foods which can cause such damage/stress include heating, irradiation, acid or alkali conditions, drying or high ionic concentrations, exposure to preservatives, freezing and freeze-drying. Cells entering a food as an environmental contaminant may be starved, desiccated or previously exposed to disinfectants or antibiotics. In addition, cells that have been exposed to relatively mild stresses such as chilling and changes in oxygen level may also show evidence of sublethal damage/stress.

The present invention concerns reagents for use in the recovery and growth of microorganisms that have particular, but not exclusive, application as a recovery medium for use in a pre-enrichment step.

Summary of the Invention

The present invention provides reagents for use in the recovery and growth of microorganisms, comprising

(a) growth medium generating low levels of toxic oxidising species;

(b) a hydrogen donor; and

(c) freeze-dried OXYRASE enzyme.

Toxic oxidising species include hydrogen peroxide, hydroxyl radicals, superoxide anions and singlet oxygen. Toxic oxidising species may be generated internally by cells by various different mechanisms, or may be generated in the growth medium. The expression "generating low levels of toxic oxidising species" means that levels of toxic oxidising species generated in use of the medium are below those to which healthy cells of the microorganism(s) of interest are sensitive. It is difficult to quantify the expression "generating low levels of toxic oxidising species" by reference to the specific amounts of hydrogen peroxide, hydroxyl radicals, superoxide anions, singlet oxygen etc generated. However, attempts can instead be made to quantify compounds that are a source of toxic oxidising species and to measure the total amount of oxidising species.

Riboflavin is an important source of toxic oxidising species, generating such species on exposure to light. An optimised growth medium has been produced which contains 0.045mg/l riboflavin, and experiments involving adding riboflavin to the optimised medium have shown that performance of the medium is not significantly affected until more than 0.2mg/l riboflavin is added. On this basis, the growth medium desirably contains not more than 0.245 mg riboflavin per litre of suspension. The main sources of riboflavin in conventional dehydrated culture media are yeast and liver peptone, so it is appropriate for the growth medium to include such ingredients in small amounts and/or in forms low in riboflavin.

Riboflavin only produces toxic oxidising species on exposure to light, so an alternative approach is to maintain the growth medium in light- free conditions during storage and use.

Riboflavin levels are conveniently determined by high performance liquid chromatography (HPLC).

The level of hydrogen peroxide equivalent of the growth medium is preferably less than about 0.03mM, more preferably less than about 0.02mM.

Other vitamins, particularly B vitamins, may also act as sources of toxic oxidising species, so these should be present in the growth medium at suitably low levels.

It is also known that high levels of phosphates and amino acids in the presence of sugars contribute further toxic oxidising species, so these should be kept to a minimum.

Exposure to high temperatures, for example during autoclaving, can also generate toxic oxidising species, so filter sterilisation, eg using a membrane with pores 0.2μ in diameter, is preferred, at least with media containing high levels of sugars and phosphates. Alternatively sugars and phosphates can be separated and removed prior to autoclaving.

By using a growth medium generating low levels of toxic oxidising species, oxidative stress of microorganisms is reduced, thus leading to improved microorganism recovery and growth.

OXYRASE enzyme is a known recovery agent that aids recovery of stressed or damaged cells. OXYRASE is an enzyme composition which is made from sterilised bacterial membrane fragments, and which is know to be an effective oxygen-reducing enzyme used to produce anaerobic conditions. The OXYRASE enzyme is described in technical bulletins distributed by Oxyrase, Inc. of Ohio and is further described by Adler et al in J. Bacteriology, August 1981, 326-332, and in a paper by H I Adler in Critical Reviews of Biotechnology 10: 118 (1990) entitled "The Use of Microbial Membranes to Achieve Anaerobiosis". See also US Patents Nos. 4476224, 4996073 and 5204853 and WO88/04319. The word OXYRASE is a trade mark of Oxyrase, Inc, from whom OXYRASE enzyme is available as a frozen suspension.

OXYRASE enzyme is used in freeze-dried form, for ease of handling and storage and to increase shelf life. OXYRASE enzyme may be freeze-dried, e.g. using conventional freeze-drying techniques. For freeze-drying it is necessary to use the OXYRASE enzyme in a form without lactate and succinate, as freeze-drying is otherwise not possible. If necessary, lactate and succinate must be removed from the OXYRASE enzyme, eg by centrifuging, prior to freeze-drying.

