NOTES
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Comparison of the effects of three fluorocarbons on certain bacteria1 0. W. VAN AUKENA N D J. HEALY Department of Pl~ysicaland Biological Sciences, Soitth~vestResearch Institltte, P.O. Drarver 28510, Son Antonio, Texas 78284 AND
A . J. KAUFMANN Department of Life Sciences, St. Mary's University, San Antonio. Texas 78228 Accepted September 24, 1974 1975. Comparison of the effects of three VANAUKEN,0. W., J. HEALY,and A. J. KAUFMANN. fluorocarbons on certain bacteria. Can. J. Microbiol. 21: 221-226. Three fluorocarbons were tested to determine their effect on bacterial growth. Freon I1 and 21 in various concentrations had an inhibitory effect on selected test organisms, but Freon 22 had no effect. Both aerobic and anaerobic microorganisms, as well as gram-positive and gram-negative species, were included among the bacteria tested. Freon I I and 21 caused a similar response with Freon I I being more inhibitory to some species and Freon 21 more inhibitory to others. Inhibition was dependent on the concentration of the halocarbon and resulted in decreased respiration rates at all concentrations tested. Results reported here indicate that the action of the fluorocarbons tested is bactericidal rather than bacteriostatic. Serratia marcescens and Clostridi~rmbot~rlit~rtm were the species most sensitive to the halocarbons tested. VANAUKEN,0. W., J. HEALYet A. J. KAUFMANN. 1975. Comparison of the effects of three fluorocarbons on certain bacteria. Can. J. Microbiol. 21: 221-226. On aetudit I'effet de trois fluorocarbones sur lacroissance bacttrienne. Le Frton I I et le Frton 21 a diverses concentrations ont un effet inhibiteur envers les organismes ttudits, tandis que le Frton 22 n'a aucun effet. Une variete de bacteries gram-positives et gram-ntgatives, atrobies et anatrobies a t t t ttudite. Le Frton I 1 et le Frton 2 1 ont des effets semblables; le Frton 11 est plus actifenvers certaines esptces alors que le Freon 2 1 I'est plus envers d'autres. L'inhibition dtpend de la concentration de I'halocarbone; on observe une diminution dans les taux de respiration a toutes les concentrations Ctudiees. Les rtsultats obtenus indiquent que Ies fluorocarbones Btudits ont une action bacttricide plutbt que bacteriostatique. Serratia marcescens et Clostridiltm botltlinlrm sont les esptces les plus sensibles aux halocarbones Btudies. [Traduit par le journal]
In recent years considerable information has become available on the effect of chloroalkanes on plant, animal, and microbial systems (1, 5, 13). However, there exists a paucity of data on a closely related group of compounds, the fluorocarbons. One compound, fluoroacetate, has been investigated extensively and its toxicity and mode of action is well known (4), but the fluoroalkanes (Freons and Genetronsm) have not been studied as well and consequently their modes of action and metabolism are not well understood. Freon compounds are organic compounds which contain fluorine, and in some cases chlorine, bromine, and hydrogen in addition to a carbon skeleton. They have many desirable characteristics which include a high degree of chemical stability and relatively low 'Received April 9, 1974.
toxicity, and they are nonflammable. Freon compounds have found many applications ranging from use as propellants to refrigerants and to solvents. Few studies have been carried out concerning the effects of fluoroalkanes on microorganisms. Early work by Lie (M.S. Thesis, University of Wisconsin, Madison, 1966) showed that Freon 11 was toxic to Pseudomonas striata, but Reed and Dychdala (12) did not find any adverse effects of Freon 12 or Freon 114 on several species of microorganisms. More recent work (9, 11) has shown that dichlorodifluoromethane (Freon 12), 1,l-difluoro-1-chloroethane(Freon 141b), and several other fluoroalkanes greatly reduced the number of microorganisms when they were exposed to these gases. The metabolism of Staphylococcus aureus is also affected by exposure to fluorocarbons (lo), with dichloro-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
222
CAN. J.
MICROBIOL. VOL.
fluoromethane (Freon 21) causing the greatest reduction of population, as well as reducing cellular metabolism. We have shown in previous work enhancement of food preservation by certain concentration of Freon 11 and 21 (14). This preservation was thought to be due to the bactericidal action of the fluorocarbons. The present investigation, which is an extension of the previous work, shows some of the lethal effects of three fluoroalkanes on eight different species of bacteria. The compounds used were Freon 11 (trichlorofluoromethane), Freon 21 (dichlorofluoromethane), and Freon 22 (monochlorodifluoromethane). Parameters investigated included inhibition of colony development, concentration effects, alterations of growth rates, bacteriostatic or bactericidal properties, as well as respiration changes. Laboratory strains of Escherichia coli (ATCC' 8739), Salmonella typhimurium, Serratia marcescens, Clostridium botulinum, type C-ATCC 11772, Clostridium sporogenes, Bacillus subtilis, Staphylococcus aureus, and Sarcina lutea were used in this study. Cultures of anaerobic organisms were kept at 4°C on nutrient agar slants containing alkaline pyrogallic acid. All other organisms were carried on brain heart infusion agar (BHI, Difco) slants and stored at the same temperature. Cultures of E. coli and B. subtilis were inoculated into flasks containing 250 ml of BHI broth (Difco) and allowed to grow for 24 h with agitation at 37°C. A growth curve for each organism was constructed by removing 5.0-ml samples from each culture, at specified time intervals during the 24-h period, and plating various dilutions using the pour-plate technique on BHI agar. Optical density measurements were also made on all samples using a Cary-14 spectrophotometer set at 550 nm. The test organisms were exposed to the fluorocarbons in two different systems. The first system involved bacteria added to pour plates, and the second system consisted of 250-ml liquid shake cultures. 1n the first test, the organisms were inoculated into BHI broth and growth was allowed to proceed until the desired optical density was reached as determined by the growth curve. Samples were taken and diluted serially in BHI broth. A 0.1-ml sample of the appropriate 'ATCC, American Type Culture Collection.
