Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm2 tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log10 cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus, which was non-culturable in broth culture, attained population densities of 109 cells ml-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm2 resulted in increased cell densities

Baetz, A.

Dickson, James

Manke, Tamara

Wesley, I. V.

Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm2 tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log10 cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus, which was non-culturable in broth culture, attained population densities of 109 cells ml-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm2 resulted in increased cell densities.This article is from Letters in Applied Microbiology 22 (1996): 195, doi:10.1111/j.1472-765X.1996.tb01141.x.</p

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Letters in Applied Microbiology 1996, 22, 195-1 98 Biphasic culture of Arcobacter spp. J.S. Dickson, T.R. Manke, I.V. Wesley' and A.L. Baetz' Department of Microbiology, Immunology and Preventive Medicine, Iowa State University, and 'United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA, USA FS/234: received 9 September 1994, revised 10 July 1995 and accepted 2 August 1995 J .S .  D I C K S O N ,  T . R .  M A N K E ,  I .V.  W E S L E Y  A N D  A .L .  B A E T Z .  1996. Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm2 tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log,,, cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus, which was non-culturable in broth culture, attained population densities of lo9 cells ml-' in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm2 resulted in increased cell densities. INTRODUCTION Arcobacter spp. were first described in the late 1970s, although they were initially identified as Campylobacter (Neil1 et al. 1985). They are microscopically similar to Campylobacter, but differ physiologically in their ability to grow aerobically at 30°C, while Campylobacter spp. require higher temperatures and microaerophilic atmospheres. However, Arcobacter. spp. grow poorly on Campylobacter isolation media, making cul- tivation difficult. In 1991, Vandamme proposed the genus Arcobacter for these aerotolerant Campylobacters, based on immunotyping and DNA hybridization studies (Vandamme et al. 1991). Arcobacter spp. are fastidious bacteria that are difficult to cultivate. Some species grow poorly on blood agar plates (Vandamme et al. 1992), and most are difficult to grow on high cell densities in liquid culture systems. For many bac- teriological studies, it is advantageous to have concentrated cultures of the specific bacterium and to able to produce these cultures consistently. The biphase culture technique consists of a solid phase of appropriate agar overlayed with a liquid phase of saline, buffer or broth. An early reference to biphase culture techniques reported the enhanced bacterial production of levansucrase (Hestrin et al. 1943), and this technique was formally docu- Correspondence to : Dr3. S. Dickson, Department of Microbiology, Immunology and Preventive Medicine, 207 Science I,  Iowa State University, Ames, IA 50011, USA. Q 1996 The Society for Applied Bacteriology mented for routine blood cultures in 1947 (Castaneda 1947). Biphase culture techniques have been used to produce con- centrated cultures of many types of bacteria (Tyrrell et al. 1958; Hall et al. 1979). In addition, a biphase culture tech- nique has been described to enhance the growth and cell numbers of Campylobacter spp. (Rollins et al. 1983). The specific objectives of this study were to determine if biphase culture techniques could enhance the growth of Arcobacter spp., and if the inclusion of fetal bovine serum into the liquid phase would augment growth. MATERIALS AND METHODS Bacterial cultures Five strains of Arcobacter spp., A .  butzleri, A .  skirrowii 3699, A .  skirrowii 3700, A. c yaerophilus 3252 and A. cyaerophilus 3256 (ATCC 49615) were obtained from the USDA-ARS National Animal Disease Center, Ames, IA, USA. The lyophilized cultures were revived in brain heart infusion broth (BHI ; Accumedia, Baltimore, MD, USA) and maintained in biphasic culture (see following description). Culture techniques Liquid media consisted of either BHI broth or BHI+ 10% (v/v) inactivated fetal bovine serum (BHI + FBS). Solid 196 J.S. DICKSON E T A L .  