The hydrogen donor should be present in sufficient amount for functioning of the enzymes contained in OXYRASE. Suitable donors include lactic acid, succinic acid, formic acid, alpha glycerol phosphate and salts of these materials. A mixture of hydrogen donors may be used. The currently preferred hydrogen donor is succinate, e.g. sodium succinate. A millimolar concentration of the hydrogen donor, in use, is generally ample to remove all dissolved oxygen. Good results have been obtained with hydrogen donor at a concentration of 15mM.

The hydrogen donor may be included as an ingredient of the growth medium, providing a 2 component recovery system, or may be presented as a separate reagent, eg in the form of a vial of liquid reagent, providing a 3 component recovery system. In the latter case, the growth medium may be more versatile and multi-purpose.

As a further possibility, the reagents may be presented in solid form, eg impregnated into agar, typically by adding agar to reagent broth in an amount 15g of agar per litre of broth. In this case it may be appropriate to use OXYRASE at increased concentration, eg at up to 10 times the concentration that would otherwise be appropriate.

OXYRASE enzyme causes the cells to grow anaerobically thus bypassing any internal pathways that produce toxic oxidising species such as hydrogen peroxide. OXYRASE enzyme also contains catalase which removes external hydrogen peroxide which is why when it is added to poor peptones at high concentrations it is still effective at improving recovery. Additionally, free nucleic acid and lipid material in die OXYRASE preparation may absorb toxic oxidising species. Additional catalase may optionally be included in the reagents.

For optimised microorganism recovery and growth it is necessary to optimise the nature and amount of growth medium and the amount of OXYRASE enzyme. Poorer performing growth medium combined wiui high concentrations of OXYRASE enzyme, and optimised growth medium combined with lower concentrations of OXYRASE enzyme, will achieve similar results in terms of speed and extent of microorganism recovery and growth. OXYRASE enzyme is expensive so it is therefore beneficial to use it at the lowest concentration possible. For this reason, use of an optimised growth medium is highly desirable. The combination of use of a growth medium generating low levels of toxic oxidising species together with OXYRASE enzyme enables significantly improved recovery, i.e. better and faster recovery, of stressed microorganism cells than is possible with conventional growth media. Not only are more stressed cells recoverable, but also recovery of all cells takes place in less time than under traditional conditions. Both the recovery medium and OXYRASE act to reduce oxidative stress, which is believed to be involved in cell recovery in a number of ways:

a) The growth medium contributes only low levels of toxic oxidising species, e.g. hydrogen peroxide, hydroxyl radicals, superoxide anions and singlet oxygen, to the medium, possibly generated by exposure to oxygen, light and/or high temperatures, eg on autoclaving.

b) Stressed cells are likely to be generating intracellular toxic oxidising species such as those mentioned above in a way that is independent of external levels. This explains why adding external catalase does not give maximum improvement to recovery. It is also possible that internal toxic oxidising species are generated at different rates in the different peptones.

c) As explained above, OXYRASE causes the cells to grow anaerobically thus bypassing any internal pathways that provide toxic oxidising species such as hydrogen peroxide. OXYRASE also contains catalase which removes external hydrogen peroxide which is why when it is added to poor growth media at high concentration it is still effective at improving recovery. Additionally, free nucleic acid and lipid material in the OXYRASE preparation may absorb toxic oxidising species.

The growth medium may be otherwise generally conventional in formulation. Typical ingredients include peptone, sodium chloride, buffer such as orthophosphate.

It is believed the reagents of the invention will work for a very wide range of microorganisms, bodi facultative and obligate anaerobes. It is also expected that the reagents of the invention will be effective with cells exposed to may different types of stress: so far they have been shown to be effective with cells damaged by heat, acid/salt combinations, hypochlorite and irradiation.

The reagents may be used for enriching the population of a target microorganism in a sample, which is the conventional first step in detecting a target microorganism of interest, e.g. Salmonella, in a sample such as a sample of a foodstuff, beverage or an environmental sample.

