THE GROWTH OF ESCHERICHIA COLI IN BUFFER SUBSTRATE AND DISTILLED WATER

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1 THE GROWTH OF ESCHERICHIA COLI IN BUFFER SUBSTRATE AND DISTILLED WATER ELLEN I. GARVIE National Institute for Research in Dairying, Univer8ity of Reading, England Received for publication September 8, 1954 Heinmets, Taylor, and Lehman (1954) treated a suspension of cells of Escherichia coli strain B/r with disifectant and found that after treatment none or only a few viable organisms were present. However, when the liquid (containing the cells which had been exposed to the disinfectant) was mixed with a solution of a metabolite in phosphate buffer and incubated overnight at 37 C, a large number of viable cells was found. (Similar results were obtained with several disinfectants, including sodium hypochlorite.) Heinmets et al. suggest that the increase in numbers was due to reactivation of the damaged, but not dead, cells and was not due to multiplication of a few viable cells remaining in the buffer substrate. This would necesitate a radical alteration in the present method of assay of disinfectants. If surfaces after treatment with a disinfectant were likely to come in contact with a fluid, such as milk, containing a selection of metabolites, the action of the disinfectant might be nullified. Garvie and Clarke (1955, in press) used a strain (28.D.10) of E. colt for estimating the bactericidal properties of disinfectants, and, in order to examine the validity of the results they obtained, the work of Heinmets et al. was repeated as nearly as possible using, however, E. coli strain 28.D.10. It became evident that this strain could grow in the buffer substrate, and the work was continued to study some of the conditions for bacterial growth. EXPERIMENTAL METHODS Growth after treatment with sodium hypochorite. The test orgam E. coli strain 28.D.10 was grown on a nutrient agar slant (peptone, 1 per cent; "lemco", 1 per cent; NaCl, 0.5 per cent; agar, 2 per cent) and subcultured daily for at least 3 days. A suspension of cells from a 24 hr slant was prepared in phosphate buffer (0.1 M solution KH2PO4, NaHP04; ph 7.0) and exposed to a solution of sodium hypochlorite also in 393 phosphate buffer. The action of the sodium hypochlorite was stopped by adding an equal volume of 0.2 per cent sodium thiosulfate. Portions of this liquid were diluted 1:1 with 0.2 per cent solutions of metabolites in phosphate buffer, and incubated at 37 C overnight. The metabolites selected were sodium citrate, lactic acid, and malic acid. The number of cells present before and after incubation in metabolite solution was estimated by plate colony counts using 1.5 per cent nutrient agar. The growth of organisms in buffer. The buffer solution was tubed in 10 ml quantities, sterilized by boiling for 10 min, and used on the day of preparation. The ph was about 7; it was not adjusted because additional food materials might be present in any NaOH and HCl added. Lactic acid was added, when required, to give a final concentration of 0.1 per cent. Two test organisms were studied, E. coli strain 28.D.10 and P8eudomonas fluorescens strain N.C.T.C The suspensions for use were from an 18 hr slant; the growth was suspended in phosphate buffer, washed twice, and resuspended in buffer for use. The number of cells inoculated was estimated by a plate colony count on a control tube. The amount of growth was estimated by the same method. Dead cells were prepared by boiling the suspensions for 10 min. Plates and tubes of E. coti were incubated at 37 C and those of P. fluorescens at 30C (all for 3 days). RESUIITS Growth after treatment with sodium hypochlorite. The results, given in table 1, show that after incubation in the presence of lactic acid or malic acid, an increased number of viable cells was usually present, but generally no or only a slight increase in numbers was recorded after incubation in the presence of buffer or buffer + sodium citrate.

