Long-term storage of Campylobacter pylori

Vol. 27. No. 7

JOURNAL OF CLINICAL MICROBIOLOGY. July 1989. p. 1655-1656

0095-1137/89/071655-02$02 .00/0 Copyright (O 1989. American Society for Microbiology

NOTES

Long-Term Storage of Campylobacter pylori B. DRUMM* AND P. SHERMAN

Division

of`Gastrroetteiuology,,

Department of Pediatiniûs, ResearcIt Institute, T/e Hospital for Sick Chljidreiz, Tornonto, Ontario M5G IX8, Canada

University oJf Tononto,

Received 26 January 1989/Accepted 21 March 1989

A reproducible method for long-term storage of Campylobacter pylori has not been previously described. We cultured 10 strains of C. pylori in brucella broth that was supplemented with 10% fetal bovine serum. After incubation for 24 h, 10% glycerol and an additional 10% fetal bovine serum were added to the cultures, which were then held in storage at -70°C. After 6 months, each of the strains remained viable. This technique, therefore, represents an excellent method for long-term storage of C. pylori isolates.

yielded 108 viable CFU/ml of inoculated broth. Following incubation, the liquid growth was examined by phase-contrast microscopy to confirm the morphology of the organisms and to exclude the possibility of contaminating organisms. Contamination of liquid broth cultures was occasionally observed. Following supplementation of brucella broth with vancomycin (10 p.g/ml) and trimethoprim (5 p.g/ml), the problem of contamination was eliminated. For storage, 1.2 ml of sterile 100%c glycerol and 1.2 ml of sterile fetal bovine serum were added to the broth culture of C. pylori. After being mixed, 0.5-ml portions were placed into sterile cryovials (Simport Plastics, Belloeil, Quebec, Canada) and held in storage at -70°C. To rule out the possibility of contaminating bacteria. 0.1 ml of the broth culture was inoculated onto two blood agar plates composed of Columbia blood agar base (GIBCO) supplemented with 7% defibrinated horse blood (3). One plate was incubated at 37°C under microaerophilic conditions, and the second plate was held under ambient environmental conditions of 37°C. Initially, three strains of C. pvloni were grown and stored by these methods. Each of the three strains stored after growth in broth was successfully recultured at 1, 2, 4, and 6 months after placement into storage at -70°C. On each occasion, even after 6 months, there were 108 CFU/ml cultured from the stored vials. The organisms were demonstrated by both phase-contrast microscopy and Gram stain to have a curved or spiral shape identical to that seen with primary isolates of C. p/lo)i. Under the phase-contrast microscope. the organisms had a darting motion typical of campylobacters. The organisms produced urease, catalase. and oxidase, and they were negative for nitrate reduction and hippurate hydrolysis (7). They were resistant to nalidixic acid but sensitive to cephalothin. When viewed under the transmission electron microscope, after being stained with 2% phosphotungstic acid, the organisms had smooth coats and multiple sheathed polar flagella typical of C. p/loni. Since then, seven more strains have been successfully stored for periods of at least 6 months. The three initial strains are viable 1 year after storage. In contrast, when the original three C. pvl/oi isolates were placed directly from agar plates into either glycerol or glycerol supplemented with fetal bovine serum, they were not viable even after storage at -70°C for only 1 month. These findings indicate that this method is suitable for

