APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p. 1222-1224
Copyright © 1977 American Society for Microbiology
Vol. 33, No. 5 Printed in U.S.A.
Medium for the Selective Isolation of Members of the Genus Pseudomonas from Natural Habitats MICHAEL A. GRANT* AND JOHN G. HOLT Department of Bacteriology, Iowa State University, Ames, Iowa 50011 Received for publication 13 December 1976
A new medium was developed that was superior to four others tested in selecting for members of the genus Pseudomonas.
The genus Pseudomonas has received widespread attention due to its medical and phytopathogenic importance and its catabolic versatility (4, 11). In recent years, the genetics of certain species has also been studied, and others are under consideration for use in singlecell protein production (1, 8). The interest in Pseudomonas has led to the development of media selective and/or differential for pseudomonads, but most of these were designed specifically for P. aeruginosa. A medium selective for some marine pseudomonads (6) and another selective for fluorescent soil pseudomonads (7) have been among the few media designed to select for more than one species of Pseudomonas. The importance of the genus Pseudomonas, plus the lack of an effective medium to allow selective isolation of all species in natural habitats, led us to develop the new medium. The effects of 48 dyes, antibiotics, and chemicals on 30 bacterial strains were initially tested. Each compound was incorporated individually and in various combinations into Trypticase soy agar (TSA) (Baltimore Biological Laboratory, Cockeysville, Md.). These plates were inoculated with all 30 strains, using the capillary tube technique of Hartman and Pattee (3). The bacterial strains used were: seven Pseudomonas soil isolates, seven Pseudomonas plant isolates, two Pseudomonas food isolates, P. aeruginosa, P. stutzeri, Xanthomonas campestris, Xanthomonas sp., Staphylococcus aureus, Bacillus subtilis, B. cereus, Arthrobacter sp., two strains of Acinetobacter calcoaceticus, Aeromonas liquefaciens, Escherichia coli, Klebsiella sp., and Citrobacter sp. The soil isolates were obtained from local soils, the plant isolates came from J. M. Dunleavy of the Department of Botany and Plant Pathology at Iowa State University, the food isolates were obtained from A. A. Kraft of the Department of Food Technology at Iowa State University, and all other strains were obtained from the Bacteriology Department culture collection.
The selective agents that inhibited various nonspeudomonads, with the least detrimental effect on pseudomonads, were combined in a selective medium that was field tested with soil samples. Soil samples were taken from rhizosphere soil in two natural prairie areas in Ames, Iowa. Serial dilutions of field samples were made in 0.5% peptone (10). Selective media and control plates containing unamended TSA were spread inoculated with 0.1-ml portions and incubated at 30°C for 36 h. Colonies were identified as Psuedomonas if the bacteria met the following criteria: motile, gram negative, slender rods, strict aerobes, oxidative metabolism, catalase and oxidase positive, no growth at pH 4.5, growth in the presence of 0.1% 2,3,5-triphenyl tetrazolium chloride (TTC), and no chains, flocs, or pleomorphic forms (2). Between 29 and 100% (X = 80%) of the colonies growing on plates of selective media were randomly selected and identified. The percentage that were Pseudomonas was termed the selective index. This parameter reflected the ability of the media to inhibit all genera except Pseudomonas. The total available Pseudomonas recovered (TAPR) values were calculated as follows. The total number of Pseudomonas per gram of sample was determined by plate counts on both selective media and TSA control plates. The value obtained with selective media was divided by the value obtained with control plates, and the result was multiplied by 100. The TAPR value reflected the ability of the media to allow the growth of the majority of Pseudomonas within the samples. The range of TAPR values was determined by using standard methods for determining confidence limits for a proportion (9). Comparisons between the selective indexes and TAPR of our medium and four other Pseudomonas media involved standard techniques of analysis of variance (9). Our Pseudomonas medium contained 9 gg of basic fuchsin (Matheson, Coleman, and Bell, Norwood, Ohio) per ml, 0.09% cycloheximide
1222
VOL. 33, 1977