2 394 ELLEN I. GARVI19 [VOL. 69 TABLE 1 Estimated numbers of Escherichia coli after treatment with sodium hypochlorite and incubation in the presence of various buffer substrates Test 1 Test 2 Sodium hypochlorite, per cent Sodium hypochlorite, per cent Contact time... 1 mi 4 m 2 min 4 min Before treatment with hypochlo- 30,800,000 30,800,000 50,000,000 20,000,000 rite After treatment with hypochlorite After incubation in: Buffer ,010 0 Buffer + sodium citrate , Buffer + lactic acid 50, , Buffer + malic acid 30, ,000 >500, Cell suspensions treated with sodium hypochlorite and then incubated overnight in the presence of 0.1 M KH2PO4-Na1HPO0. Metabolites when present as 0.1 per cent solutions. Numbers estimated by plate colony counts. TABLE 2 Comparison of plate colony count, dilution counts, and counts after incubation of Escherichia coli in various buffer substrates. Culture treated with sodium hypochlorite per cent for I min Test I Test 2 Dilution count Plate count after Dilution count Plate count after peptone water incubation overnight peptone water incubation overnight + metabolite buffer + metabolite + metabolite buffer + metabolite Litmus milk 2,400 _ 24,000 Buffer 540,000-1,580,000 Sodium citrate 4, ,000 4,300 1,790,000 Lactic acid 24,000 2,000,000 2,400 8,440,000 Malic acid 24,000 5,000,000 43,000 10,160,000 Plate count before treatment with 6,000,000 40,000,000 hypochlorite Plate count after treatment with 8,700 18,300 hypochlorite Cell suspensions exposed to sodium hypochlorite and then incubated in 0.1 M solution of KH2PO.- Na2HPO. Metabolites when present as 0.1 per cent solutions. In later experiments a further estimate of the number of cells, viable immediately after treatment with the disinfectant, was made using dilution counts in metabolite-peptone water (peptone, 0.1 per cent; NaCl, 0.5 per cent; metabolite, 0.1 per cent) (table 2). The probable numbers were estimated from Hoskins (1934) tables. The plate colony counts showed an increase in viable cells after incubation in buffered metabolite. If reactivation was occurring, the dilution counts would agree with plate colony counts made after incubation in the presence of the metabolites; on the other hand, agreement between the dilution count and the number of cells present before incubation in the metabolite solution would indicate that growth was occurring in the buffer substrate. Table 2 confirms the second alternative. That E. coli can grow in phosphate buffer is further demonstrated by the results shown in table 3. The spension was not exposed to sodium hypochlorite but was inoculated directly

3 19551 GROWTH OF E. COLI IN BUFFER SUBSTRATE TABLE 3 The growth of Escherichia coli in buffer, buffer + metabolite, and di8tilled water 395 Live Cells Dead Cells Count after Incubation for Substrate Inoculated Inoculated 24 hr 48 hr 72 hr Buffer ,100 1,350,000 2,110,000 Buffer + citrate ,000 _ Buffer + lactic acid ,000 5,150,000 5,580,000 Buffer + malic acid ,720,000 - Buffer ,000,000 1,790, Buffer 890 8,900, ,000 Buffer 89 89,000, ,000 3,500,000 2,800,000 Buffer 89 8,900,000 5,200 Buffer + lactic acid ,000,000 5,330,000 - Buffer + lactic acid 890 8,900,000 4,300,000 Buffer + lactic acid 89 89,000,000 7,200,000 7,010,000 8,000,000 Buffer + lactic acid 89 8,900,000 1,240,000 _ Distilled water 1,580 1,580,000,000 12,960,000 Distilled water 1, ,000, ,000 Distilled water 1,580 15,800, Washed suspensions of cells inoculated into 0.1 M solution of KH2PO4-Na2HPO4. Metabolites when present as 0.1 per cent solutions. Numbers of cells estimated by plate colony counts. into the buffer substrate. From the results it is seen that E. coli grows well in the presence of malic and lactic acids, slowly in the presence of sodium citrate, and more slowly in phosphate buffer. Growth, as estimated by cell multiplication, occurs more rapidly when dead cells are present, and the last three results show that, providing there is a sufficient number of dead organisms, growth can occur even in distilled water. (The suspension used for inoculating the distilled water was washed twice in the same fluid before use.) In no case was an increase in turbidity observed in the tubes during incubation because the numbers of organisms, limited presumably by the food supply, failed to reach a level where they would cause visible cloudiness. The growth of organinsm in buffer. It was found that phosphate buffer also supported the growth of P. fluorescens, and tests were made to find whether the source of food utilized by both E. coli and P. fluorescens could be easily removed. In table 4 the growth of the test organisms in substrate A is compared with the growth in substrate B. Substrate A was prepared from "Analar" chemicals and glass distilled water. The solutions were contained in tubes plugged with cotton wool and normally in use in the laboratory. Substrate B was prepared from "Analar" salts which were purified by recrystallization; they were dissolved in resin filtered glass distilled water. The lactic acid used was a sample of high purity which happened to be available (see appendix). The solution was put into new glassware and the tubes closed with TABLE 4 The growth in buffer substrate prepared from purifled inaredients Live Count after 3 Days Inc-Inoculated lated Control Prfe Substrate Aimgredients Substrate CeiUs Dead Cells Prfe I Escherichia coli ~~~~~Substrate B Buffer 60, , ,000 60,000 2,000, , ,000 Buffer , ,000,000 2,000,000 per cent lactic acid Pseudomona8 fluorescens Buffer 40, ,000,000 3,000,000 Washed suspensions of cells inoculated into 0.1 M KH2PO4-Na2HPO4. Substrate A prepared from glass distilled water and "Analar" chemicals. Substrate B prepared from purified water, recrystallized "Analar" salts, and high grade lactic acid. Numbers of cells estimated by plate colony counts.

