A Thin-Layer Liquid Culture Technique for the Growth of Helicobacter pylori

Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐la...

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Published inHelicobacter (Cambridge, Mass.) Vol. 15; no. 4; pp. 295 - 302
Main Authors Joo, Jung-Soo, Park, Kyung-Chul, Song, Jae-Young, Kim, Dong-Hyun, Lee, Kyung-Ja, Kwon, Young-Cheol, Kim, Jung-Min, Kim, Kyung-Mi, Youn, Hee-Shang, Kang, Hyung-Lyun, Baik, Seung-Chul, Lee, Woo-Kon, Cho, Myung-Je, Rhee, Kwang-Ho
Format Journal Article
LanguageEnglish
Published Oxford, UK Blackwell Publishing Ltd 01.08.2010
Subjects
Alcohol dehydrogenase
bacterial growth
Brucella
Catalase
Cell culture
Cell density
Culture Media - metabolism
Culture Techniques - methods
Cytotoxins
g-Glutamyltransferase
Growth curves
H. pylori
Helicobacter pylori
Helicobacter pylori - growth & development
Helicobacter pylori - metabolism
Liquid culture
Media (culture)
Nuclease
reductase
Superoxide dismutase
Urease
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Abstract Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods:  A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results:  Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD600 with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD600 contained 1.3 ± 0.1 × 109 CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions:  Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
AbstractList Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori.BACKGROUND AND AIMSSeveral attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori.A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined.METHODSA thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined.Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours.RESULTSMaximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours.Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.CONCLUSIONSThin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori. Methods:  A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results:  Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD600 with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD600 contained 1.3 ± 0.1 × 109 CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions:  Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
AbstractBackground and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori.Methods: A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined.Results: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-b-cyclodextrin (200 kg-mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD600 with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD600 contained 1.3 c 0.1 x 109 CFU-mL. g-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours.Conclusions: Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.
Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori . However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin‐layer liquid culture technique for the growth of H. pylori . Methods:  A thin‐layer liquid culture system was established by adding liquid media to a 90‐mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Results:  Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI‐1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum‐free RPMI‐1640 supported the growth of H. pylori when supplemented with dimethyl‐β‐cyclodextrin (200 μg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD 600 with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD 600 contained 1.3 ± 0.1 × 10 9  CFU/mL. γ‐Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin‐layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Conclusions:  Thin‐layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori .
Author Kang, Hyung-Lyun
Kwon, Young-Cheol
Youn, Hee-Shang
Kim, Dong-Hyun
Lee, Woo-Kon
Park, Kyung-Chul
Joo, Jung-Soo
Rhee, Kwang-Ho
Song, Jae-Young
Lee, Kyung-Ja
Kim, Kyung-Mi
Cho, Myung-Je
Baik, Seung-Chul
Kim, Jung-Min
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  surname: Rhee
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Publisher Blackwell Publishing Ltd
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Hazell SL, Markesich DC, Evans DJ, Evans DG, Graham DY. Influence of media supplements on growth and survival of Campylobacter pylori. Eur J Clin Microbiol Infect Dis 1989;8:597-602.
Hazell SL, Evans DJ Jr, Graham DY. Helicobacter pylori catalase. J Gen Microbiol 1991;137:57-61.
Kusters JG, Van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449-90.
Stevenson TH, Castillo A, Lucia LM, Acuff GR. Growth of Helicobacter pylori in various liquid and plating media. Lett Appl Microbiol 2000;30:192-6.
Chauhan R, Mande SC. Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis. Biochem J 2002;367:255-61.
Buck GE, Smith JS. Medium supplementation for growth of Campylobacter pyloridis. J Clin Microbiol 1987;25:597-9.
Li Y, Schellhorn HE. Rapid kinetic microassay for catalase activity. J Biomol Tech 2007;18:185-7.
Coudron PE, Stratton CW. Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media. J Clin Microbiol 1995;33:1028-30.