medium consisted of 10% bovine blood in Tryptose Blood Agar Base (BLA; Difco; Detroit, MI, USA). Liquid culture was conducted in screw capped tubes. Biphase culture sys- tems were constructed as described by Krieg and Gerhardt (1994) using 25 or 75 cm2 tissue culture flasks, by adding 7.0 or 15.0 ml of molten BLA, respectively, to the flask and allowing it to solidify on the surface. After the agar solidified, 4.5 ml BHI or 9.0 ml BHI + FBS was added as a liquid phase overlay. Experimental design The objective of experiment 1 was to determine the effect on bacterial populations of biphase culture in comparison to conventional liquid culture. The  liquid medium was also supplemented with fetal bovine serum to determine if this enhanced growth. The  bacteria were inoculated inde- pendently with a loopful of a 24 h culture into 9.0 ml tubes of BHI broth and thoroughly mixed. After mixing, 4.5 ml of the inoculated broth was transferred to biphase cultures constructed in 25 cmz tissue culture flasks. This technique assured that the initial populations in both the broth and biphase cultures were equivalent. The experiments were also replicated using comparable liquid and biphase culture sys- tems prepared with BHI+FBS broth. The  culture systems were incubated at 30°C in a gyrorotatory incubator a t  50 rev min-I for 48 h in ambient air. Biphase culture systems were incubated horizontally, with the liquid phase overlaying the solid phase. The  objective of experiment 2 was to determine if the surface area of the biphase culture system had an effect on bacterial populations. Briefly, biphase culture systems were constructed with 25 or 75 cm2 tissue culture flasks, and inocu- lated and incubated as previously described. The  objective of experiment 3 was to determine the gen- eration times for A. skirrowii 3699 in both conventional liquid and biphase culture systems. Approximately 500 cells of the bacterium were inoculated into either 9 ml tubes or 75 cmz flasks and incubated at 30°C at 50 rev min-I. Samples were evaluated for both optical density (650 nm) and viable cell population by plating. Bacterial enumeration The populations of A. butzleri and A. skirrowii 3699 were enumerated by serially diluting in buffered peptone water (Accumedia) and surface plating on BLA. The plates were incubated at 30°C for 24-48 h in ambient air. The populations of A.  skirrowii 3700, A. cryaerophilus 3252 and A. cryaerophilus 3256 produced extremely small colonies on BLA plates which could not be enumerated without the aid of a low mag- nification microscope, and these cultures were subsequently enumerated by direct microscopic count with a Petroff- Hauser counting chamber. Statistical analysis Each experiment was independently replicated twice, and the populations of bacteria were converted to log,, colony- forming units (cfu) ml-'. The  data were analysed using the general linear models procedure of Statistical Analysis Sys- tem (Anon. 1985). Generation times were calculated using the formula : C t  = t / N ;  where t equals time in hours, and N is calculated using the formula : N = 3.3 log,, (x/xo) ; where x, equals the initial bacterial population and x equals the population after t hours (Pelczar et al. 1977). RESULTS AND DISCUSSION Biphase culture systems significantly ( P  < 0.05) increased the populations of all of the Arcobacter species studied (Table 1). This increase ranged from approximately 1 log,, cycle for A. cryaerophilus 3256 to approximately 2 log,, cycles for A. butzleri. The extreme case was A. cryaerophilus 3252, which showed no detectable growth in the liquid culture system after 48 h, but produced a population of approximately 9 log,, cells ml-I in the biphase culture system. Arcobacter spp. require a nutrient-rich medium, usually containing blood or albumin, for growth. Fetal bovine serum appeared to be a reasonable component to add to liquid media to enhance the growth of the organisms. However, the addition of 10% FBS resulted in no significant ( P  > 0.1) increase in the populations of Arcobacter spp. for a given culture system (Table 1). An increase in the surface area of a biphase culture system can increase the population of cells in the liquid phase for some bacteria (e.g. ,  Salmonella, Escherichia coli ; Tyrrell et al. 1958 ; Dickson, unpublished data). Increasing the surface area from 25 to 75 cm2 significantly ( P  < 0.05) increased the populations of A. butzleri by approximately 0.75 log,, cycles (Table 2). The larger surface area numerically increased the average populations of the other four species, but these results were not significant. Increasing the surfaces area results in improved oxygen diffusion into the liquid medium. However, since Arcobacter spp. are aerotolerant bacteria, increasing the oxygen diffusion into the media would not necessarily enhance their growth. The growth curves of A. skirrowii 3699 confirmed that the maximum population density of this bacterium was approxi- mately 2 log,, cycles less in liquid culture than in biphase culture. The generation times were 0.869 h in biphase culture and 1.470 h in liquid culture, based on viable plate counts. The interpretation of the optical density readings for the biphase culture was different, since the bacterium apparently hydrolysed the solid phase, releasing red blood cells into the 0 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 1951 98 