The reagents of the invention may, for example, be used as the pre-enrichment medium in methods of enriching the population of a target microorganism in a sample as disclosed in the specification of our copending British Patent Application No. 9714594.0 filed 11th July 1997 (pursued in a PCT application), which conveniently also involves the use of timed-release capsules containing selective agents.

The reagents of the invention also find use in other techniques requiring recovery and growth of microorganisms, particularly microorganisms in stressed, damaged or debilitated condition.

The reagents of the invention could also be used as an alternative to the traditional Buffered Peptone Water (BPW) in a pre-enrichment step either in a conventional 20-24h incubation period or in a shortened incubation period followed by an earlier transfer to selective enrichment media. They could be used with die most sensitive rapid detection techniques such as PCR whereby low numbers of target organism (where low numbers still means greater than 10³ cells/ml for successful PCR detection) can be detected in a very dense background flora of non-target organisms, thus possibly removing the need for any form of selective enrichment. Generation of such levels of target organisms is not possible in all situations with current pre-enrichment because of the Jameson Effect preventing increase in the target population before recovery is complete. With faster recovery of all stressed cells, using the reagents of the invention, this may enable high enough numbers of target cells to be generated before the Jameson Effect takes place.

The reagents of the invention find application in many areas in addition to the area of foodborne pathogen isolation. The reagents will be of interest for research purposes as well as for use in commercial testing in the recovery of so called "viable but non- culturable" microorganisms, particularly those present in soil and water samples. There is much interest in this field of microbiology from a medical perspective and there are likely to be many potential areas for application where the recovery of stressed/injured cells is important. Analysis of blood samples is one such area.

Another potential application area is in the study of preservation/disinfection treatments. Survival of new organisms against existing treatments and survival of existing organisms against new treatments are all measured by counting numbers of surviving viable cells against time of exposure. The existing techniques commonly involve plating out samples onto agar and counting colonies appearing after incubation. It is in the interests of the operators of these survival experiments to use the medium best able to recover all damaged as well as undamaged cells that may cause problems if such a treatment was used in everyday life. It is worth pointing out here that the recovery conditions created by the low oxidative stress/OXYRASE combination described in this specification are not by any means artificial nor do they give an unfair advantage to stressed cells that would otherwise not survive. In fact, the conditions created by use of reagents of the invention are highly realistic when compared to conditions micToorganisms might be exposed to in natural environments. Apart from when encountering a host defence system that uses oxidative stress as a means to kill invading cells it is unlikely for a microorganism to be expected to recover and grow in the presence of riboflavin and hydrogen peroxide. The recovery conditions created by use of the reagents of the invention very closely resemble those of an animal intestine.

The invention will be further described by way of illustration, in the following Examples and with reference to the accompanying Figures, in which:

Figures 1 and 2 are graphs of absorbance (600nm) versus time (h) illustrating the recovery of heat- injured Salmonella typhimurium using a conventional medium (Figure 1) and reagents in accordance with the invention (Figure 2); Figure 3 is a bar chart illustrating the effect of recovery conditions on the recovery of low levels of heat injured Salmonella typhimurium, showing the number of cells recovered relative to those obtained using a standard conventional medium (Buffered Peptone Water (BPW) from Oxoid Limited) expressed as difference in log_I0 cells recovered/ml; and

Figure 4 is a further bar chart illustrating the effect of recovery conditions on the recovery of low levels of heat injured Salmonella typhimurium, showing the number of cells recovered relative to those obtained using a standard conventional medium (Buffered Peptone Water (BPW) from Oxoid Limited) expressed as difference in log₁₀ cells recovered/ml.

Example 1

Optimised reagents for use in the recovery and growth of microorganisms comprise

(a) dehydrated optimised growth medium; and

(b) freeze-dried OXYRASE enzyme.

The growth medium (a) is intended to be dissolved in distilled water in the amount of 4.275g of medium in 225ml of water to produce a broth having the following composition:

(g per litre) Meat-based Peptone 10.0

Sodium chloride 5.0

Di-potassium hydrogen orthophosphate 3.5

Potassium di-hydrogen orthophosphate 1.5

Sodium succinate (anhydrous) 2.4

pH 7.2 ± 0.2

The peptone component of the pre-enrichment broth formulation is a meat based product comprising: % dry weight meat meal 82.3 tryptone 13.9 yeast extract 2.5 disodium hydrogen orthophosphate 1.3

The riboflavin content of the growth medium was measured by the technique described in Example 3, and was determined to be 0.045 mg/1.

