APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1977, p. Copyright C) 1977 American Society for Microbiology
Vol. 33, No. 1 Printed in U.S.A.
213-214
Chemically Defined Medium for Rothia dentocariosa R. J. LESHERI *
AND S. J. DEAL Department of Microbiology, West Virginia University Medical Center, Morgantown,
West Virginia 26506
Received for publication 9 August 1976
A chemically defined medium for Rothia dentocariosa has been established. All strains tested, including the type strain, grew well in this medium. Rothia strains had a generation time of 3.5 to 4.0 h, with a yield of 62 mg (dry weight) per 100 ml after 72 h of growth in the chemically defined medium.
Rothia dentocariosa, a common inhabitant of the oral cavity, has been the subject of recent characterization studies (1, 2, 4). However, except for a glycolipid and major cell wall antigen being reported (3, 5), extensive physiological characterization ofRothia is lacking. The objective of this study was to establish a chemically defined medium for Rothia to facilitate its physiological characterization. Representative strains from the established serotypes of Rothia and the fluorescent-antibody-negative group (4) were used in this study. These strains included: (i) the type strain, 1489 (ATCC 17931, serotype 1); (ii) strain 477 (serotype 2); (iii)) strain 936 (serotype 3); and (iv) strain 804 (fluorescent-antibodynegative group). The source and previous designation ofthese strains were reported previously (4). All strains were maintained by monthly transfer on tryptic soy agar (Difco) slants. All strains were first grown in tryptic soy broth (Difco) for 24 h at 370C under aeration by agitation. A 5% inoculum (used throughout this study) from this culture was inoculated into 200 ml of a medium (medium no. 1) containing 5% glucose (Sigma), 1% acid-hydrolyzed casein (Difco), 0.1% yeast extract (Difco), Ltryptophan (200 mg), cysteine-hydrochloride (100 mg), and 2.5 ml of a salts solution (2.5 ml of salts A [25 g of K2HPO4 and 25 g of KH2PO4 per 250 ml of distilled water] and 2.5 ml of salts B [10 g of MgSO4 and 0.5 g of MnSO4 per 250 ml of distilled water] added to 100 ml of distilled water) and incubated for 48 h at 37°C under aeration by agitation. Growth for all experiments in this study was determined spectrophotometrically with a Beckman model 2400 spectrophotometer. As little as 0.01% yeast extract substituted for 0.1% yeast extract in medium no. 1 supported the growth of Rothia. Strains of Rothia tested in medium no. 1 entered the logarithmic phase approximately 13 h after initial
inoculation and remained in this phase of growth for approximately 18 h. The generation time for Rothia in medium no. 1 was 3 h, with a yield of 110 mg (dry weight) per 100 ml of medium. In another experiment, the 0.1% yeast extract was replaced by 16 different vitamins, and the 1% acid-hydrolyzed casein was replaced by 18 different amino acids at concentrations similar to those used by Takazoe (6). After vitamins and amino acids were eliminated one by one, a chemicaly defined medium (medium no. 2) containing the essential growth-promoting factors for Rothia was established (Table 1). These are not to be interpreted as the minimal essential nutritional requirements for this bacterium. All strains tested were grown in 100 ml of medium no. 2 (Table 1) under various atmospheric conditions (aerobic, aeration by agitation; or
1 Present address: Department of Microbiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642.
TABLE 1. Chemical composition of medium no. 2a Amt Component A g Glucose ........................ 50 Glutamic acid .................. 0.5 g 0.100 g Cysteine-hydrochloride ......... 0.200 g L-Tryptophan ..................
MgSO4.........................
0.2
g
K2HPO4 ....................... KH2PO4 ....................... NaCl ......................... (NH4)2SO4 .....................
4.0 2.0 2.0 1.3 0.036
g g
Pyridoxamine-hydrochloride .....
g
B 0.23 mg 0.07 mg 0.09 mg 0.5 mg Thiamine-hydrochloride ......... 1.98 mg Riboflavin ..................... Calcium pantothenate .......... 11.8 mg a All components were added to 1,000 ml of distilled water, adjusted to pH 7.2, and filter sterilized with a 0.45- Am pore size membrane filter (Millipore Corp). Components listed under part B were made at a concentration of lOOx and then diluted accord-
Nicotinic acid .................. Biotin ......................... p-Aminobenzoic acid ............