21, 1975
dilution was used to prepare pour plates for testing with various concentrations of the three Freons. Duplicate plates were used and after the medium solidified, the plates were placed in a 4-liter glass, air tight chamber before the addition of the fluorocarbon. The top was then attached and the test gas (Freon 11, 21, 22, or N,) was added using a displacement procedure. Anaerobes used for gas treatment were grown for 24-36 h in thioglycolate medium, appropriately diluted in BHI broth, and then a 0.1-ml sample was added to each pour plate. The anaerobes were treated in a fashion similar to the aerobes, except the plates were placed in a commercial gas pack (BBL) for Freon additions. On completion of the addition of the gas, the flow was stopped and the chamber was sealed. The chambers were placed in the incubator for 24 h at 37"C, removed the next day, and degassed with compressed air. The plates were removed and the colonies counted. Two independent experiments were carried out for each organism, and percentage of survival was calculated. Freon was added as a gas to each chamber according to the following schedule: loo%',,lo%, I%, and 0.00% with a 100% concentration being equal to about 5.0 g/liter of the fluorocarbon. In the second system, effects of the fluorocarbons were tested on log-phase cultures of both E. coli and S. aureus. Ten milliliters of inoculum from a 24-h culture of either bacterium was added to 250 ml of sterile, BHI medium. The new culture was incubated for 1-2 h at 35°C and monitored spectrophotometrically until the OD was equal to about 0.4, at which time an aliquot of Freon 11 or 21 was added. The culture was capped and incubation was continued. Fivemilliliter samples were taken at 30-min intervals for further spectrophotometric analysis. The first shake-culture experiments with the fluorocarbons were carried out with E. coli at concentrations ranging from 3.6 to 35.6 g/500 ml. The results of these experiments showed all concentrations to be completely inhibitory; consequently, the experiments with S. aureus were made using lower levels of the inhibitor (0.43.6 g/500 ml). All plating and transfers were carried out with standard aseptic procedures. Respiration measurements were carried out at 25°C on log-phase cultures suspended in BHI medium and incubated for 2 min with various concentrations of fluorocarbon. A Clark type
NOTES
TABLE 1 The effect of various concentrations of Freon 11, 21, and 22 and N z on the percentage survival of E. coli, B. subtilis, S. aureus, S. ryphimurium, S. marcescens, S . lurea, C. sporogerzes, and C. borulinum Concentration '% (v/v)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Organism
Gas
0.0
21 22 Nz 11
100 100 100 100
103.3I6.8 93.0k0.7 102.5t3.5 98.0k8.8
Nz 11 21 22 Nz 21 22 21 21 22
I00 100 100 100 100 100 100 100 100 100
106.1~10.4 98.0k2.1 85.0k9.2 91.0k0.9 103.5k3.4 78.0k9.6 100.0 100.0 86.5k6.4 98.2k8.2
1 .O
10
100*
E. coli
S. aureus
S. marcescens
B. subrilis
S. typhimurium
S. lurea
C. sporogenes5 C. borulinurn§
21.0220.0 103.0k2.1 101 .O+ 1 . 4 90.Ot14.5
0.0 95.0k3.2 91.3k14.1 0.0
'A 100% concentration of the Freons was equal to 5.0 rnglrnl. TPercentage of control. $ 1 standard deviation. §Cells grown in a gas pack under anaerobic conditions.
+
electrode was used for measurement of 0, utilization. Standard methods (6) were used in setting up and calibrating the electrode system. The effects of various concentrations of Freon 11, 21, and 22 on E. coli, B. subtilis, S. typhimurium, S . aureus, S. marcescens, S. lutea, C. sporogenes, and C. botulinum are shown in Table 1. Freon 11 and 21 show the greatest inhibition of bacterial growth with Freon 22 having little effect on the bacteria at the concentration used. Serratia marcescens was the organism most sensitive to the gases tested, being inhibited by Freon 11 at 1% and lo%, and by Freon 21 at 10%. Clostridium botulinum was sensitive to Freon 21, being 13% inhibited by the gas at 1% concentration; but C. sporogenes was not inhibited at all at the 10% level of Freon 21. Escherichia coli, B. subtilis, S. typhimurium, S. aureus, and C. sporogenes were all very similar in their response to the gases tested (Table 1). There was a slight inhibition by Freon 11 and 21 at the 10% concentration, and a complete
inhibition at 100%. The growth of these organisms was not affected by a 100% N2 atmosphere except in the case of S. marcescens and S. typhimurium, in which case the inhibition was only slight (Table 1). Freon 22 had little or no inhibitory effect on the above-mentioned species of bacteria. The inhibition of bacterial colony development, as depicted above (Table I), was due to the bactericidal effects of the three halocarbons tested. An additional 24-h incubation of the plates after degassing did not allow the development of additional colonies. Noninhibited cells streaked on control plates after they had been gassed with the fluorocarbon for 24 h and then degassed for 24 h in air showed complete colony growth. Another experiment was carried out to determine for certain if some of the fluorocarbons were actually bactericidal. Freon 21 was tested in this manner, and Table 2 shows the results of this experiment. Four plates were poured, and
CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 2 Bactericidal effects of Freon 21 on E. coli
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Colonies/plate Plate No.