4 396 ELLEN I. GARVIE [vol. 69 TABLE 5 The effect of repeated subcultures through buffer Substrate Inoculum Tube 1 Tube 2 Tube 3 Tube 4 Escherichia coli Buffer 8, ,000 95,000 6,400 12,400 Buffer per cent 8,000 39,300,000 38,500,000 55,200,000 22,000,000 lactic acid Pseudomonas fluorescenre Buffer 24,000 3,000,000 7,300,000 5,000,000 5,000,000 Washed suspensions of cells inoculated into 0.1 M KH2PO4-Na2HPO4. Each tube was incubated for 3 days, and 0.01 ml used to inoculate 10 ml of substrate in the subsequent tube. Numbers of cells estimated by plate colony counts. aluminum caps. The new glassware for substrate B was boiled in soda. The glassware for both substrates A and B and the aluminum caps were cleaned in chromic acid before use, and the final rinse was with the appropriate distilled water. The results show that as the growth was the same in both substrates, the source of food and energy had not been removed by purifying the ingredients of the substrate. P. fluorescens grows in phosphate buffer, but although on some occasions growth occurred in buffer, E. coli required an additional carbon source (i.e., lactic acid). Growth of P. fluorescens became visible only when both lactic acid and (NH4)H2PO were added to the phosphate buffer solutions; therefore lack of both carbon and nitrogen was restricting growth; growth of E. coli became visible when (NH4)H2PO4 was present. During the course of the work a number of tests was made, and with either organism, irrespective of the number of cells present initially, the number after 3 days of incubation varied between 1 and 50 million, but was generally about 10 million and did not increase on further incubation. Table 5 shows the effect of successive transfers of both E. coli and P. fluorescens through buffer substrate (B). Tube 1 was prepared as described and incubated for 72 hr. At that time an estimation of the number of cells present was made, and 0.01 ml of tube 1 inoculated into tube 2 containing 10 ml of substrate. The process was repeated. The number of organisms present in tube 4 after incubation was the same as recorded in tube 1; each tube, therefore, contained sufficient nutrient to support the same amount of growth. Had the nutrients been carried from the initial suspension into the first tube, successive transfers would have resulted in these nutrients becoming more dilute, and a decrease in the number of cells would have been expected. A culture of E. coli was transferred at 6 hourly intervals during a period of 24 hr and the suspension prepared from 6 hr growth on an agar slant. The suspension probably contained a high proportion of live cells and a low proportion of dead cells; when it was used for inoculation into phosphate buffer substrate, growth occurred. It was, therefore, unlikely that growth was supported by nutrients obtained from dead cells. A similar test was made using P. fluorescens, but making the subculture at 9 hourly intervals, again growth occurred. It seems certain, therefore, that the nutrients supporting growth were contained either in the water supply, buffer salts, or were being leached from the surface of the glas. DISCUSSION It has been shown that a strain of P. fluorescens and a strain of E. coli will grow when cell suspensions are inoculated into phosphate buffer, and this was also found to occur when the buffer was prepared from ingredients of greater purity than normally used in the laboratory. The difficulty of preparing media free of traces of impurities has been discussed by others (Waring and Werkman, 1942; Young, Begg, and Pentz, 1944; and Pinsent, 1954). A million bacterial cells contain less than 0.1,ug of nitrogen and