Lin YL, Lee N, Chan EC. Determination of optimal liquid medium for enzyme expression by Helicobacter pylori. J Clin Pathol 1996;49:818-20.
Salmela KS, Roine RP, Koivisto T, Höök-Nikanne J, Kosunen TU, Salaspuro M. Characteristics of Helicobacter pylori alcohol dehydrogenase. Gastroenterology 1993;105:325-30.
Henriksen TH, Lia A, Schoyen R, Thoresen T, Berstad A. Assessment of optimal atmospheric conditions for growth of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 2000;19:718-20.
Cussac V, Ferrero RL, Labigne A. Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. J Bacteriol 1992;174:2466-73.
Lior H, Patel A. Improved toluidine blue-DNA agar for detection of DNA hydrolysis by campylobacters. J Clin Microbiol 1987;25:2030-1.
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Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford NG, Henderson PJ. Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol Microbiol 2007;64:396-406.
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Kitsos CM, Stadtlander CT. Helicobacter pylori in liquid culture: evaluation of growth rates and ultrastructure. Curr Microbiol 1998;37:88-93.
Morgan DR, Freedman F, Depew CE, Kraft WG. Growth of Campylobacter pylori in liquid media. J Clin Microbiol 1987;25:2123-5.
Hazell SL, Graham DY. Unsaturated fatty acids and viability of Helicobacter (Campylobacter) pylori. J Clin Microbiol 1990;28:1060-1.
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Testerman TL, McGee DJ, Mobley HL. Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture. J Clin Microbiol 2001;39:3842-50.
Xia HX, English L, Keane CT, O'Morain CA. Enhanced cultivation of Helicobacter pylori in liquid media. J Clin Pathol 1993;46:750-3.
Tomb JF, White O, Kerlavage AR, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997;388:539-47.
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Ansorg R, Von Recklinghausen G, Pomarius R, Schmid EN. Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter pylori. J Clin Microbiol 1991;29:51-3.
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2007; 18
1993; 46
1993; 61
2006; 11
1995; 33
1989; 8
2006; 19
1993; 105
1991; 137
1997; 388
1987; 25
1998; 37
1991; 29
2000; 19
1986; 168
2000; 38
1992; 174
2001; 6
2002; 184
1990; 28
2002; 367
1993; 31
2000; 30
2005; 73
1994; 77
1988; 23
2001; 39
1967; 242
2007; 64
1996; 49
1995; 141
1998; 36
1994; 6
Heins JN (e_1_2_6_23_2) 1967; 242
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Ansorg R (e_1_2_6_12_2) 1991; 29
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Morgan DR (e_1_2_6_15_2) 1987; 25
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Li Y (e_1_2_6_21_2) 2007; 18
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Kamiya S (e_1_2_6_25_2) 1994; 6
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Albertson N (e_1_2_6_29_2) 1998; 36
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Buck GE (e_1_2_6_7_2) 1987; 25
Lior H (e_1_2_6_36_2) 1987; 25
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Rhee KH (e_1_2_6_26_2) 1988; 23
e_1_2_6_27_2
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– reference: Kamiya S, Kai M, Ozawa A, Kobayashi H, Shirai T, Harasawa S, Miwa T. Characteristics of vacuolating toxin produced by Helicobacter pylori. Eur J Gastroenterol Hepatol 1994;6(Suppl. 1):S23-7.
– reference: Stevenson TH, Castillo A, Lucia LM, Acuff GR. Growth of Helicobacter pylori in various liquid and plating media. Lett Appl Microbiol 2000;30:192-6.
– reference: Albertson N, Wenngren I, Sjöström JE. Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78. J Clin Microbiol 1998;36:1232-5.
– reference: Shibayama K, Wachino J, Arakawa Y, Saidijam M, Rutherford NG, Henderson PJ. Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol Microbiol 2007;64:396-406.
– reference: Marcus EA, Scott DR. Cell lysis is responsible for the appearance of extracellular urease in Helicobacter pylori. Helicobacter 2001;6:93-9.