Growth in air 24 h
Treatment, 24 h
1 2 3 4
48 46 42 45
100% Freon 21 Air 100% Freon 21 Air
Streak plates of* original colonies after exposure to Freon 21 or air
-t
+ +
'Four random colonies were picked from each orizinal plate. t A ( ) indicates no colony development o n any o f the streak plates after an additional 24-h incubation after treatment and a (+) indicates colony development o n all o f the streak plates alter incubation.
growth was determined after a 24-h incubation. Plates 1 and 3 were then exposed to a 100% Freon 2 1 atmosphere for an additional 24 h, and plates 2 and 4 were exposed to air for this additional time period. Next, four random-surface colonies from each plate were picked and streaked on BHI agar and incubated for 24 h. The results in Table 2 show that the bacteria exposed to the fluorocarbons were killed and those used as controls were not. This experiment also presents evidence to show that the inhibition of colony development is not due to residual adsorption of the fluorocarbon on the agar. Transferred cells will not grow on fresh media that is completely devoid of fluorocarbon, thus suggesting that residual fluorocarbon levels do not have any effect on the cells, but that the cells are killed by the gas treatment. Growth rates of E. coli and S. aureus, whose response to the fluorocarbons on solid media were similar (Table l), were also investigated in liquid culture. High concentrations of Freon 11 and 21 were used with the E. coli cultures (Fig. 1, A and B) and these concentrations completely stopped further growth as measured by the lack of change in OD. The OD increased slightly after the fluorocarbon addition and then decreased (due to cell clumping) and finally remained the same at the end of the experiment. Stapl~ylococcusaureus, an organism showing similar sensitivity, was tested at lower concentrations of the inhibitor to determine if any intermediate effects occurred. At the lowest concentrations used, Freon 1 1 and 21 had little or no effect on the growth rate of S. aureus. The rate was not changed nor was the maximum cell density. Intermediate concentrations changed the growth rates and also limited the cell density
T I M E (hours1
T I M E (hours1
FIG. 1. The effect of the addition of various concentrations of Freon I1 (A and C) and Freon 21 (€3 and D) on the growth rate of E. coli (A and B ) and S. aur-elm (C and D). The fluorocarbons were added after 1.5 h growth (see arrow). The concentrations of Freon 11 and 21 used in A and B were 0.0 g/500 ml ( a ) , 3.6 g/500 ml (A), 17.8 g/500 mi ( O ) , and 35.6 g/500 ml ( 0 ) . The concentrat~ons of Freon 11 and 21 used in C and D were 0.0 g/500 ml ( a ) , 0.4 g/500 ml (A), 1.8 91500 ml ( O ) , and 3.6 g/500 ml(0).
(Fig. 1, C and D). Both inhibitors had about the same effect on the growth rates of S. aureus. Respiration rates of E. coli were measured as a function of inhibitor concentration. As the concentrations of Freon 21 increased, the respiratory rate, or the rate of 0, uptake, decreased (Table 3).
NOTES
TABLE 3 Effect of Freon 21 on the 0, uptake rate of E. coli
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Freon 21 concn., mg/ml
mmol O,/min at 25'C Trial 1
"Standard deviation
Trial 2
Trial 3
Averages
SD*
of' the mean.
The results reported in this paper show that some of the fluoroalkane gases are inhibitory to bacterial growth and actually bactericidal at certain concentrations. Freon 11 (trichlorofluoromethane) and Freon 21 (dichlorofluoromethane) are the most inhibitory, while Freon 22 (monochlorodifluoromethane) has little, if any, effect. Some of these effects have been reported before for other fluorocarbons and for other species of bacteria and fungi (7, 9-1 1). Oujesky and Bhagat (10) have also reported that Freon 21 or Genetron 21 is very inhibitory to S. aureus, a finding confirmed in this paper. Most species tested were not inhibited by 1% of any of the Freons tested, 10% was slightly inhibitory, and 100% was completely inhibitory except for Freon 22. The actual concentration required to kill the different species of bacteria lies between 10% and 100% and is probably between 10 and 50% for Freon 11 and 21. The fact that some of the fluoroalkanes actually kill bacteria has been established, but their mode of action is not known. It has been suggested that the fluorinated alkanes block the Kreb's cycle (9) and possibly alter the glycolytic pathway (10). Other authors suggest that the lipid components of the cells are affected (1 l), orthat the fluorocarbon adsorbs to the lipoprotein cell membrane, thus interfering with cell permeability (2, 3). Coupling parameters in oxidative phosphorylation are also known to be altered (15). Our data (Table 3) show that there is a definite inhibition of cellular respiration by Freon 21 at all concentrations tested. Oxygen exclusion cannot explain the inhibition observed in these experiments. Nitrogen controls were run and little or no inhibition of colony development occurred. All of the organisms studied were either facultative or anaerobic, and
thus would be unaffected by low levels of 0 , . Prior et a[. (1 1) reached this conclusion in their studies on the effects of Freon 12 and 142b on certain species of bacteria and fungi. Harris (8) also found that the toxic effects of the Freons were considerably different from N, anoxia in mammalian cardiac investigations. Perhaps the fluorocarbons are interacting with the membrane lipoproteins causing alteration in respiratory rates, blockage of glycolysis and the tricarboxylic acid (TCA) cycle, and changing cell permeability. Acknowledgments For technical assistance and critical evaluation, we thank Dr. J. N. Bollinger, Mr. R. Estefan, Mrs. A. Henderson, Miss R. Lee, Dr. H. Oujesky, Mr. D. Perrotta, and Dr. J. R. Rowlands. This project was supported by an internal research award at Southwest Research Institute. 1. ANDERSON, J. D. 1973. Dichloromethane and lettuce seed germination. Science (Wash.), 179: 94-95. 2. BENNETT,P. B., and A. N. DOSSETT. 1970. Mechanism and prevention of inert as narcosis and anaesthesia. Nature (Lond.), 228: 13f7-1318. 3. BENNETT, P. B., and A. J. HAYWARD. 1967. Electrolyte imbalance as the mechanism for inert gas narcosis and anaesthesia. Nature (Lond.), 213: 938-939. 4. CLAYTON, J . W., JR. 1967. Fluorocarbon toxicity and biological action. Fluorine Chem. Rev. 1: 197-252. 5. DYKES,M. H. M. 1970. Halogenated hydrocarbon ingestion. Int. Anesthesiol. Clin. 8: 357-368. 6. ESTABROOK, R. W. 1967. Mitochondria1 respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47. 7. FUERST,R., and S. STEPHENS. 1970. Studies of effects of gases and gamma irradiation on Neurospora crassa. Dev. Ind. Microbiol. 11: 301-310. 8. HARRIS,W. S. 1972. Cardiac effects of halogenated hydrocarbons. I n An appraisal of halogenated fire extinguishing agents, Proceedings of a sy mposium, April 11-12, 1972. Edired by W. J. Christian and R. C. Wands. National Academy of Sciences, Washington. pp. 114-126.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
226
CAN. 1.
