R. Gorman and C.C. Adley Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland

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1 Letters in Applied Microbiology 2004, 38, doi: /j x x An evaluation of five preservation techniques and conventional freezing temperatures of )20 C and )85 C for long-term preservation of Campylobacter jejuni R. Gorman and C.C. Adley Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland 2003/0402: received 14 May 2003, revised 9 January 2004 and accepted 22 January 2004 ABSTRACT R. G O R M A N A N D C.C. A D L E Y Aims: This study aimed to identify a simple, inexpensive preservation technique that will allow a quick and reliable recovery of Campylobacter jejuni following long-term periods of preservation. Methods and Results: Preservation techniques include (i) Cryobank microbial preservation system using hypertonic cryopreservative solution and glass beads, (ii) Cryobank microbial preservation system using defibrinated lysed horse blood and glass beads, (iii) FBP medium, (iv) 15% glycerol/85% nutrient broth no. 2 culture, and (v) 50% glycerol/50% nutrient broth no. 2 culture. Each preservation technique was evaluated over a 1-year period at conventional freezing temperatures of )20 C and )85 C. Replacement of cryopreservative fluid in commercially prepared vials of glass beads with lysed horse blood increased the duration of preservation of Camp. jejuni by up to 6 months. Conclusions: FBP medium proved the most successful preservation technique with 100 and 80% recovery after 1 year at )85 C and )20 C, respectively. Significance and Impact of the Study: This study demonstrated a simple inexpensive preservation method for long-term storage of Camp. jejuni. Keywords: Campylobacter jejuni, cryopreservation, FBP medium, lysed horse blood. INTRODUCTION Campylobacter spp. can cause a wide spectrum of infections including, human diarrhoeal disease, reproductive disorders in domestic animals and opportunistic infections in immuno-compromised human patients (Park et al. 1991). There have been many reports of an association between Campylobacter jejuni (Camp. jejuni) enteritis and Guillain- Barré syndrome (Ang et al. 2000; Hadden and Gregson 2001). Globally Camp. jejuni has been recognized as a leading cause of human gastroenteritis (Frost et al. 1996; Altekruse et al. 1999; Whyte and Igoe 1999). This worldwide recognition has generated considerable interest in the development of special selective techniques, for optimal Correspondence to: Dr Catherine C. Adley, Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland ( Catherine.Adley@ul.ie). growth and isolation of Campylobacter from clinical and environmental sources (Butzler and Skirrow 1979; Park et al. 1991; Tran 1998). However, the preservation of Campylobacter spp. long term has posed problems. A number of published reports have described simple short-term preservation techniques for Campylobacter (Amies 1967; Wang et al. 1980; Amos 1981; Luechtefeld et al. 1981; Rogol et al. 1990). However, long-term preservation is required for storage of quality control strains, teaching, research, epidemiological purposes and quantitative and qualitative analyses (White and Sands 1985; Rogol et al. 1990). Extended preservation of Campylobacter is hindered by its sensitivity to oxygen (Hoffman et al. 1979; Bolton et al. 1984; Lee et al. 1988) and the formation of a viable but non-culturable form of the bacteria (Jones et al. 1991; Saha and Sanyal 1991). Long-term preservation of Campylobacter described in the literature involves liquid ª 2004 The Society for Applied Microbiology

2 LONG-TERM PRESERVATION OF CAMP. JEJUNI 307 drying (Malik and Lang 1996), liquid nitrogen and freezedrying (Mills and Gherna 1988), however, these methods require equipment not available in all laboratories. This study was an observation of the potential of five preservation methods to preserve Campylobacter spp. during storage by conventional freezing at )20 C and )85 C to detect which methods were most effective. MATERIALS AND METHODS Materials Bacteriological agar, Columbia agar base, nutrient broth no. 2, yeast extract, CampyGen TM microaerophilic sachets and FBP campylobacter growth supplement [0Æ025% sodium pyruvate (w/v), 0Æ025% sodium metabisulphite (w/v), 0Æ025% ferrous sulphate (w/v)] were purchased from Oxoid (Basingstoke, UK). Defibrinated lysed horse blood was purchased from Unitech (Dublin, Ireland). The commercial cryobank microbial preservation system was purchased from MAST diagnostics (Merseyside, UK). Glycerol was purchased form R.B Chemicals (Tallaght, Dublin, Ireland). The medium was cooled to 50 C and FBP enrichment supplement added aseptically. The medium was gently mixed and 4-ml volumes dispensed aseptically into sterile 15-ml universal tubes. A quantity of 500 ll of fresh Camp. jejuni (Mc Farland 3 4) were inoculated into each of two 4-ml FBP vials. One vial was stored at )20 C and the other at )85 C. Recovery of the organism required complete thawing of the medium. A sterile 10-ll loop of culture was streaked onto a CBA plate, and incubated at 37 C for 48 h under microaerophilic conditions. iv 15% glycerol/85% nutrient broth no. 2 culture. A quantity of 850 ll of fresh Camp. jejuni culture (Mc Farland 3 4) was aseptically transferred into two vials of 150-ll sterile glycerol. The mixtures were emulsified by vortexing and one stored at )20 C and the other at )85 C. Organisms were recovered as described in (iii) above). v 50% glycerol/50% nutrient broth no. 2 culture. This procedure is as described previously for (iv) above except that, 500 ll of culture broth was emulsified with 500 ll of sterile glycerol. Bacterial strains and culture conditions The strains studied were five fresh isolates of Camp. jejuni that were obtained from the Microbiology Laboratory at the Mid-Western Regional Hospital, Limerick, Ireland. These strains were isolated from patients suffering from gastroenteritis. All five cultures were stored using five different preservation methods, at both )20 C (Tricity) and )85 C (Nuaire )85 C Ultralow freezer). Each strain was monitored for viability at 1, 2, 3, 6, 9 and 12-month intervals by subculturing on Columbia Blood Agar (CBA) and incubated at 37 C for 48 h under microaerophilic conditions (5% O 2, 10% CO 2, 85% N 2 ) (Gorman and Adley 2002). Preservation methods i Cryobank microbial preservation system using hypertonic cryopreservative solution and glass beads. This procedure was performed according to manufacturer s instructions. ii Cryobank microbial preservation system using defibrinated lysed horse blood and glass beads. This procedure is as described previously for (i) above with the following exception, prior to harvesting the hypertonic cryopreservative solution was replaced with 750 ll defibrinated lysed horse blood. iii FBP medium. This medium was prepared by autoclaving nutrient broth no. 2, 0Æ12% [w/v] bacteriological agar, 15% [v/v] glycerol and 0Æ1% [w/v] yeast extract. RESULTS Following incubation, CBA plates were examined for culture growth. An estimate of growth of the test strains was made based on the following scale: confluent growth, 3+; semi-confluent growth, 2+; <10 colonies in the primary inoculated area, 1+; no visible growth, ). Results of Camp. jejuni viability over a 12-month period following storage at )20 C and )85 C are summarized in Table 1. DISCUSSION White and Sands (1985) and Feltham et al. (1978) both studied cryopreservation of bacteria using glass beads. This method eliminated the detrimental effects that freezethawing can have on bacteria. This method was not assessed using Campylobacter strains but recovery of sensitive organisms was successful (White and Sands 1985). In our laboratory, strains of bacteria such as E. coli and Salmonella Typhimurium have been recovered from glass beads when stored at )85 C for more than 1 year. Blood is an undefined medium containing iron and detoxifying enzymes such as catalase, peroxidase and superoxide dismutase, which have been shown to reduce toxicity of media (Hoffman et al. 1979). Blood has been recognized as an excellent supplement in the growth and recovery of Campylobacter spp. (Wang et al. 1980; Bolton and Coates 1983; Bolton et al. 1984; Rogol et al. 1990). This study has optimized the advantages of blood and cryogenic glass beads in maintaining viability of Camp. jejuni

3 308 R. GORMAN AND C.C. ADLEY Table 1 Recovery of Campylobacter jejuni over a 12-month period following storage at )20 C and )85 C No. of months Preservation temperature ( C) Preservation method* Isolate no. )20 )85 )20 )85 )20 )85 )20 )85 )20 )85 )20 )85 (i) 1 ) ) ) ++ ) ) ) + ) ) ) ) ) ) ) ) ) 3 ) ++ ) ++ ) ) ) + ) ) ) ) ) ) ) ++ ) + ) ) ) ) ) + ) + ) + ) ) ) ) % Recovery (ii) 1 ) +++ ) +++ ) +++ ) +++ ) ) ) ) +++ ) +++ ) +++ ) +++ ) ++ ) ) +++ ) +++ ) +++ ) ++ ) ) ) +++ ) +++ ) + ) ) +++ ) +++ ) +++ ) +++ % Recovery (iii) ) ) % Recovery (iv) ) +++ ) ) ) ) +++ ) +++ ) +++ % Recovery (v) ) ) ) ) ) ) ) ) ) ) ) ) +++ ) ) ) ) ++ ) ) ) ++ ) +++ ) ) ) ) +++ ) +++ ) ) ) ++ ) +++ ) +++ ) ) % Recovery Confluent growth, 3+; semi-confluent growth, 2+; <10 colonies in the primary inoculated area, 1+; no visible growth, ). *Preservation methods: (i) Cryobank microbial preservation system using hypertonic cryopreservative solution (MAST diagnostics); (ii) Cryobank microbial preservation system using defibrinated lysed horse blood; (iii) FBP medium; (iv) 15% glycerol/85% nutrient broth no. 2 culture; (v) 50% glycerol/50% nutrient broth no. 2 culture. Indicates a test period where no growth was observed but recovery of Campylobacter was found at the next test period. when stored at low temperatures for long periods of time. Replacement of the cryopreservative fluid in the preprepared vial of glass beads with lysed horse blood increased the duration of preservation of Camp. jejuni by up to 6 months when stored at )85 C. During preparation, each individual bead within the vial becomes coated with the inoculated cryopreservative fluid or the inoculated lysed horse blood. Camp. jejuni recovery from cryogenic glass beads stored at )20 C showed poor growth using both these cryopreservative methods. In contrast however, when stored at )85 C a substantial difference in the preservation capability of the cryogenic glass beads for storage of Camp. jejuni when cryopreservative fluid was replaced with lysed horse blood was observed. In our laboratory, strains of Camp. jejuni stored at )85 C have been recovered after 20 months using the cryogenic glass beads replaced with blood method. Difficulty in finding one individual component to replace blood as a supplement in Campylobacter media has been recognized (Bolton and Coates 1983). In a study of 22 supplements screened by Bolton and Coates (1983), only seven were found to facilitate aerotolerance, the most effective being blood. No other individual supplement was as good as lysed horse blood.