A recently identified gram-negative organism, currently referred to as Campylobactei pvloni, has been associated with both gastritis and peptic ulcer disease in adults (1, 8). We have previously shown an association of the presence of C. pvloni on the antrum of children with both primary gastritis and duodenal ulcer disease (2, 3). Further experimental work to evaluate its etiologic role in disease requires that human C. pvlori isolates be easily available to multiple research laboratories. However, it has been our experience, and that of other investigators, that it is difficult to store C. pylori (T. U. Westblom, J. S. Barthel, A. D. Havey, F. J. Gonzalez, E. F. Tarka, and E. Everett, Letter, J. Clin. Pathol. 40:353, 1987). C. pvloni is not consistently recovered after storage in liquid broth supplemented with 15%c glycerol. Thus, unlike other campylobacters, these organisms may be very sensitive to freezing (Westblom et al., Letter). We now describe a simple and reliable method for successful longterm storage of C. pvloni. In addition, this method allows for storage of large numbers of organisms even in the face of scant growth of bacteria from the primary culture of human antral mucosa. In this study, 10 primary cultures of C. pylori were isolated from antral biopsy material (3, 5) obtained from 10 patients undergoing upper gastrointestinal endoscopy. Growth of these human C. pylori isolates in liquid broth medium was achieved by using a method similar to that recently described by Morgan et al. (6). Brucella broth (10 ml; GIBCO Laboratories, Madison, Wis.) supplemented with 10% fetal bovine serum (Bocknek Laboratories Inc., Rexdale, Ontario, Canada) was added to a sterile disposable Erlenmeyer flask (Corning, N.Y.). Scrapings from the primary growth of C. p/loni were inoculated into the broth. The flask, with a loosely fitted screw top, was then placed into an evacuation jar (GasPak 100 anaerobic system. vented; John's Scientific Inc., Toronto, Ontario, Canada). The jar was partially evacuated and then flushed with carbon dioxide and hydrogen to create an environment approximating 7% oxygen, 10% carbon dioxide, 58% hydrogen, and 25% nitrogen (4). The sealed evacuation jar was held at 37°C for 24 h while being shaken at 70 rpm (American Rotator V; American Dade, Miami, Fla.). Culture of C. pvloni in liquid broth for 24 h repeatedly * Corresponding author. 1655

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successful long-term storage of C. pylori. The organisms stored in this manner appeared biochemically, morphologically, and ultrastructurally identical to primary isolates of C. pylori. Morgan et al. previously reported that C. pylori grown in liquid medium appears similar to the organism in vivo (6). In contrast, C. pylori cultured on agar plates has a different morphology (6). In addition, this method provides a way to store, from a single primary culture, multiple portions, each of which contains large numbers of viable organisms. The availability of large amounts of C. pylori isolated from the same patient should prove valuable for moredetailed investigations of identical strains in multiple research laboratories. P. Sherman is the recipient of a Career Scientist Award from the Ontario Ministry of Health Research Development Program. We thank Mohamed Karmali for his advice and support.

LITERATURE CITED 1. Blaser, M. J. 1987. Gastric campylobacter-like organisms, gastritis, and peptic ulcer disease. Gastroenterology 93:371-383.

J. CLIN. MICROBIOL.

2. Drumm, B., A. O'Brien, E. Cutz, and P. Sherman. 1987. Campylobacter pyloridis-associated primary gastritis in children. Pediatrics 80:192-195. 3. Drumm, B., P. Sherman, E. Cutz, and M. Karmali. 1987. Association of Canmpylobacter pylori on the gastric mucosa with antral gastritis in children. N. Engl. J. Med. 316:1557-1561. 4. Karmali, M. A., A. E. Simor, M. Roscoe, P. C. Fleming, S. S. Smith, and J. Lane. 1986. Evaluation of a blood-free, charcoalbased, selective medium for the isolation of Campylobacter organisms from feces. J. Clin. Microbiol. 23:456-459. 5. Marshall, B. J., H. Royce, D. I. Annear, C. S. Goodwin, J. W. Pearman, J. R. Warren, and J. A. Armstrong. 1984. Original isolation of Campylobacter pyloridis from human gastric mucosa. Microb. Lett. 25:83-88. 6. Morgan, D. R., R. Freedman, C. E. Depew, and W. G. Kraft. 1987. Growth of Campylobacter pylori in liquid media. J. Clin. Microbiol. 25:2123-2125. 7. Penner, J. L. 1988. The genus Campylobacter: a decade of progress. Clin. Microbiol. Rev. 1:157-172. 8. Warren, J. R., and B. J. Marshall. 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i: 1273-1275.