A Medium for the Isolation and Enumeration of Oral Actinomycetaceae from Dental Plaque D. BEIGHTON and G. COLMAN* Department of Conservative Dentistry, School
of Dental Science, University of Melbourne, Melbourne, 3000, Victoria, Australia
A supplemented, infusion-based medium that supports the growth of most oral Actinomycetaceae is described. The inclusion of NaF and colistin sulfate enabled actinomyces to be isolated from dental plaque in the presence of few other oral microorgan-
isms. The isolation and enumeration of oral Actinomycetaceae from dental plaque has previously been possible only when they represented a major component of the plaque flora.' They have been isolated under relatively nonselective conditions,l on infusion medium alone,2 or on infusion medium supplemented with blood or serum.' The enumeration of Actinomycetaceae may be influenced by the presence of other organisms in the dental plaque, particularly the acidogenic oral streptococci. When tlhe ratio of streptococci to Actinomycetaceae is large, the cultivable proportion of Actinomycetaceae in a sample becomes difficult to estimate accurately. The enumerationi of oral Actinomycetaceae may then be facilitated by the inhibition of the streptococci and other oral commensal bacteria, such as gram-negative bacteria. A method has not previously been described that inhibits the streptococci but allows the growth of the Actinomycetaceae. A medium has now been developed that allows the isolation and facilitates the accurate enumeration of oral Actinomycetaceae. It is
an infusion-based medium supplemented with 250 jig/ml of NaF to inhibit the oral streptococci, and 5 jcg/ml of colistin sulfate to inhibit the commensal gram-negative bacteria.3
Materials and Methods The basal culture medium (BYS medium) was prepared as follows: 3.7 gm Brain Heart Infusion broth,a 0.5 gm yeast extract powder,a I gm polyvinylpryrrolidone,b 0.1 gm cysteine HCl,e and 1.5 gm agard were added to 100 ml of distilled water, autoclaved for 15 minutes at 10 psi, cooled to 45 C, and 5 ml of sterile horse serume added. The enrichment medium (FC medium) was prepared by the addition of 1 ml of a sterile 25 mg/ml NaF solution and 0.5 ml of a sterile 1 mg/ml colistin sulfatef solution per 100 ml of BYS medium. The antimicrobial agents were sterilized by autoclaving at 10 psi for 15 minutes. Changes in the batch and source of NaF did not affect the efficiency of the FC medium. The ability of the FC medium to support the growth of various gram-positive organisms was established. The organisms were put into three groups: oral streptococci; oral Actinomycetaceae, and organisms occasionally isolated in low numbers from dental plaque. The groups are given in Table 1. The organisms were grown for 24 to 48 Oxoid, Abott Laboratories, Melbourne, Australia. May and Baker Ltd, Melbourne, Australia. Ajax Chemicals Ltd, Auburn, New South Wales, Australia. d Davis Gelatine (NZ) Ltd, New Zealand. e Commonwealth Serum Laboratories, Melbourne, Australia. f W. R. Warner & Co. Pty. Ltd, Villawood, New South Wales, Australia. a
b
This investigation was supported by the Milk Board of Victoria. Received for publication June 30, 1975. Accepted for publication March 12, 1976. 0 Present Address: Research Establishment, Royal College of Surgeons of England, Downe, near Orpington, Kent BR 7JJ, Eng.
e
Downloaded from jdr.sagepub.com at Harvard Libraries on July 14, 2015 For personal use only. No other uses without permission.