4 LONG-TERM PRESERVATION OF CAMP. JEJUNI 309 However, the combination of charcoal, a detoxifying agent, ferrous sulphate and sodium pyruvate (CFP) was found to be effective (Bolton and Coates 1983). George et al. (1978) first described FBP supplement in Campylobacter media to enhance aerotolerance and growth of Camp. fetus. The FBP medium used in this study was the most successful preservation technique examined, after periodic testing over 12 months, 100 and 80% recovery was obtained from strains stored at )85 C and )20 C, respectively. Although the use of cryogenic glass beads eliminates the effects of freeze-thawing, the FBP medium, which required thawing at each test period, was superior at maintaining Camp. jejuni viability at )20 C than the cryogenic glass beads with or without the use of lysed horse blood, and was as effective as the cryogenic glass beads with lysed horse blood following storage at )85 C. Furthermore, in our laboratory Camp. jejuni stored at )20 C in this FBP medium have been recovered after 20 months of storage. The incorporation of FBP in the basal medium, with or without additional supplements, has previously being demonstrated as very successful in maintaining viability of Campylobacter at various temperatures (Rogol et al. 1990; Saha and Sanyal 1991). Temperature is a very important factor for preservation of micro-organisms, and various storage temperatures for preservation of Campylobacter spp. have been studied and employed (Amos 1981; Luechtefeld et al. 1981; Lee et al. 1988; Rogol et al. 1990; Saha and Sanyal 1991). Freezing methods have been utilized for mid-term and long-term preservation of Campylobacter. Mills and Gherna (1988) have carried out a long-term cryopreservation study of Campylobacter using 10% glycerol and Brucella Albimi broth as preservation media. Results from this study found liquid nitrogen to be the most efficient method for maintaining Campylobacter viability. Slow rate freeze-drying was the next most successful, followed by conventional freezing at )65 C. Each method showed viability for up to 2 years. Storage at )20 C in 10% glycerol maintained Campylobacter viability for 7 months. Similarly, results from our study using 15% glycerol maintained 100% Campylobacter viability for 12 months when stored at )85 C. Furthermore, stocks of Camp. jejuni have been recovered after 20 months under such conditions in our laboratory. Malik and Lang (1996) describe the very successful preservation of Campylobacter spp. by liquid-drying under anaerobic conditions with very good recovery rates following storage at both )30 C and )80 C for 12 months. Mills and Gherna (1988) identified liquid nitrogen as an excellent method surpassing conventional freezing conditions for long-term storage of Campylobacter for up to 4 years. Although the uses of liquid nitrogen and freeze-drying have been very successful with regard to long-term storage of Campylobacter, not all laboratories possess the equipment necessary. Saha and Sanyal (1991) have found that at a higher temperature of )10 C Camp. jejuni stored in 15% glycerol loses viability after just 45 days. Similarly, in this study, recovery of samples stored at )20 C gave 100% recovery at 3 months and just 20% recovery at 12 months. Although 50% glycerol stocks are widely used for longterm storage of bacteria such as Salm. Typhimurium, E. coli and Staphylococcus aureus, variable results were observed during Camp. jejuni preservation. In summary, this study has adapted the success of commercial cryogenic glass beads (Feltham et al. 1978; White and Sands 1985). Supplements including lysed horse blood and FBP and the cryopreservative agent glycerol to conventional freezing temperatures of )20 C and )85 C, one of which is available in most scientific laboratories. Five preservation methods were studied for their potential for long-term storage of Campylobacter spp. using two conventional freezing temperatures and identified just one i.e. FBP medium, that could be recommended as a simple and reliable method for long-term storage of Camp. jejuni at )20 C. Additionally, where deep-freezers at much lower temperatures of )85 C are available a selection or combination of FBP medium, the commercial Cryobank microbial preservation system using defibrinated lysed horse blood and glass beads, or 15% glycerol/85% nutrient broth no. 2 culture, can be recommended as successful methods for long-term preservation of Camp. jejuni. ACKNOWLEDGEMENTS The authors would like to acknowledge the assistance afforded by the personnel of the Microbiology Laboratory at the Mid-West Regional Hospital, Limerick, Ireland. This work was funded by an Irish American Partnership and Strategic Research Grant (no. ST99/043). 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