MICROBIOL. VOL.
9. LANDRY, M. M., and R. FUERST.1968. Gas ecology of bacteria. Dev. Ind. Microbiol. 9: 370-380. 10. OUJESKY,H., and I. BHAGAT.1973. Response of Staphylococcus aureus to atmospheres of Freons and Genetrons. Dev. Ind. Microbiol. 14: 229-237. ~ 1970. 11. PRIOR,B. A,, 0. F E N N E M AE., ~H.~ MARTH. Effect of gas hydrate formers on microorganisms. Appl. Microbiol. 20: 139-144. 12. REED,A. B., JR., and G. R. DYCHDALA. 1964. Microbiological activity of aerosol propellants. Soap Chem. Spec. 40: 204,208,210, and 290. 13. RUFENER, W. H., JR., and M. J. WOLIN.1968. Effect
21. 1975
of CC14 on CH4 and volatile acid production in continuous cultures of rumen organisms and in a sheep rumen. Appl. Microbiol. 16: 1955-1956. 0.W., J. HEALY, and A. J. KAUFMANN. 14. VANAUKEN, 1974. Food preservation and antibacterial properties of four fluoroalkane compounds. Can. J. Microbiol. 20: 891-896. 15. VAN AUKEN,0. W., and R. H. WILSON. 1973. Halogenated hydrocarbon induced reduction in coupling parameters of rabbit liver and mung bean mitochondria. Natunvissenschaften, 60: 259.
Photomicrography of nalidixic acid treated Hyphomicrobium neptunium: inhibition of bud formation and bud separation1 MARCIAA. BLACK MAN^ A N D RONALDM. WEINER Department of Microbiology, University of Maryland, College Park, Maryland 20742
Accepted October 11, 1974 BLACKMAN, M. A,, and R. M. WEINER.1975. Photomicrography of nalidixic acid treated Hyphomicrobium neptunium: inhibition of bud formation and bud separation. Can. J. Microbiol. 21: 226-230. Hyphomicrobiltm neptunium proceeds through a number of morphological stages during the course of its life cycle, but apparently only the bud formation and the bud separation stages are inhibited by nalidixic acid. BLACKMAN, M. A., et R. M. WEINER.1975. Photomicrography of nalidixic acid treated Hyphomicrobilrm neptunirrm: inhibition of bud formation and bud separation. Can. J. Microbiol. 21: 226-230. Le cycle vital de Hypllomicrobi~rmneptunium comporte plusieurs stades morphologiques, mais il semble que seule la formation de bourgeons et la skparationde ces derniers soient inhibees par I'acide nalidixque. [Traduit par le journal]
We have previously shown that nalidixic acid (nal) selectively inhibits deoxyribonucleic acid (DNA) synthesis in Hyphomicrobium neptunium (7). From observations of cells grown in batch cultures, it appeared that bud formation did not occur when cells were exposed to nal. It could not be determined, using batch cultures, whether bud separation was also blocked. When we examined individual cells embedded in agar we were able to establish the effects of nal on the developmental cycle of H. neptunium. These observations, described in this report, reveal that bud formation and bud release are 'Received August 16, 1974. ZPresent address: Metabolism Branch of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014.