BlPHASlC CULTURE OF ARCOBACTER 197 Table 1 Effect of biphasic culture and fetal bovine serum on the growth of Log,, colony forming units ml-' Arcobacter spp. Bacterium Liquid* Bi-phase? BHI BHI+FBS BHI BHI+FBS A. butzleri 8.699 8.749 10.151 l O . l q  A. skirrowii 3699 7.749 7.679 9.261 9.281 A. skirrowii 37001 3.239 3-449 9,137 9.521 A. cryaerophilus 32521 NDG NDG 9.179 8.80§ A. cryaerophilus 32561 7.939 7.529 8.737 8.761 *4.5 ml liquid culture. t25 cm2 tissue culture flask ; solid phase 10% bovine blood agar. fBacterial populations enumerated by direct microscopic count. fj1Means within rows with different symbols are significantly ( P  < 0.05) different. BHI, Brain Heart Infusion Broth ; BHI + FBS, Brain Heart Infusion Broth + 10°/o fetal bovine serum ; NDG, no detectable growth. liquid phase. As such, the optical density readings could not be directly correlated with an increase in cell numbers. Although the mechanisms by which biphase culture sys- tems increase the bacterial populations are unknown, it is speculated that these culture systems enhance bacterial cell numbers by providing the organisms access to nutrients in the solid phase (Rollins et al .  1983). Biphase culture systems have been shown to have applications to culturing clinically important bacteria (Castaneda 1947; Hall et al .  1979), and have also been useful in culturing other bacteria to high concentrations (Tyrrell et al .  1958). This culture technique appears to have application to producing concentrated liquid cultures of Arcobacter spp. Table 2 Effect of surface area of biphasic culture on the growth of Arcobacter spp. Bacterium Log,, colony forming units m1-I 25 cm2 flask* 75 cm* flask A. butzleri 9.261 9.995 A. skirrowii 3699 9.061 9.211 A. skirrowii 3700t 9.181 9.561 A. cyaerophilus 3252t 9.171 9.271 A. cryaerophilus 32561. 8.861 8.891 *25 or 75 cm2 tissue culture flask; solid phase 10% bovine blood agar, liquid phase Brain Heart Infusion Broth. +Bacterial populations enumerated by direct microscopic count. f$Means within rows with different symbols are significantly ( P  < 0.05) different. ACKNOWLEDGEMENTS The authors acknowledge the advice and support of Mr  Clifford Collins and Dr Elsa A. Murano, Department of Microbiology, Immunology and Preventive Medicine, Iowa State University. This research was funded in part by the Agriculture and Home Economics Experiment Station at Iowa State University, Ames, Iowa Project number 3250. REFERENCES Anon. (1985) GLM. In Statistical Analysis System User's Guide : Statistics, Ver. 5 edition. pp. 433-506. Cary, NC : SAS Institute, Inc. Castaneda, M.R. (1947) A practical method for routine blood cul- tures in Brucellosis. Proceedings of  the Society f i r  Experimental Biology and Medicine 64, 114-115. Hall, M.M., Mueske, C.A., Ilstrup, D.M. and Washington, J.A. (1979) Evaluation of a biphase medium for blood cultures.3ournal of  Clinical Microbiology 10, 673476. Hestrin, S., Avineri-Shapiro, S. and Aschner, M. (1943) The enzymatic production of levan. BiochemicalJournal37,45@456. Krieg, N.R. and Gerhardt, P. (1994) Solid, liquid/solid and semi- solid culture. In Methods for  General and Molecular Bacteriology, ed. Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. pp. 216-223. Washington, DC : American Society for Microbiology. Neill, S.D. Campbell, J.N., O'Brien, J.J. Weatherup, S.T.C. and Ellis, W.A. (1985) Taxonomic position of Campylobacter c y a -  erophila sp. nov. International Journal of Systematic Bacteriology Pelczar, M.J., Reid, R.D. and Chan, E.C.S. (1977) Reproduction and growth. In Microbiology 4th ed. pp. 118-134. New York: McGraw-Hill. Rollins, D.M., Coolbaugh, J.C., Walker, R.I. and Weiss, E. (1983) 35, 342-356. 6 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 195-1 98 198 J.S. DICKSON ETAL.  Biphasic culture system for rapid Campylobacter cultivation. Applied and Environmental Microbiology 45, 28C289. Tyrrell, E.A., MacDonald, R.E. and Gerhardt, P. (1958) Biphasic system for growing bacteria in concentrated culture. Journal of Bacteriology 75, 1 4 .  Vandamme, P., Falsen, E., Rossau, R., Hoste, B., Segers, P., Tytgat, R. and De Ley, J. (1991) Revision of Campylobacter, Helicobacter, and Wolinella taxonomy : emendation of generic descriptions and proposal of Arcobacter gen. nov. International3ournal of Systematic Bacteriology 41, 88-103. Vandamme, P., Vancanneyt, M., Pot, B., Mels, L., Hoste, B., Dewettinck, D., Vlaes, L., VanDenBorre, C., Higgens, R., Hommez, J., Kersters, K., Butzler, J.P. and Goossens, H. (1992) Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary speci- mens. International Journal of Systematic Bacteriology 42, 344- 356. Q 1996 The Society for Applied Bacteriology, Letters in Applied Microbiology 22, 195-1 98 

Biphasic Culture of Arcobacter spp.

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Biphasic Culture of Arcobacter spp.

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