ingly. 213
g
g
214
NOTES
anaerobic, by GasPak incubation), temperatures (room temperature, 25 and 37°C), and pH (medium pH, 6.0 to 8.0). The optimal conditions for growth were incubation for 72 h at 37°C under aeration by agitation with the medium adjusted to pH 7.2. Strains of Rothia tested in the chemically defined medium entered the logarithmic phase approximately 18 h after initial inoculation and remained in this phase of growth for approximately 17 h. The generation time for Rothia in the chemically defined medium was 3.5 to 4.0 h, with a yield of 62 mg (dry weight) per 100 ml of medium. Ten serial transfers of Rothia in this medium were successful. Also, the morphology of all strains tested was a mixture of coccobacillary and filamentous forms and remained as such for the entire growth cycle in the chemically defined medium. Although we are currently working on resolving the essential minimal nutritional requirements for Rothia, we feel that medium no. 2 (Table 1) will provide a helpful tool for continued physiological studies with this bacterium.
APPL. ENVIRON. MICROBIOL. This study was supported by Public Health Service training grant 5T01-DE 00249-06 and research grant DE 04132 from the National Institute of Dental Research. R. J. L. is a Postdoctoral Fellow, Department of Microbiology, University of Rochester, School of Medicine and Dentistry. We would like to thank Robert E. Marquis for his help in the preparation of this manuscript. LITERATURE CITED 1. Brown, J. M., L. K. Georg, and L. C. Waters. 1969. Laboratory identification of Rothia dentocariosa and its occurrence in human clinical materials. Appl. Microbiol. 17:150-156. 2. Georg, L. K., and J. M. Brown. 1967. Rothia, gen. nov. an aerobic genus of the family Actinomycetaceae. Int. J. Syst. Bacteriol. 17:79-88. 3. Hammond, B. F. 1970. Isolation and serological characterization of a cell wall antigen ofRothia dentocariosa. J. Bacteriol. 103:634-640. 4. Lesher, R. J., M. A. Gerencser, and V. F. Gerencser. 1974. Morphological, biochemical, and serological characterization of Rothia dentocariosa. Int. J. Syst. Bacteriol. 24:154-159. 5. Pandhi, P. N., and B. F. Hammond. 1975. A glycolipid from Rothia dentocariosa. Arch. Oral Biol. 20:399-401. 6. Takazoe, I. 1969. A synthetic medium for Bacterionema matruchotii. Bull. Tokyo Dent. Coll. 10:97-105.
Vol. 33, No. 1 Printed in U.S.A.
213-214
Chemically Defined Medium for Rothia dentocariosa R. J. LESHERI *
AND S. J. DEAL Department of Microbiology, West Virginia University Medical Center, Morgantown,
West Virginia 26506
Received for publication 9 August 1976
A chemically defined medium for Rothia dentocariosa has been established. All strains tested, including the type strain, grew well in this medium. Rothia strains had a generation time of 3.5 to 4.0 h, with a yield of 62 mg (dry weight) per 100 ml after 72 h of growth in the chemically defined medium.
Rothia dentocariosa, a common inhabitant of the oral cavity, has been the subject of recent characterization studies (1, 2, 4). However, except for a glycolipid and major cell wall antigen being reported (3, 5), extensive physiological characterization ofRothia is lacking. The objective of this study was to establish a chemically defined medium for Rothia to facilitate its physiological characterization. Representative strains from the established serotypes of Rothia and the fluorescent-antibody-negative group (4) were used in this study. These strains included: (i) the type strain, 1489 (ATCC 17931, serotype 1); (ii) strain 477 (serotype 2); (iii)) strain 936 (serotype 3); and (iv) strain 804 (fluorescent-antibodynegative group). The source and previous designation ofthese strains were reported previously (4). All strains were maintained by monthly transfer on tryptic soy agar (Difco) slants. All strains were first grown in tryptic soy broth (Difco) for 24 h at 370C under aeration by agitation. A 5% inoculum (used throughout this study) from this culture was inoculated into 200 ml of a medium (medium no. 1) containing 5% glucose (Sigma), 1% acid-hydrolyzed casein (Difco), 0.1% yeast extract (Difco), Ltryptophan (200 mg), cysteine-hydrochloride (100 mg), and 2.5 ml of a salts solution (2.5 ml of salts A [25 g of K2HPO4 and 25 g of KH2PO4 per 250 ml of distilled water] and 2.5 ml of salts B [10 g of MgSO4 and 0.5 g of MnSO4 per 250 ml of distilled water] added to 100 ml of distilled water) and incubated for 48 h at 37°C under aeration by agitation. Growth for all experiments in this study was determined spectrophotometrically with a Beckman model 2400 spectrophotometer. As little as 0.01% yeast extract substituted for 0.1% yeast extract in medium no. 1 supported the growth of Rothia. Strains of Rothia tested in medium no. 1 entered the logarithmic phase approximately 13 h after initial
inoculation and remained in this phase of growth for approximately 18 h. The generation time for Rothia in medium no. 1 was 3 h, with a yield of 110 mg (dry weight) per 100 ml of medium. In another experiment, the 0.1% yeast extract was replaced by 16 different vitamins, and the 1% acid-hydrolyzed casein was replaced by 18 different amino acids at concentrations similar to those used by Takazoe (6). After vitamins and amino acids were eliminated one by one, a chemicaly defined medium (medium no. 2) containing the essential growth-promoting factors for Rothia was established (Table 1). These are not to be interpreted as the minimal essential nutritional requirements for this bacterium. All strains tested were grown in 100 ml of medium no. 2 (Table 1) under various atmospheric conditions (aerobic, aeration by agitation; or