(Sigma Chemical Co., St. Louis, Mo.), 0.014% TTC (General Biochemicals, Chagrin Falls, Ohio), 10 ,ug of nitrofurantoin (Eaton Laboratories, Norwich, N.Y.) per ml, and 23 ,ug of nalidixic acid (Sigma) per ml. All ingredients were combined in a TSA base at a final pH of 7.2. Basic fuchsin and TTC could be added to the TSA base before autoclaving at 121°C for 15 min. Reduction of TTC in the autoclave did not affect its selective properties. Stock solutions of nalidixic acid and nitrofurantoin were filter sterilized with a 0.45-,um membrane filter (Millipore Corp., Bedford, Mass.) and added aseptically to the autoclaved TSA base. Nalidixic acid was dissolved in an alkaline solution. Cycloheximide could be filter sterilized or added as the unsterile powder to the sterilized medium. Basic fuchsin was incorporated to inhibit gram-positive bacteria, although TTC was also inhibitory to certain gram-positive organisms, such as Arthrobacter. Cycloheximide was included as an antifungal agent. Nalidixic acid was primarily responsible for the inhibition of enteric bacteria and gramnegative cocci, especially Acinetobacter. Nitrofurantoin and TTC were important in preventing the growth of Aeromonas and aerobic gram-negative rods other than Pseudomonas. The optimum concentration of selective agents was determined by testing a variety of concentrations in both the field and laboratory. Pseudosel agar (BBL), Pseudomonas agar P (Difco), and Pseudomonas agar F (Difco) were prepared according to the instructions of the manufacturer. The ACC medium of Simon and Ridge was prepared according to their instructions (7). Our medium supported good growth of 16 of the 18 Pseudomonas laboratory isolates and inhibited all 12 nonpseudomonad isolates. To determine its effectiveness in selecting for Pseudomonas in natural habitats, the medium was field tested (Table 1). The selective properties of this medium were examined in three more soil tests. In these tests it was compared to Pseudosel agar, Pseudomonas agar P (Difco), Pseudomonas agar F (Difco), and ACC medium of Simon and Ridge. The results of these comparative tests are shown in Table 2. Statistical analysis of the mean selective index values in Table 2 showed that the least-significant difference at the 95% confidence level was 6.8%. The difference between the mean selective index value of our medium and that of Pseudosel, with the second highest selective index, was 10.7%. The superiority of the selective index of our medium was therefore statistically verified. The least-significant difference
NOTES
1223
TABLE 1. Effectiveness of the new medium in selecting for Pseudomonas in two soil samples Sample n.
°lO.
Selective available Pseuindex Total ) domonas/g
1 2
90 100
4.6 1.7
x x
TAPR M
105 106
100 100
Range TAPRof (%) 95-100 95-100
TABLE 2. Mean values for three tests comparing medium effectiveness Medium
Selective index
TAPR (
Range of TAPR (%)
(%)
70.1 65.1-75.1 97.5 Test mediuma 86.8 26.4 21.2-32.0 Pseudosel 30.3-41.0 12.5 35.1 Pseudomonas pb 9.2 71.7 66.7-76.7 Pseudomonas Fc 72.8-81.0 53.3 76.9 ACCd aMedium described in text. b Pseudomonas agar P (Difco). c Pseudomonas agar F (Difco). d ACC, Ampicillin-cycloheximide-chloramphenicol medium of Simon and Ridge (7).
between the TAPR values, at the 95% confidence level, was 17.3%. Thus, although ACC medium and Pseudomonas agar F (Difco) had slightly higher TAPR values, the superiority was not statistically significant. Our medium was superior to all other media tested in inhibiting all nonspeudomonads (selective index). Our medium was as efficient as Pseudomonas agar F (Difco) and ACC medium in allowing the growth of the majority of Pseudomonas in the samples (TAPR) and was superior to Pseudosel and Pseudomonas agar P (Difco) in this regard. The average selective index of our medium for all five soil tests was 96.2%. The remaining 3.8% differed from Pseudomonas only in their alkaline oxidative reaction in the Hugh-Leifson oxidation-fermentation test (5). A portion of this fraction may have been P. alcaligenes or P. pseudoalcaligenes. In the five soil tests, an average of 82% of all Pseudomonas in the samples grew on the new selective medium (82% TAPR). The new medium was effective in allowing selective isolation of Pseudomonas from rhizosphere soils. It was also effective in selecting Pseudomonas from habitats containing large numbers of nonpseudomonad gram-negative bacteria, since a medium containing the same ingredients, at slightly different concentrations, had an average 95% selective index and 77% TAPR when tested with raw sewage samples. Although the new medium was not cheaper or easier to prepare than the others
1224
tested, it displayed superior selective properties, with a short incubation time and minimal inhibition of Pseudomonas. 1.