The freeze-dried OXYRASE enzyme (b) is produced by freeze-drying in known manner OXYRASE enzyme obtained from Oxyrase, Inc. , after removal of succinate and lactate if present. For use with growth medium of the composition and in the amount described above, it is appropriate to use 50 units of OXYRASE (as measured by the method described in OXYRASE product literature). This amount of freeze-dried OXYRASE enzyme should be rehydrated with 2mls of sterile distilled water and mixed gently to avoid frothing.

The sodium succinate in the growth medium acts as a hydrogen donor for the enzymes contained in the OXYRASE, and is present in an amount that equates to 15mM.

Example 2

In a variant of Example 1 the sodium succinate is omitted from the growth medium and is present as a separate reagent in the form of a vial of liquid sodium succinate. The reagents thus constitute a 3 component recovery system comprising:

(a) dehydrated optimised growth medium;

(b) sodium succinate solution; and

(c) freeze-dried OXYRASE enzyme.

The reagents are otherwise the same as in Example 1, and are used generally the same way, but with me sodium succinate being added to me growth medium after rehydration. Alternatively, the sodium succinate solution may be used to rehydrate the freeze-dried OXYRASE enzyme.

Example 3

High Performance Liquid Chromatographic Method for the Quantification of Riboflavin in Yeast Extract.

1 - CHROMATOGRAPHIC CONDITIONS

Column :Phenomenex Prodigy 5μ C8 150MM*4.60mm Mobile phase :Eluent A: 5mM Hexanesulphonate, 20mM Potassium Dihydrogen Phosphate pH2.8 Eluent B: Methanol

Gradient Conditions :0-0.71 mins 10%B,, 0.71-4.71mins 10%-28%B, 4.71-6.71 mins 28%B, 6.21-7.21 mins 28 -32%B, 7.21-9.21 mins 32%B, 9.21- 10 mins 32%-40%B, 10-12.51 mins 40%B, 12.51-12.71 mins 40%- 50%B, 12.71-15.71 mins 50%B, 15.71-16.21 mins 50%-90%B, 16.21-22.71 mins 90%B, 22.71-26 mins 10%B

Temperature Ambient Flow rate 1.0 m minute Detection UV at 272nm Sensitivity 0.1 AUFS Inject volume 2Ojul Run time 30 minutes

2-PREPARATION OF SOLUTIONS

2.1 Dissolution Solvent

18.2MΩ Water. 2.2 Preparation of Mobile Phase.

Weigh out 0.96g of hexanesulphonic acid (Sigma 98% MW 188.2g) and 2.74g potassium dihydrogen phosphate (BDHG 99.5% MW 136.09) and dissolve in 600ml 18.2MΩ water. Adjust pH to 2.8 with phosphoric acid. Filter through 0.22 i filter. Degas with helium.

2.3 Preparation of Standard Solutions.

Dissolve 0.005g (5dp) riboflavin (Sigma) in 100ml dissolution solvent volumetrically in amber glassware.

Prepare 9 standards and filter through 0.22μ filter units into amber vials.

2.4 Preparation of Sample Solutions.

Volumetrically prepare a 5% (w/v 4dp) solution of sample in dissolution solvent. Condition an Alltech C8 cartridge with 5ml methanol followed by 5ml 18.2MΩ water. Accurately load 1ml of sample onto cartridge. Hush off interferences with 4ml 18.2MΩ water. Accurately load 2ml methanol onto cartridge and collect eluent in amber vial. Evaporate methanol off in oven. Reconstimte in 1ml 18.2MΩ water.

3-sγsτEM SUΓΓABΓJ ΠΎ

3.1 Inject one standard solution six times, calculate RSD (relative standard deviation) for the response areas of the six injections. The RSD should not be greater than 1.0% .