both suppressed in the presence of the DNA inhibitor. A slide-chamber technique devised by Noller and Durham (6) and by Casida (2) was used to facilitate maintenance and observations of selected viable cells over an extended period of time. The slide chamber was modified so that the teflon membrane was under, rather than on top of, the slide as detailed in Fig. 1. Therefore, the agar block was thicker and less susceptible to drying. At intervals, several fields of the slides were observed and photographed using a Zeiss I microscope, with a phase-contrast objective lens (N.A. 1.32), X-8 photo-setting, and Kodak Tri-X film. The cells were examined for 7 h and control cells (no nal) multiplied normally throughout this period. At 5 h virtually 100% of the nal-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
This article has been cited by: 1. Trichlorofluoromethane [MAK Value Documentation, 1990] . [CrossRef] 2. Trichlorfluormethan [MAK Value Documentation in German language, 1989] . [CrossRef] 3. S Kawachi, T Arao, Y Hara, Y Suzuki, K Tamura. 2010. Effects of compression with ethane, ethylene and their fluorinated derivatives on yeast growth. Journal of Physics: Conference Series 215, 012168. [CrossRef] 4. Norbert Winker, Wolfgang Klein, Peter Weniger, Elisabeth Ott, Heinz Hofer. 1995. UDS-test with freon 11 (R-11). Environmental Science and Pollution Research 2:4, 233-236. [CrossRef] 5. S. Sousa, J. A. Akkara, D. L. Kaplan. 1987. Effects of Freon-113 on the survival of bacteria. Journal of Applied Microbiology 63:6, 559-564. [CrossRef]
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Comparison of the effects of three fluorocarbons on certain bacteria1 0. W. VAN AUKENA N D J. HEALY Department of Pl~ysicaland Biological Sciences, Soitth~vestResearch Institltte, P.O. Drarver 28510, Son Antonio, Texas 78284 AND
A . J. KAUFMANN Department of Life Sciences, St. Mary's University, San Antonio. Texas 78228 Accepted September 24, 1974 1975. Comparison of the effects of three VANAUKEN,0. W., J. HEALY,and A. J. KAUFMANN. fluorocarbons on certain bacteria. Can. J. Microbiol. 21: 221-226. Three fluorocarbons were tested to determine their effect on bacterial growth. Freon I1 and 21 in various concentrations had an inhibitory effect on selected test organisms, but Freon 22 had no effect. Both aerobic and anaerobic microorganisms, as well as gram-positive and gram-negative species, were included among the bacteria tested. Freon I I and 21 caused a similar response with Freon I I being more inhibitory to some species and Freon 21 more inhibitory to others. Inhibition was dependent on the concentration of the halocarbon and resulted in decreased respiration rates at all concentrations tested. Results reported here indicate that the action of the fluorocarbons tested is bactericidal rather than bacteriostatic. Serratia marcescens and Clostridi~rmbot~rlit~rtm were the species most sensitive to the halocarbons tested. VANAUKEN,0. W., J. HEALYet A. J. KAUFMANN. 1975. Comparison of the effects of three fluorocarbons on certain bacteria. Can. J. Microbiol. 21: 221-226. On aetudit I'effet de trois fluorocarbones sur lacroissance bacttrienne. Le Frton I I et le Frton 21 a diverses concentrations ont un effet inhibiteur envers les organismes ttudits, tandis que le Frton 22 n'a aucun effet. Une variete de bacteries gram-positives et gram-ntgatives, atrobies et anatrobies a t t t ttudite. Le Frton I 1 et le Frton 2 1 ont des effets semblables; le Frton 11 est plus actifenvers certaines esptces alors que le Freon 2 1 I'est plus envers d'autres. L'inhibition dtpend de la concentration de I'halocarbone; on observe une diminution dans les taux de respiration a toutes les concentrations Ctudiees. Les rtsultats obtenus indiquent que Ies fluorocarbones Btudits ont une action bacttricide plutbt que bacteriostatique. Serratia marcescens et Clostridiltm botltlinlrm sont les esptces les plus sensibles aux halocarbones Btudies. [Traduit par le journal]
In recent years considerable information has become available on the effect of chloroalkanes on plant, animal, and microbial systems (1, 5, 13). However, there exists a paucity of data on a closely related group of compounds, the fluorocarbons. One compound, fluoroacetate, has been investigated extensively and its toxicity and mode of action is well known (4), but the fluoroalkanes (Freons and Genetronsm) have not been studied as well and consequently their modes of action and metabolism are not well understood. Freon compounds are organic compounds which contain fluorine, and in some cases chlorine, bromine, and hydrogen in addition to a carbon skeleton. They have many desirable characteristics which include a high degree of chemical stability and relatively low 'Received April 9, 1974.
toxicity, and they are nonflammable. Freon compounds have found many applications ranging from use as propellants to refrigerants and to solvents. Few studies have been carried out concerning the effects of fluoroalkanes on microorganisms. Early work by Lie (M.S. Thesis, University of Wisconsin, Madison, 1966) showed that Freon 11 was toxic to Pseudomonas striata, but Reed and Dychdala (12) did not find any adverse effects of Freon 12 or Freon 114 on several species of microorganisms. More recent work (9, 11) has shown that dichlorodifluoromethane (Freon 12), 1,l-difluoro-1-chloroethane(Freon 141b), and several other fluoroalkanes greatly reduced the number of microorganisms when they were exposed to these gases. The metabolism of Staphylococcus aureus is also affected by exposure to fluorocarbons (lo), with dichloro-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
222
CAN. J.
MICROBIOL. VOL.
fluoromethane (Freon 21) causing the greatest reduction of population, as well as reducing cellular metabolism. We have shown in previous work enhancement of food preservation by certain concentration of Freon 11 and 21 (14). This preservation was thought to be due to the bactericidal action of the fluorocarbons. The present investigation, which is an extension of the previous work, shows some of the lethal effects of three fluoroalkanes on eight different species of bacteria. The compounds used were Freon 11 (trichlorofluoromethane), Freon 21 (dichlorofluoromethane), and Freon 22 (monochlorodifluoromethane). Parameters investigated included inhibition of colony development, concentration effects, alterations of growth rates, bacteriostatic or bactericidal properties, as well as respiration changes. Laboratory strains of Escherichia coli (ATCC' 8739), Salmonella typhimurium, Serratia marcescens, Clostridium botulinum, type C-ATCC 11772, Clostridium sporogenes, Bacillus subtilis, Staphylococcus aureus, and Sarcina lutea were used in this study. Cultures of anaerobic organisms were kept at 4°C on nutrient agar slants containing alkaline pyrogallic acid. All other organisms were carried on brain heart infusion agar (BHI, Difco) slants and stored at the same temperature. Cultures of E. coli and B. subtilis were inoculated into flasks containing 250 ml of BHI broth (Difco) and allowed to grow for 24 h with agitation at 37°C. A growth curve for each organism was constructed by removing 5.0-ml samples from each culture, at specified time intervals during the 24-h period, and plating various dilutions using the pour-plate technique on BHI agar. Optical density measurements were also made on all samples using a Cary-14 spectrophotometer set at 550 nm. The test organisms were exposed to the fluorocarbons in two different systems. The first system involved bacteria added to pour plates, and the second system consisted of 250-ml liquid shake cultures. 1n the first test, the organisms were inoculated into BHI broth and growth was allowed to proceed until the desired optical density was reached as determined by the growth curve. Samples were taken and diluted serially in BHI broth. A 0.1-ml sample of the appropriate 'ATCC, American Type Culture Collection.