875
876
J Dent Res September-October 1976
BEIGHTON AND COLMAN
TABLE 1 RECOVERY OF STOCK AND TYPED STRAINS OF VARIOUS GRAM-POSITIVE ORGANISMS ON THE FC AND BYS MEDIA Medium
Organisms
Oral streptococci S mutans PK1 S mutans FAI S milleri OG3771 S mitior FW 75 S salivarius FW135 S sanguis GC S sanguis NCTC 7864 S bovis NCTC 8177 Oral actinomycetaceae A viscosus WVU626 A odontolyticus NCTC 9931 A naeslundii NCTC 10301 A israelii NCTC 10215 A israelii NCTC 10236 Actinomyces sp HO2+ A ctinomyces sp HO4+ Actinomyces sp HO7+ Ar proprionica ATCC 14157 B matruchotii ATCC 14266$ R dentocariosa ATCC 17931 Rothia sp Fil 3+
Minor oral gram-positive organisms Clostridium perfringens ATCC 8034 Bifidobacterium bifidum NCTC 10471 Lactobacillus sp FW 208
Proprionibacterium arabinosum Staphylococcus aureus FDA strain S faecalis E 10
Candida albicans N asteroides NCTC 6761 *
FC
BYS
of Dental Science, University of Melbourne, Melbourne, 3000, Victoria, Australia
A supplemented, infusion-based medium that supports the growth of most oral Actinomycetaceae is described. The inclusion of NaF and colistin sulfate enabled actinomyces to be isolated from dental plaque in the presence of few other oral microorgan-
isms. The isolation and enumeration of oral Actinomycetaceae from dental plaque has previously been possible only when they represented a major component of the plaque flora.' They have been isolated under relatively nonselective conditions,l on infusion medium alone,2 or on infusion medium supplemented with blood or serum.' The enumeration of Actinomycetaceae may be influenced by the presence of other organisms in the dental plaque, particularly the acidogenic oral streptococci. When tlhe ratio of streptococci to Actinomycetaceae is large, the cultivable proportion of Actinomycetaceae in a sample becomes difficult to estimate accurately. The enumerationi of oral Actinomycetaceae may then be facilitated by the inhibition of the streptococci and other oral commensal bacteria, such as gram-negative bacteria. A method has not previously been described that inhibits the streptococci but allows the growth of the Actinomycetaceae. A medium has now been developed that allows the isolation and facilitates the accurate enumeration of oral Actinomycetaceae. It is
an infusion-based medium supplemented with 250 jig/ml of NaF to inhibit the oral streptococci, and 5 jcg/ml of colistin sulfate to inhibit the commensal gram-negative bacteria.3
Materials and Methods The basal culture medium (BYS medium) was prepared as follows: 3.7 gm Brain Heart Infusion broth,a 0.5 gm yeast extract powder,a I gm polyvinylpryrrolidone,b 0.1 gm cysteine HCl,e and 1.5 gm agard were added to 100 ml of distilled water, autoclaved for 15 minutes at 10 psi, cooled to 45 C, and 5 ml of sterile horse serume added. The enrichment medium (FC medium) was prepared by the addition of 1 ml of a sterile 25 mg/ml NaF solution and 0.5 ml of a sterile 1 mg/ml colistin sulfatef solution per 100 ml of BYS medium. The antimicrobial agents were sterilized by autoclaving at 10 psi for 15 minutes. Changes in the batch and source of NaF did not affect the efficiency of the FC medium. The ability of the FC medium to support the growth of various gram-positive organisms was established. The organisms were put into three groups: oral streptococci; oral Actinomycetaceae, and organisms occasionally isolated in low numbers from dental plaque. The groups are given in Table 1. The organisms were grown for 24 to 48 Oxoid, Abott Laboratories, Melbourne, Australia. May and Baker Ltd, Melbourne, Australia. Ajax Chemicals Ltd, Auburn, New South Wales, Australia. d Davis Gelatine (NZ) Ltd, New Zealand. e Commonwealth Serum Laboratories, Melbourne, Australia. f W. R. Warner & Co. Pty. Ltd, Villawood, New South Wales, Australia. a
b
This investigation was supported by the Milk Board of Victoria. Received for publication June 30, 1975. Accepted for publication March 12, 1976. 0 Present Address: Research Establishment, Royal College of Surgeons of England, Downe, near Orpington, Kent BR 7JJ, Eng.
e
Downloaded from jdr.sagepub.com at Harvard Libraries on July 14, 2015 For personal use only. No other uses without permission.
875
876
J Dent Res September-October 1976
BEIGHTON AND COLMAN
TABLE 1 RECOVERY OF STOCK AND TYPED STRAINS OF VARIOUS GRAM-POSITIVE ORGANISMS ON THE FC AND BYS MEDIA Medium
Organisms
Oral streptococci S mutans PK1 S mutans FAI S milleri OG3771 S mitior FW 75 S salivarius FW135 S sanguis GC S sanguis NCTC 7864 S bovis NCTC 8177 Oral actinomycetaceae A viscosus WVU626 A odontolyticus NCTC 9931 A naeslundii NCTC 10301 A israelii NCTC 10215 A israelii NCTC 10236 Actinomyces sp HO2+ A ctinomyces sp HO4+ Actinomyces sp HO7+ Ar proprionica ATCC 14157 B matruchotii ATCC 14266$ R dentocariosa ATCC 17931 Rothia sp Fil 3+
Minor oral gram-positive organisms Clostridium perfringens ATCC 8034 Bifidobacterium bifidum NCTC 10471 Lactobacillus sp FW 208
Proprionibacterium arabinosum Staphylococcus aureus FDA strain S faecalis E 10
Candida albicans N asteroides NCTC 6761 *
FC
BYS