3.2 Inject a test solution twice. Calculate the R, between the sample peak and any ingredient peaks R, > 1.5. Example 4

Measurement of hydrogen peroxide levels in media

Hydrogen peroxide levels in pre-enrichment media were analysed using a YSI Model 2700 Select Biochemistry Analyser (Yellow Springs Instrument Company, Ohio, U.S.A.). The sensor probe is covered by a membrane which in instruments for detecting glucose and otiier similar metabolites would contain immobilised oxidase enzyme. Sample material containing the appropriate metabolite would diffuse into die membrane where the oxidase enzyme would rapidly oxidise the text metabolite and produce hydrogen peroxide which in turn would be detected at the electrode. For direct hydrogen peroxide measurements the oxidase membrane was replaced with a non-enzymatic protein membrane. This allowed hydrogen peroxide in the sample to diffuse directly through to the electrode. The electrode consists of a platinum anode and a silver cathode. Hydrogen peroxide is oxidised at the platinum electrode producing a flow of detectable electrons:

H₂O₂ → 2H⁺ + O₂ + 2e^*

The platinum anode is capable of oxidising many substances other than hydrogen peroxide. To prevent these reducing agents from contributing to sensor current, the membrane contains an inner layer consisting of a very thin film of cellulose acetate. The film readily passes hydrogen peroxide but excludes chemical compounds with molecular weights above approximately 200 Daltons. The film, however, can still be penetrated by compounds such as hydrogen sulphide, low molecular weight mercaptens, hydroxylamines, hydrazines, phenols and anilines.

For analysis of the respective pre-enrichment media, 50μl samples were drawn into the analyser for testing on two electrodes, one with die protecting cellulose acetate film and one without. Results were presented in m.mol l^'1 concentrations. Example 5

To compare the performance of reagents in accordance with the invention with conventional recovery media, tests were carried out using a newly devised technique based on recovery times of heat-injured Salmonella cells. The technique involves the generation of a standard heat-injured culture, serial dilution of this culture to near extinction, inoculation of the serial dilutions across many microtitre plates and measurement of the subsequent recovery and growth using an automated turbidometric analyser.

MATERIALS AND METHODS

Inoculum preparation

A vial of freeze-dried Salmonella typhimurium (Colworth House Microbiology Culture Collection No. 3073) was opened aseptically and its contents rehydrated in 1 ml of Heart Infusion Broth (HUB, Difco, 0038-17-7). A quantity (0.5ml) of the reconstituted culture was inoculated into 90 ml of fresh HIB and incubated shaking (80 rev min^"1) in a 250 ml conical flask in a waterbath at 37° C. After 20 h a sterile loop was used to remove a small volume of this broth culture and inoculate a slope of Heart Infusion Agar (HIA, Difco, 0044-17). This was incubated for 20 h at 37°C to give a dense lawn of growth and then stored at 4°C until required. Fresh slopes were made every 14 days.

The day before an experiment was to be carried out, a mid-exponential phase culture of Salmonella typhimurium was set up by transferring, by loop, a small portion of the slope culture into 9 ml of HIB. This was then incubated at 37 °C for 20 h without shaking. On the day of die experiment, 1 ml of this stationary phase culture was used to inoculate 90 ml of pre-warmed HIB in a 250 ml conical flask held in a shaking waterbath (80 rev min ¹) at 37°C. This culture was incubated for 3-4 h until its optical density (OD) at 600 nm against a HIB blank was 0.300. This indicated (when compared to a complete growth curve under these conditions) that die Salmonella population was in mid-exponential phase. A portion of this culture was then serially diluted in HIB and plated onto HIA to estimate cell numbers. Plates were incubated at 37 °C for 24 h before counting. Heat-injury

Approximately 10 ml of the mid-exponential phase culture was heat-injured at 53.5 °C for 15 minutes using a submerged-coil heating apparatus. This comprised a narrow bore stainless steel coil submerged in a thermostatically controlled waterbath, generally as described in Cole and Jones 1990, Letters in Applied Microbiology 11, 233-235. Before every experiment the coil was sterilized, flushed through several times with sterile distilled water and pre-heated to the appropriate temperature. The temperature was verified using a calibrated electronic probe held in the waterbath.