5 19551 GROWTH OF E. COLI IN BUFFER SUBSTRATE 397 carbon, and it is therefore difficult to purify material so that it will not support the growth of microorganisms with simple food requirements. When other strains of organisms were studied, Lactobacilus acidophilus, Streptococcus lactis, Streptococcus faecalis, and Staphylococcus aureus decreased in number, while Bacillus cereus and Proteus vulgaris remained static, showing neither an increase nor a decrease in number. A second strain of E. coli and a strain of Aerobacter aerogenes behaved like E. coli strain 28.D.10. Bigger and Nelson (1941) found that E. coli would grow in distilled water if talc was added, and they suggested that the talc was making the carbon dioxide and nitrogen in the atmosphere available to the bacteria. If this were so, however, E. coli was growing autotrophically. In the present study, whatever the food supply it can only be utilized in the presence of oxygen because, incubated anaerobically, E. coli failed to grow. Heinmets et al. found that E. coli was not reactivated in phosphate buffer but generally needed an added carbon source for growth. Allen, Cooper, and Rose (1954) showed that in the absence of alternative supplies of carbon and under aerobic conditions E. coli was able to utilize breakdown products of glucose. It is important to know whether bacteria can be reactivated after treatment by disinfectants; no evidence so far available proves that in fact they can be. ACKNOWLEDGMENTS The author wishes to thank Dr. W. G. Wearmouth for supplying the resin treated water and special lactic acid, Dr. L. A. Mabbitt for recrystallizing the buffer salts, and both for giving much helpful advice. SUMMARY A strain of Ewcherichia coli after exposure to sodium hypochlorite was found to grow in a solution of a metabolite in phosphate buffer. When inoculated directly into buffer a strain of Pseudomonas fluoreses was found to grow and a strain of Escherichia coli (type 1) in phosphate buffer to which a source of carbon had been added. Growth took place only under aerobic conditions, and the maximum number of organisms was between 1 and 10 million cells per ml. Growth occurred even when very pure ingredients were used, and it appears probable that the food was not transferred with the bacterial suspension when the tubes were inoculated, nor was it obtained from dead cells. It is suggested that traces of impurities either in the chemicals, in the water, or on the glassware were supplying the necessary food and energy. APPENDIX W. G. WEARMOUTH National Institute for Research in Dairying, University of Reading, England The lactic acid used in the experiments was specially purified. Its ultraviolet absorption spectrum was measured on a "unicam S.P." spectrophotometer in the region 200 to 350 m,u. The wavelength for maximum absorption was 287 mju, and in a 12.5 per cent concentration in resin treated distilled water the acid showed an extinction coefficient K = The specially purified water was double distilled and then passed through a resin column filled with "biodeminrolit resin" supplied by Messrs. Permutit, Ltd., of London. It had an / WAVELE(4GTH ' Figure 1. Broken line: optical density of ordinary distilled water; solid line: optical density of specially purified distilled water.

6 398 ELLEN I. GARVIE [VOL. 69 optical density of 0.22 at 200 m,u, which is a considerable improvement on the more usual value of 0.43 for ordinary distilled water (figure 1). REFERENCES ALLEN, L. A., COOPER, A. H., AND ROSE, J. NAOMI 1954 Microbiological changes in carbohydrate media: Effect of aeration on ph value and on oxidation and assimilation. J. Appl. Bacteriol., 17, BIGGER, J. W., AND NELSON, J. H The growth of coliform bacilli in distilled water. J. Pathol. Bacteriol., 53, HEINMETS, F., TAYLOR, W. W., AND LEHMAN, J. J The use of metabolites in the restoration of the viability of heat killed and chemically inactivated Escherichia coli. J. Bacteriol., 67, HoSKINs, J. K Most probable numbers for evaluation of coli-aerogenes tests by fermentation tube method. Public Health Repts. (U. S.)., 49, PINSENT, JANE 1954 The need for selenite and molybdate in the formation of formic dehydrogenase by members of the coli-aerogenes group of bacteria. Biochem. J. (London), 57, WARING, W. S., AND WERKMAN, C. H Growth of bacteria in an iron-free medium. Arch. Biochem., 1, YOUNG, E. G., BEGG, R. W., AND PENTZ, E. IRENE 1944 The inorganic nutrient requirements of Escherichia coli. Arch. Biochem., 5, Downloaded from on March 11, 2019 by guest