2.
3. 4. 5.
APPL. ENVIRON. MICROBIOL.
NOTES
LITERATURE CITED Chakrabarty, A. M., G. Chou, and I. C. Gunsalus. 1973. Genetic regulation of octane dissimilation plasmid in Pseudomonas. Proc. Natl. Acad. Sci. U.S.A. 70:11371140. Doudoroff, M., and N. J. Palleroni. 1974. The genus Pseudomonas, p. 217-243. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology. Williams & Wilkins Co., Baltimore, Md. Hartman, P. A., and P. A. Pattee. 1968. Improved capillary-action replicating apparatus. Appl. Microbiol. 16:151-153. Horvath, R. S. 1972. Microbial co-metabolism and the degradation of organic compounds in nature. Bacteriol. Rev. 36:146-155. Hugh, R., and E. Leifson. 1953. The taxonomic signifi-
6.
7.
8. 9.
10. 11.
cance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66:24-26. Noseworthy, J. E., and G. Moskovits. 1974. Applicability of centrimide, nitrofurantoin, TTC, and sodium arsenate to the isolation of marine Pseudomonas. Can. J. Microbiol. 20:1561-1563. Simon, A., and E. H. Ridge. 1974. The use of ampicillin in a simplified selective medium for the isolation of fluorescent Pseudomonas. J. Appl. Bacteriol. 37:459460. Slater, L. E. 1974. SCP: the methanol way. Food Eng. 46:68-72. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, 6th ed. The Iowa State University Press, Ames. Straka, R. P., and J. L. Stokes. 1957. Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol. 5:21-25. Wilson, G. S., and A. Miles. 1975. Topley and Wilson's principles of bacteriology and immunity, 6th ed. Williams & Wilkins Co., Baltimore, Md.
Copyright © 1977 American Society for Microbiology
Vol. 33, No. 5 Printed in U.S.A.
Medium for the Selective Isolation of Members of the Genus Pseudomonas from Natural Habitats MICHAEL A. GRANT* AND JOHN G. HOLT Department of Bacteriology, Iowa State University, Ames, Iowa 50011 Received for publication 13 December 1976
A new medium was developed that was superior to four others tested in selecting for members of the genus Pseudomonas.
The genus Pseudomonas has received widespread attention due to its medical and phytopathogenic importance and its catabolic versatility (4, 11). In recent years, the genetics of certain species has also been studied, and others are under consideration for use in singlecell protein production (1, 8). The interest in Pseudomonas has led to the development of media selective and/or differential for pseudomonads, but most of these were designed specifically for P. aeruginosa. A medium selective for some marine pseudomonads (6) and another selective for fluorescent soil pseudomonads (7) have been among the few media designed to select for more than one species of Pseudomonas. The importance of the genus Pseudomonas, plus the lack of an effective medium to allow selective isolation of all species in natural habitats, led us to develop the new medium. The effects of 48 dyes, antibiotics, and chemicals on 30 bacterial strains were initially tested. Each compound was incorporated individually and in various combinations into Trypticase soy agar (TSA) (Baltimore Biological Laboratory, Cockeysville, Md.). These plates were inoculated with all 30 strains, using the capillary tube technique of Hartman and Pattee (3). The bacterial strains used were: seven Pseudomonas soil isolates, seven Pseudomonas plant isolates, two Pseudomonas food isolates, P. aeruginosa, P. stutzeri, Xanthomonas campestris, Xanthomonas sp., Staphylococcus aureus, Bacillus subtilis, B. cereus, Arthrobacter sp., two strains of Acinetobacter calcoaceticus, Aeromonas liquefaciens, Escherichia coli, Klebsiella sp., and Citrobacter sp. The soil isolates were obtained from local soils, the plant isolates came from J. M. Dunleavy of the Department of Botany and Plant Pathology at Iowa State University, the food isolates were obtained from A. A. Kraft of the Department of Food Technology at Iowa State University, and all other strains were obtained from the Bacteriology Department culture collection.