1 Present address: Department of Microbiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642.
TABLE 1. Chemical composition of medium no. 2a Amt Component A g Glucose ........................ 50 Glutamic acid .................. 0.5 g 0.100 g Cysteine-hydrochloride ......... 0.200 g L-Tryptophan ..................
MgSO4.........................
0.2
g
K2HPO4 ....................... KH2PO4 ....................... NaCl ......................... (NH4)2SO4 .....................
4.0 2.0 2.0 1.3 0.036
g g
Pyridoxamine-hydrochloride .....
g
B 0.23 mg 0.07 mg 0.09 mg 0.5 mg Thiamine-hydrochloride ......... 1.98 mg Riboflavin ..................... Calcium pantothenate .......... 11.8 mg a All components were added to 1,000 ml of distilled water, adjusted to pH 7.2, and filter sterilized with a 0.45- Am pore size membrane filter (Millipore Corp). Components listed under part B were made at a concentration of lOOx and then diluted accord-
Nicotinic acid .................. Biotin ......................... p-Aminobenzoic acid ............
ingly. 213
g
g
214
NOTES
anaerobic, by GasPak incubation), temperatures (room temperature, 25 and 37°C), and pH (medium pH, 6.0 to 8.0). The optimal conditions for growth were incubation for 72 h at 37°C under aeration by agitation with the medium adjusted to pH 7.2. Strains of Rothia tested in the chemically defined medium entered the logarithmic phase approximately 18 h after initial inoculation and remained in this phase of growth for approximately 17 h. The generation time for Rothia in the chemically defined medium was 3.5 to 4.0 h, with a yield of 62 mg (dry weight) per 100 ml of medium. Ten serial transfers of Rothia in this medium were successful. Also, the morphology of all strains tested was a mixture of coccobacillary and filamentous forms and remained as such for the entire growth cycle in the chemically defined medium. Although we are currently working on resolving the essential minimal nutritional requirements for Rothia, we feel that medium no. 2 (Table 1) will provide a helpful tool for continued physiological studies with this bacterium.
APPL. ENVIRON. MICROBIOL. This study was supported by Public Health Service training grant 5T01-DE 00249-06 and research grant DE 04132 from the National Institute of Dental Research. R. J. L. is a Postdoctoral Fellow, Department of Microbiology, University of Rochester, School of Medicine and Dentistry. We would like to thank Robert E. Marquis for his help in the preparation of this manuscript. LITERATURE CITED 1. Brown, J. M., L. K. Georg, and L. C. Waters. 1969. Laboratory identification of Rothia dentocariosa and its occurrence in human clinical materials. Appl. Microbiol. 17:150-156. 2. Georg, L. K., and J. M. Brown. 1967. Rothia, gen. nov. an aerobic genus of the family Actinomycetaceae. Int. J. Syst. Bacteriol. 17:79-88. 3. Hammond, B. F. 1970. Isolation and serological characterization of a cell wall antigen ofRothia dentocariosa. J. Bacteriol. 103:634-640. 4. Lesher, R. J., M. A. Gerencser, and V. F. Gerencser. 1974. Morphological, biochemical, and serological characterization of Rothia dentocariosa. Int. J. Syst. Bacteriol. 24:154-159. 5. Pandhi, P. N., and B. F. Hammond. 1975. A glycolipid from Rothia dentocariosa. Arch. Oral Biol. 20:399-401. 6. Takazoe, I. 1969. A synthetic medium for Bacterionema matruchotii. Bull. Tokyo Dent. Coll. 10:97-105.