21, 1975
dilution was used to prepare pour plates for testing with various concentrations of the three Freons. Duplicate plates were used and after the medium solidified, the plates were placed in a 4-liter glass, air tight chamber before the addition of the fluorocarbon. The top was then attached and the test gas (Freon 11, 21, 22, or N,) was added using a displacement procedure. Anaerobes used for gas treatment were grown for 24-36 h in thioglycolate medium, appropriately diluted in BHI broth, and then a 0.1-ml sample was added to each pour plate. The anaerobes were treated in a fashion similar to the aerobes, except the plates were placed in a commercial gas pack (BBL) for Freon additions. On completion of the addition of the gas, the flow was stopped and the chamber was sealed. The chambers were placed in the incubator for 24 h at 37"C, removed the next day, and degassed with compressed air. The plates were removed and the colonies counted. Two independent experiments were carried out for each organism, and percentage of survival was calculated. Freon was added as a gas to each chamber according to the following schedule: loo%',,lo%, I%, and 0.00% with a 100% concentration being equal to about 5.0 g/liter of the fluorocarbon. In the second system, effects of the fluorocarbons were tested on log-phase cultures of both E. coli and S. aureus. Ten milliliters of inoculum from a 24-h culture of either bacterium was added to 250 ml of sterile, BHI medium. The new culture was incubated for 1-2 h at 35°C and monitored spectrophotometrically until the OD was equal to about 0.4, at which time an aliquot of Freon 11 or 21 was added. The culture was capped and incubation was continued. Fivemilliliter samples were taken at 30-min intervals for further spectrophotometric analysis. The first shake-culture experiments with the fluorocarbons were carried out with E. coli at concentrations ranging from 3.6 to 35.6 g/500 ml. The results of these experiments showed all concentrations to be completely inhibitory; consequently, the experiments with S. aureus were made using lower levels of the inhibitor (0.43.6 g/500 ml). All plating and transfers were carried out with standard aseptic procedures. Respiration measurements were carried out at 25°C on log-phase cultures suspended in BHI medium and incubated for 2 min with various concentrations of fluorocarbon. A Clark type
NOTES
TABLE 1 The effect of various concentrations of Freon 11, 21, and 22 and N z on the percentage survival of E. coli, B. subtilis, S. aureus, S. ryphimurium, S. marcescens, S . lurea, C. sporogerzes, and C. borulinum Concentration '% (v/v)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Organism
Gas
0.0
21 22 Nz 11
100 100 100 100
103.3I6.8 93.0k0.7 102.5t3.5 98.0k8.8
Nz 11 21 22 Nz 21 22 21 21 22
I00 100 100 100 100 100 100 100 100 100
106.1~10.4 98.0k2.1 85.0k9.2 91.0k0.9 103.5k3.4 78.0k9.6 100.0 100.0 86.5k6.4 98.2k8.2
1 .O
10
100*
E. coli
S. aureus
S. marcescens
B. subrilis
S. typhimurium
S. lurea
C. sporogenes5 C. borulinurn§
21.0220.0 103.0k2.1 101 .O+ 1 . 4 90.Ot14.5
0.0 95.0k3.2 91.3k14.1 0.0
'A 100% concentration of the Freons was equal to 5.0 rnglrnl. TPercentage of control. $ 1 standard deviation. §Cells grown in a gas pack under anaerobic conditions.
+
electrode was used for measurement of 0, utilization. Standard methods (6) were used in setting up and calibrating the electrode system. The effects of various concentrations of Freon 11, 21, and 22 on E. coli, B. subtilis, S. typhimurium, S . aureus, S. marcescens, S. lutea, C. sporogenes, and C. botulinum are shown in Table 1. Freon 11 and 21 show the greatest inhibition of bacterial growth with Freon 22 having little effect on the bacteria at the concentration used. Serratia marcescens was the organism most sensitive to the gases tested, being inhibited by Freon 11 at 1% and lo%, and by Freon 21 at 10%. Clostridium botulinum was sensitive to Freon 21, being 13% inhibited by the gas at 1% concentration; but C. sporogenes was not inhibited at all at the 10% level of Freon 21. Escherichia coli, B. subtilis, S. typhimurium, S. aureus, and C. sporogenes were all very similar in their response to the gases tested (Table 1). There was a slight inhibition by Freon 11 and 21 at the 10% concentration, and a complete
inhibition at 100%. The growth of these organisms was not affected by a 100% N2 atmosphere except in the case of S. marcescens and S. typhimurium, in which case the inhibition was only slight (Table 1). Freon 22 had little or no inhibitory effect on the above-mentioned species of bacteria. The inhibition of bacterial colony development, as depicted above (Table I), was due to the bactericidal effects of the three halocarbons tested. An additional 24-h incubation of the plates after degassing did not allow the development of additional colonies. Noninhibited cells streaked on control plates after they had been gassed with the fluorocarbon for 24 h and then degassed for 24 h in air showed complete colony growth. Another experiment was carried out to determine for certain if some of the fluorocarbons were actually bactericidal. Freon 21 was tested in this manner, and Table 2 shows the results of this experiment. Four plates were poured, and
CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 2 Bactericidal effects of Freon 21 on E. coli
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Colonies/plate Plate No.