The selective agents that inhibited various nonspeudomonads, with the least detrimental effect on pseudomonads, were combined in a selective medium that was field tested with soil samples. Soil samples were taken from rhizosphere soil in two natural prairie areas in Ames, Iowa. Serial dilutions of field samples were made in 0.5% peptone (10). Selective media and control plates containing unamended TSA were spread inoculated with 0.1-ml portions and incubated at 30°C for 36 h. Colonies were identified as Psuedomonas if the bacteria met the following criteria: motile, gram negative, slender rods, strict aerobes, oxidative metabolism, catalase and oxidase positive, no growth at pH 4.5, growth in the presence of 0.1% 2,3,5-triphenyl tetrazolium chloride (TTC), and no chains, flocs, or pleomorphic forms (2). Between 29 and 100% (X = 80%) of the colonies growing on plates of selective media were randomly selected and identified. The percentage that were Pseudomonas was termed the selective index. This parameter reflected the ability of the media to inhibit all genera except Pseudomonas. The total available Pseudomonas recovered (TAPR) values were calculated as follows. The total number of Pseudomonas per gram of sample was determined by plate counts on both selective media and TSA control plates. The value obtained with selective media was divided by the value obtained with control plates, and the result was multiplied by 100. The TAPR value reflected the ability of the media to allow the growth of the majority of Pseudomonas within the samples. The range of TAPR values was determined by using standard methods for determining confidence limits for a proportion (9). Comparisons between the selective indexes and TAPR of our medium and four other Pseudomonas media involved standard techniques of analysis of variance (9). Our Pseudomonas medium contained 9 gg of basic fuchsin (Matheson, Coleman, and Bell, Norwood, Ohio) per ml, 0.09% cycloheximide
1222
VOL. 33, 1977
(Sigma Chemical Co., St. Louis, Mo.), 0.014% TTC (General Biochemicals, Chagrin Falls, Ohio), 10 ,ug of nitrofurantoin (Eaton Laboratories, Norwich, N.Y.) per ml, and 23 ,ug of nalidixic acid (Sigma) per ml. All ingredients were combined in a TSA base at a final pH of 7.2. Basic fuchsin and TTC could be added to the TSA base before autoclaving at 121°C for 15 min. Reduction of TTC in the autoclave did not affect its selective properties. Stock solutions of nalidixic acid and nitrofurantoin were filter sterilized with a 0.45-,um membrane filter (Millipore Corp., Bedford, Mass.) and added aseptically to the autoclaved TSA base. Nalidixic acid was dissolved in an alkaline solution. Cycloheximide could be filter sterilized or added as the unsterile powder to the sterilized medium. Basic fuchsin was incorporated to inhibit gram-positive bacteria, although TTC was also inhibitory to certain gram-positive organisms, such as Arthrobacter. Cycloheximide was included as an antifungal agent. Nalidixic acid was primarily responsible for the inhibition of enteric bacteria and gramnegative cocci, especially Acinetobacter. Nitrofurantoin and TTC were important in preventing the growth of Aeromonas and aerobic gram-negative rods other than Pseudomonas. The optimum concentration of selective agents was determined by testing a variety of concentrations in both the field and laboratory. Pseudosel agar (BBL), Pseudomonas agar P (Difco), and Pseudomonas agar F (Difco) were prepared according to the instructions of the manufacturer. The ACC medium of Simon and Ridge was prepared according to their instructions (7). Our medium supported good growth of 16 of the 18 Pseudomonas laboratory isolates and inhibited all 12 nonpseudomonad isolates. To determine its effectiveness in selecting for Pseudomonas in natural habitats, the medium was field tested (Table 1). The selective properties of this medium were examined in three more soil tests. In these tests it was compared to Pseudosel agar, Pseudomonas agar P (Difco), Pseudomonas agar F (Difco), and ACC medium of Simon and Ridge. The results of these comparative tests are shown in Table 2. Statistical analysis of the mean selective index values in Table 2 showed that the least-significant difference at the 95% confidence level was 6.8%. The difference between the mean selective index value of our medium and that of Pseudosel, with the second highest selective index, was 10.7%. The superiority of the selective index of our medium was therefore statistically verified. The least-significant difference
NOTES
1223
TABLE 1. Effectiveness of the new medium in selecting for Pseudomonas in two soil samples Sample n.