At intervals, 200 μl of heat-injured cells were expelled from the coil into 24 volumes of pre-enrichment media held at 20°C. The diluted cooled sample was split into two portions and one of them serially diluted in HIB and plated onto HIA and HIA with added NaCl (2.5% w/v). Plates were incubated at 37° C for 48 h before cpunting.

Pre-enrichment media

The kinetics of recovery and growth of heat-injured Salmonella were measured in a number of different pre-enrichment media; including Buffered Peptone Water, optimised peptone medium, yeast extract peptone medium and optimised peptone medium plus OXYRASE (0.2 units/ml). All media were prepared and handled according to the manufacturer's guidelines.

Automated growth curve analysis of recovery

The second portion of the 1 in 25 diluted culture of heat-injured cells was serially diluted, up to 10⁹ fold, through the pre-enrichment media under investigation. When comparing different pre-enrichment media the same first dilution was used before separately diluting the culture in each of the different broths. Each dilution was then used to inoculate up to 4 Bioscreen honeycomb plates. Up to 100 wells were filled (400 μl) for each dilution. The inoculated honeycomb plates were placed in the reading chamber of a Labsystems Bioscreen (Life Sciences International) where they were incubated at 37 °C and shaken at medium intensity for 5 sec prior to every reading. Measurements of the OD of all wells were made at a wavelength of 600 nm at 15 min intervals for 48 h. The data generated were then converted into Microsoft Excel format and where appropriate, processed into growth curves. The inoculum level was estimated from a most probable number (MPN) calculation on the number of positive and negative wells in the Bioscreen plates. Purity of Salmonella cultures, in Bioscreen plates and microtitre plates used for MPNs, were checked by streaking random well contents onto HIA and XLD agar (Oxoid CM469).

Most Probable Number analysis of recovery

The second portion of the 1 in 25 diluted culture was also used for an MPN analysis of cell numbers surviving and growing in the different pre-enrichment media. After serial dilution, the dilutions containing the fewest cells were placed into 96 well microtitre plates (200 μl well^"1). At least two plates were filled for each dilution. The plates were sealed with a plastics film (ICN How, UK) before being incubated at 37°C. After 48 h incubation the number of wells in which growth had occurred, ie. the number of turbid wells, was assessed visually, was recorded and an MPN estimate calculated using a standard MPN equation.

RESULTS

Heat-injury

For the specific Salmonella typhimurium strain and the conditions used in these experiments a heat treatment at 53.5° C was found to generate a large number of injured cells. At higher temperatures a large amount of death occurred and injury kinetics were too fast to be easily controlled, whereas at lower temperatures the time required to induce sufficient injury was too long to fit easily into the time course of the experiments. Injury was quantified by a cell being unable to grow on HIA containing added NaCl (2.5% w/v) but still being able to grow on a normal HIA plate with no added NaCl. The level of added NaCl was critical in providing a good separation of injured from healthy cells. Too much NaCl and some of die healthy cells could not grow and too little allowed some of the injured cells to grow. A heating time of 15 min was chosen as this generated a large population of injured cells but not too many dead cells. Using a controlled growth phase inoculum and the submerged coil heating apparatus this degree of injury was found to be easily reproducible (n = 13, mean = 97% injury, S.D. = 1.9).

Recovery times of heat-injured Salmonella

When recovery times of low levels of heat-injured Salmonella cells were measured a very broad distribution was commonly found. Often growth was not detected in the Bioscreen for more than 24 h. The distribution for uninjured cells at the same inoculum level, however, was much narrower with all growth curves being detected within 9 h. This was found in all the pre-enrichment media tested.

By way of illustration of the results obtained, Figures 1 and 2 are Bioscreen printouts (showing absorbance at 600nm versus time in hours) illustrating lag times of heat injured Salmonella typhimurium cells (injured by treatment at 53.5 °C for 15 minutes) incubated at 37°C with different recovery media at inoculum levels obtained by 10^"5 dilution of a 2 x 10⁸ cells/ml population of injured cells.

Figure 1 shows results using a conventional unmodified Buffered Peptone Water (BPW) from Oxoid Limited, and Figure 2 shows results using the reagents generally as described in Example 1, in the form of optimised peptone medium as described in Example 1 but not including succinate, with fresh OXYRASE (including succinate at the same level used in Example 1) added to give a working concentration of OXYRASE of 0.2 units/ml.