Growth in air 24 h
Treatment, 24 h
1 2 3 4
48 46 42 45
100% Freon 21 Air 100% Freon 21 Air
Streak plates of* original colonies after exposure to Freon 21 or air
-t
+ +
'Four random colonies were picked from each orizinal plate. t A ( ) indicates no colony development o n any o f the streak plates after an additional 24-h incubation after treatment and a (+) indicates colony development o n all o f the streak plates alter incubation.
growth was determined after a 24-h incubation. Plates 1 and 3 were then exposed to a 100% Freon 2 1 atmosphere for an additional 24 h, and plates 2 and 4 were exposed to air for this additional time period. Next, four random-surface colonies from each plate were picked and streaked on BHI agar and incubated for 24 h. The results in Table 2 show that the bacteria exposed to the fluorocarbons were killed and those used as controls were not. This experiment also presents evidence to show that the inhibition of colony development is not due to residual adsorption of the fluorocarbon on the agar. Transferred cells will not grow on fresh media that is completely devoid of fluorocarbon, thus suggesting that residual fluorocarbon levels do not have any effect on the cells, but that the cells are killed by the gas treatment. Growth rates of E. coli and S. aureus, whose response to the fluorocarbons on solid media were similar (Table l), were also investigated in liquid culture. High concentrations of Freon 11 and 21 were used with the E. coli cultures (Fig. 1, A and B) and these concentrations completely stopped further growth as measured by the lack of change in OD. The OD increased slightly after the fluorocarbon addition and then decreased (due to cell clumping) and finally remained the same at the end of the experiment. Stapl~ylococcusaureus, an organism showing similar sensitivity, was tested at lower concentrations of the inhibitor to determine if any intermediate effects occurred. At the lowest concentrations used, Freon 1 1 and 21 had little or no effect on the growth rate of S. aureus. The rate was not changed nor was the maximum cell density. Intermediate concentrations changed the growth rates and also limited the cell density
T I M E (hours1
T I M E (hours1
FIG. 1. The effect of the addition of various concentrations of Freon I1 (A and C) and Freon 21 (€3 and D) on the growth rate of E. coli (A and B ) and S. aur-elm (C and D). The fluorocarbons were added after 1.5 h growth (see arrow). The concentrations of Freon 11 and 21 used in A and B were 0.0 g/500 ml ( a ) , 3.6 g/500 ml (A), 17.8 g/500 mi ( O ) , and 35.6 g/500 ml ( 0 ) . The concentrat~ons of Freon 11 and 21 used in C and D were 0.0 g/500 ml ( a ) , 0.4 g/500 ml (A), 1.8 91500 ml ( O ) , and 3.6 g/500 ml(0).
(Fig. 1, C and D). Both inhibitors had about the same effect on the growth rates of S. aureus. Respiration rates of E. coli were measured as a function of inhibitor concentration. As the concentrations of Freon 21 increased, the respiratory rate, or the rate of 0, uptake, decreased (Table 3).
NOTES
TABLE 3 Effect of Freon 21 on the 0, uptake rate of E. coli
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
Freon 21 concn., mg/ml
mmol O,/min at 25'C Trial 1
"Standard deviation
Trial 2
Trial 3
Averages
SD*
of' the mean.
The results reported in this paper show that some of the fluoroalkane gases are inhibitory to bacterial growth and actually bactericidal at certain concentrations. Freon 11 (trichlorofluoromethane) and Freon 21 (dichlorofluoromethane) are the most inhibitory, while Freon 22 (monochlorodifluoromethane) has little, if any, effect. Some of these effects have been reported before for other fluorocarbons and for other species of bacteria and fungi (7, 9-1 1). Oujesky and Bhagat (10) have also reported that Freon 21 or Genetron 21 is very inhibitory to S. aureus, a finding confirmed in this paper. Most species tested were not inhibited by 1% of any of the Freons tested, 10% was slightly inhibitory, and 100% was completely inhibitory except for Freon 22. The actual concentration required to kill the different species of bacteria lies between 10% and 100% and is probably between 10 and 50% for Freon 11 and 21. The fact that some of the fluoroalkanes actually kill bacteria has been established, but their mode of action is not known. It has been suggested that the fluorinated alkanes block the Kreb's cycle (9) and possibly alter the glycolytic pathway (10). Other authors suggest that the lipid components of the cells are affected (1 l), orthat the fluorocarbon adsorbs to the lipoprotein cell membrane, thus interfering with cell permeability (2, 3). Coupling parameters in oxidative phosphorylation are also known to be altered (15). Our data (Table 3) show that there is a definite inhibition of cellular respiration by Freon 21 at all concentrations tested. Oxygen exclusion cannot explain the inhibition observed in these experiments. Nitrogen controls were run and little or no inhibition of colony development occurred. All of the organisms studied were either facultative or anaerobic, and
thus would be unaffected by low levels of 0 , . Prior et a[. (1 1) reached this conclusion in their studies on the effects of Freon 12 and 142b on certain species of bacteria and fungi. Harris (8) also found that the toxic effects of the Freons were considerably different from N, anoxia in mammalian cardiac investigations. Perhaps the fluorocarbons are interacting with the membrane lipoproteins causing alteration in respiratory rates, blockage of glycolysis and the tricarboxylic acid (TCA) cycle, and changing cell permeability. Acknowledgments For technical assistance and critical evaluation, we thank Dr. J. N. Bollinger, Mr. R. Estefan, Mrs. A. Henderson, Miss R. Lee, Dr. H. Oujesky, Mr. D. Perrotta, and Dr. J. R. Rowlands. This project was supported by an internal research award at Southwest Research Institute. 1. ANDERSON, J. D. 1973. Dichloromethane and lettuce seed germination. Science (Wash.), 179: 94-95. 2. BENNETT,P. B., and A. N. DOSSETT. 1970. Mechanism and prevention of inert as narcosis and anaesthesia. Nature (Lond.), 228: 13f7-1318. 3. BENNETT, P. B., and A. J. HAYWARD. 1967. Electrolyte imbalance as the mechanism for inert gas narcosis and anaesthesia. Nature (Lond.), 213: 938-939. 4. CLAYTON, J . W., JR. 1967. Fluorocarbon toxicity and biological action. Fluorine Chem. Rev. 1: 197-252. 5. DYKES,M. H. M. 1970. Halogenated hydrocarbon ingestion. Int. Anesthesiol. Clin. 8: 357-368. 6. ESTABROOK, R. W. 1967. Mitochondria1 respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47. 7. FUERST,R., and S. STEPHENS. 1970. Studies of effects of gases and gamma irradiation on Neurospora crassa. Dev. Ind. Microbiol. 11: 301-310. 8. HARRIS,W. S. 1972. Cardiac effects of halogenated hydrocarbons. I n An appraisal of halogenated fire extinguishing agents, Proceedings of a sy mposium, April 11-12, 1972. Edired by W. J. Christian and R. C. Wands. National Academy of Sciences, Washington. pp. 114-126.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
226
CAN. 1.