°lO.
Selective available Pseuindex Total ) domonas/g
1 2
90 100
4.6 1.7
x x
TAPR M
105 106
100 100
Range TAPRof (%) 95-100 95-100
TABLE 2. Mean values for three tests comparing medium effectiveness Medium
Selective index
TAPR (
Range of TAPR (%)
(%)
70.1 65.1-75.1 97.5 Test mediuma 86.8 26.4 21.2-32.0 Pseudosel 30.3-41.0 12.5 35.1 Pseudomonas pb 9.2 71.7 66.7-76.7 Pseudomonas Fc 72.8-81.0 53.3 76.9 ACCd aMedium described in text. b Pseudomonas agar P (Difco). c Pseudomonas agar F (Difco). d ACC, Ampicillin-cycloheximide-chloramphenicol medium of Simon and Ridge (7).
between the TAPR values, at the 95% confidence level, was 17.3%. Thus, although ACC medium and Pseudomonas agar F (Difco) had slightly higher TAPR values, the superiority was not statistically significant. Our medium was superior to all other media tested in inhibiting all nonspeudomonads (selective index). Our medium was as efficient as Pseudomonas agar F (Difco) and ACC medium in allowing the growth of the majority of Pseudomonas in the samples (TAPR) and was superior to Pseudosel and Pseudomonas agar P (Difco) in this regard. The average selective index of our medium for all five soil tests was 96.2%. The remaining 3.8% differed from Pseudomonas only in their alkaline oxidative reaction in the Hugh-Leifson oxidation-fermentation test (5). A portion of this fraction may have been P. alcaligenes or P. pseudoalcaligenes. In the five soil tests, an average of 82% of all Pseudomonas in the samples grew on the new selective medium (82% TAPR). The new medium was effective in allowing selective isolation of Pseudomonas from rhizosphere soils. It was also effective in selecting Pseudomonas from habitats containing large numbers of nonpseudomonad gram-negative bacteria, since a medium containing the same ingredients, at slightly different concentrations, had an average 95% selective index and 77% TAPR when tested with raw sewage samples. Although the new medium was not cheaper or easier to prepare than the others
1224
tested, it displayed superior selective properties, with a short incubation time and minimal inhibition of Pseudomonas. 1.
2.
3. 4. 5.
APPL. ENVIRON. MICROBIOL.
NOTES
LITERATURE CITED Chakrabarty, A. M., G. Chou, and I. C. Gunsalus. 1973. Genetic regulation of octane dissimilation plasmid in Pseudomonas. Proc. Natl. Acad. Sci. U.S.A. 70:11371140. Doudoroff, M., and N. J. Palleroni. 1974. The genus Pseudomonas, p. 217-243. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology. Williams & Wilkins Co., Baltimore, Md. Hartman, P. A., and P. A. Pattee. 1968. Improved capillary-action replicating apparatus. Appl. Microbiol. 16:151-153. Horvath, R. S. 1972. Microbial co-metabolism and the degradation of organic compounds in nature. Bacteriol. Rev. 36:146-155. Hugh, R., and E. Leifson. 1953. The taxonomic signifi-
6.
7.
8. 9.
10. 11.
cance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66:24-26. Noseworthy, J. E., and G. Moskovits. 1974. Applicability of centrimide, nitrofurantoin, TTC, and sodium arsenate to the isolation of marine Pseudomonas. Can. J. Microbiol. 20:1561-1563. Simon, A., and E. H. Ridge. 1974. The use of ampicillin in a simplified selective medium for the isolation of fluorescent Pseudomonas. J. Appl. Bacteriol. 37:459460. Slater, L. E. 1974. SCP: the methanol way. Food Eng. 46:68-72. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, 6th ed. The Iowa State University Press, Ames. Straka, R. P., and J. L. Stokes. 1957. Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol. 5:21-25. Wilson, G. S., and A. Miles. 1975. Topley and Wilson's principles of bacteriology and immunity, 6th ed. Williams & Wilkins Co., Baltimore, Md.