– reference: Buck GE, Smith JS. Medium supplementation for growth of Campylobacter pyloridis. J Clin Microbiol 1987;25:597-9.
– reference: Coudron PE, Stratton CW. Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media. J Clin Microbiol 1995;33:1028-30.
– reference: Burns BP, Hazell SL, Mendz GL. Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth. Microbiology 1995;141:3113-8.
– reference: Morgan DR, Freedman F, Depew CE, Kraft WG. Growth of Campylobacter pylori in liquid media. J Clin Microbiol 1987;25:2123-5.
– reference: Bury-Moné S, Kaakoush NO, Asencio C, Mégraud F, Thibonnier M, De Reuse H, Mendz G. Is Helicobacter pylori a true microaerophile? Helicobacter 2006;11:296-303.
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– reference: Li Y, Schellhorn HE. Rapid kinetic microassay for catalase activity. J Biomol Tech 2007;18:185-7.
– reference: Xia HX, English L, Keane CT, O'Morain CA. Enhanced cultivation of Helicobacter pylori in liquid media. J Clin Pathol 1993;46:750-3.
– reference: Suzuki H, Jumagai H, Tochikura T. γ-Glutamyl transpeptidase from Esherichia coli K-12: purification and properties. J Bacteriol 1986;168:1325-31.
– reference: Spiegelhalder C, Gerstenecker B, Kersten A, Schiltz E, Kist M. Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene. Infect Immun 1993;61:5315-525.
– reference: Lin YL, Lee N, Chan EC. Determination of optimal liquid medium for enzyme expression by Helicobacter pylori. J Clin Pathol 1996;49:818-20.
– reference: Chauhan R, Mande SC. Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis. Biochem J 2002;367:255-61.
– reference: Lior H, Patel A. Improved toluidine blue-DNA agar for detection of DNA hydrolysis by campylobacters. J Clin Microbiol 1987;25:2030-1.
– reference: Hazell SL, Markesich DC, Evans DJ, Evans DG, Graham DY. Influence of media supplements on growth and survival of Campylobacter pylori. Eur J Clin Microbiol Infect Dis 1989;8:597-602.
– reference: Henriksen TH, Lia A, Schoyen R, Thoresen T, Berstad A. Assessment of optimal atmospheric conditions for growth of Helicobacter pylori. Eur J Clin Microbiol Infect Dis 2000;19:718-20.
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– reference: Testerman TL, McGee DJ, Mobley HL. Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture. J Clin Microbiol 2001;39:3842-50.
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– reference: Kitsos CM, Stadtlander CT. Helicobacter pylori in liquid culture: evaluation of growth rates and ultrastructure. Curr Microbiol 1998;37:88-93.
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– reference: Kusters JG, Van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449-90.
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  doi: 10.1046/j.1365-2958.2000.01918.x
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Snippet Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H....
Background and Aims:  Several attempts have been successful in liquid cultivation of Helicobaccter pylori . However, there is a need to improve the growth of...
Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid...
AbstractBackground and Aims: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the...
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StartPage 295
SubjectTerms Alcohol dehydrogenase
bacterial growth
Brucella
Catalase
Cell culture
Cell density
Culture Media - metabolism
Culture Techniques - methods
Cytotoxins
g-Glutamyltransferase
Growth curves
H. pylori
Helicobacter pylori
Helicobacter pylori - growth & development
Helicobacter pylori - metabolism
Liquid culture
Media (culture)
Nuclease
reductase
Superoxide dismutase
Urease
Title A Thin-Layer Liquid Culture Technique for the Growth of Helicobacter pylori
URI https://api.istex.fr/ark:/67375/WNG-D30SJB5L-F/fulltext.pdf
https://onlinelibrary.wiley.com/doi/abs/10.1111%2Fj.1523-5378.2010.00767.x
https://www.ncbi.nlm.nih.gov/pubmed/20633190
https://www.proquest.com/docview/733986665
https://www.proquest.com/docview/754878187
Volume 15
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