A comparison of Figures 1 and 2 shows much more rapid and uniform recovery of heat injured cells using reagents in accordance with the invention as compared with conventional BPW.

Most probable number analysis of recovery of heat-injured Salmonella

Because of the variability found in the number of injured cells recoverable in the different pre-enrichment media, using an MPN estimation on the Bioscreen plate data, a more accurate s dy was carried out using a microtitre plate MPN method which allowed many more wells to be inoculated (192 wells dilution^"1).

Results are shown graphically in Figure 3 for:

(a) standard (unmodified) Oxoid Buffered Peptone Water, which contains approximately 0.59mg/litre riboflavin and 0.032mM hydrogen peroxide equivalent;

(b) optimised peptone medium generally as described in Example 1, without OXYRASE and without succinate (the addition of succinate does not change me performance, although not indicated here), which contains approximately 0.045mg/litre riboflavin and 0.021mM hydrogen peroxide equivalent;

(c) optimised peptone medium generally as described in Example 1 but not including succinate, with fresh OXYRASE (including succinate at the same level used in Example 1) added to give a working concentration of OXYRASE of 0.2 units/ml. Riboflavin and hydrogen peroxide levels were as in (b).

(d) a medium where the peptone component is made up entirely of yeast extract, a very rich source of riboflavin, which contains approximately 1.8mg/litre riboflavin and 0.038 hydrogen peroxide equivalent.

Riboflavin levels were measured as described in Example 3, and hydrogen peroxide equivalents were measured as described in Example 4.

The results show dramatically enhanced recovery using the reagents in accordance with the invention.

It was noticed that the medium that recovered the most injured cells also gave the least variation in recovery times at the lowest inoculum levels. Example 6

To compare the effect of riboflavin levels in growth media on the recovery times of heat- injured Salmonella cells, experiments were carried out using the technique described in Example 4 using the optimised growth medium of Example 1 (which contains riboflavin in an amount of 0.045 mg/1) ("optimised peptone", used as a control) and the optimised peptone with various amounts of riboflavin added prior to autoclaving of the media. The results are shown graphically in Figure 4. The results show that adding up to 0.2 mg/1 riboflavin has no significant effect on recovery, but at higher levels of added riboflavin recovery deteriorates significantly. On the basis of these results it is preferred that the growth medium of the invention should contain riboflavin at a concentration not exceeding 0.245 mg/1.

Comparing Figures 3 and 4 it will be noted there are apparentiy differences in performance in the same reagents in different experiments. This is commonly found because of minor variations in exact experimental conditions, particularly the extent of heat injury of the cells. This means that an absolute measure of performance is not obtainable.

Claims

1. Reagents for use in the recovery and growth of microorganisms, comprising

(a) growth medium generating low levels of toxic oxidising species;

(b) a hydrogen donor; and

(c) freeze-dried OXYRASE enzyme.

2. Reagents according to claim 1, wherein the growth medium contains not more than 0.245 mg riboflavin per litre.

3. Reagents according to claim 1 or 2, wherein the growth medium has a level of hydrogen peroxide equivalent of less than about 0.03mM.

4. Reagents according to claim 1, 2 or 3, wherein the hydrogen donor comprises one or more of lactic acid, succinic acid, formic acid, alpha glycerol phosphate and salts of these materials.

5. Reagents according to claim 4, wherein the hydrogen donor comprises succinate.

6. Reagents according to any one of the preceding claims, wherein the hydrogen donor is present at a concentration, in use, of at least one millimolar.

7. Reagents according to any one of the preceding claims, wherein the hydrogen donor is included as an ingredient of the growth medium.

8. Reagents according to any one of claims 1 to 6, wherein the hydrogen donor is supplied as a separate reagent.

9. Reagents according to any one of claims 1 to 6, wherein the reagents are presented in solid form.

10. Reagents according to claim 9, impregnated into agar.

11. Reagents according to any one of the preceding claims, wherein die growth medium includes ingredients selected from peptone, sodium chloride, buffer.