MICROBIOL. VOL.
9. LANDRY, M. M., and R. FUERST.1968. Gas ecology of bacteria. Dev. Ind. Microbiol. 9: 370-380. 10. OUJESKY,H., and I. BHAGAT.1973. Response of Staphylococcus aureus to atmospheres of Freons and Genetrons. Dev. Ind. Microbiol. 14: 229-237. ~ 1970. 11. PRIOR,B. A,, 0. F E N N E M AE., ~H.~ MARTH. Effect of gas hydrate formers on microorganisms. Appl. Microbiol. 20: 139-144. 12. REED,A. B., JR., and G. R. DYCHDALA. 1964. Microbiological activity of aerosol propellants. Soap Chem. Spec. 40: 204,208,210, and 290. 13. RUFENER, W. H., JR., and M. J. WOLIN.1968. Effect
21. 1975
of CC14 on CH4 and volatile acid production in continuous cultures of rumen organisms and in a sheep rumen. Appl. Microbiol. 16: 1955-1956. 0.W., J. HEALY, and A. J. KAUFMANN. 14. VANAUKEN, 1974. Food preservation and antibacterial properties of four fluoroalkane compounds. Can. J. Microbiol. 20: 891-896. 15. VAN AUKEN,0. W., and R. H. WILSON. 1973. Halogenated hydrocarbon induced reduction in coupling parameters of rabbit liver and mung bean mitochondria. Natunvissenschaften, 60: 259.
Photomicrography of nalidixic acid treated Hyphomicrobium neptunium: inhibition of bud formation and bud separation1 MARCIAA. BLACK MAN^ A N D RONALDM. WEINER Department of Microbiology, University of Maryland, College Park, Maryland 20742
Accepted October 11, 1974 BLACKMAN, M. A,, and R. M. WEINER.1975. Photomicrography of nalidixic acid treated Hyphomicrobium neptunium: inhibition of bud formation and bud separation. Can. J. Microbiol. 21: 226-230. Hyphomicrobiltm neptunium proceeds through a number of morphological stages during the course of its life cycle, but apparently only the bud formation and the bud separation stages are inhibited by nalidixic acid. BLACKMAN, M. A., et R. M. WEINER.1975. Photomicrography of nalidixic acid treated Hyphomicrobilrm neptunirrm: inhibition of bud formation and bud separation. Can. J. Microbiol. 21: 226-230. Le cycle vital de Hypllomicrobi~rmneptunium comporte plusieurs stades morphologiques, mais il semble que seule la formation de bourgeons et la skparationde ces derniers soient inhibees par I'acide nalidixque. [Traduit par le journal]
We have previously shown that nalidixic acid (nal) selectively inhibits deoxyribonucleic acid (DNA) synthesis in Hyphomicrobium neptunium (7). From observations of cells grown in batch cultures, it appeared that bud formation did not occur when cells were exposed to nal. It could not be determined, using batch cultures, whether bud separation was also blocked. When we examined individual cells embedded in agar we were able to establish the effects of nal on the developmental cycle of H. neptunium. These observations, described in this report, reveal that bud formation and bud release are 'Received August 16, 1974. ZPresent address: Metabolism Branch of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014.
both suppressed in the presence of the DNA inhibitor. A slide-chamber technique devised by Noller and Durham (6) and by Casida (2) was used to facilitate maintenance and observations of selected viable cells over an extended period of time. The slide chamber was modified so that the teflon membrane was under, rather than on top of, the slide as detailed in Fig. 1. Therefore, the agar block was thicker and less susceptible to drying. At intervals, several fields of the slides were observed and photographed using a Zeiss I microscope, with a phase-contrast objective lens (N.A. 1.32), X-8 photo-setting, and Kodak Tri-X film. The cells were examined for 7 h and control cells (no nal) multiplied normally throughout this period. At 5 h virtually 100% of the nal-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/17/12 For personal use only.
This article has been cited by: 1. Trichlorofluoromethane [MAK Value Documentation, 1990] . [CrossRef] 2. Trichlorfluormethan [MAK Value Documentation in German language, 1989] . [CrossRef] 3. S Kawachi, T Arao, Y Hara, Y Suzuki, K Tamura. 2010. Effects of compression with ethane, ethylene and their fluorinated derivatives on yeast growth. Journal of Physics: Conference Series 215, 012168. [CrossRef] 4. Norbert Winker, Wolfgang Klein, Peter Weniger, Elisabeth Ott, Heinz Hofer. 1995. UDS-test with freon 11 (R-11). Environmental Science and Pollution Research 2:4, 233-236. [CrossRef] 5. S. Sousa, J. A. Akkara, D. L. Kaplan. 1987. Effects of Freon-113 on the survival of bacteria. Journal of Applied Microbiology 63:6, 559-564. [CrossRef]
