commercial and biological interest. The water usually contains which have been tested tolerate no more than 6 per cent salt and

Transcription

1 THE BACTERIOSTATIC AND BACTERICIDAL ACTION OF GREAT SALT LAKE WATER CLAUDE E. ZOBELL AND D. QUENTIN ANDERSON Scripps Institution of Oceanography, University of California AND W. WHITNEY SMITH Bacteriology Department, University of Utah Received for publication August 31, 1936 Great Salt Lake in northern Utah is an extensive and extremely saline body of water having considerable geological, historical, commercial and biological interest. The water usually contains more than 20 per cent salt and in December 1935 when the water for the experiments reported below was collected, the salinity was 27.6 per cent. In reviewing the literature of bacterial life in strong brines Hof (1935) shows that most species which have been tested tolerate no more than 6 per cent salt and he concludes that 15 per cent salt limits the activities of halophiles with very few exceptions. Previous to the work of Daines (1917) who recovered as many as 625 bacteria per milliliter of water from Great Salt Lake, it was believed to be sterile. However, inasmuch as he isolated these bacteria on 2.5 per cent NaCl nutrient agar, his investigations failed to show whether the bacteria were halophiles indigenous to the lake or if they were merely halotolerant contaminants. In a survey covering a large number of samples collected during the last year from different depths and stations, Smith (1936) almost invariably found a few hundred bacteria per milliliter of lake water which developed on lake water media. Using a direct microscopic technique Smith and ZoBell (1936) have demonstrated the occurrence of autochthonous bacteria in Great Salt Lake which are capable of multiplying to form microcolonies on glass slides submerged in the brine. In continuing these studies it is desirable to know if the bacteria found in the 253

2 254 C. E. ZOBELL, D. Q. ANDERSON AND W. W. SMITH lake are obligate halophiles which are foreign to other habitats or if the water also contains appreciable numbers of halotolerant transplants from soil, sewage or other less saline environments. The problem also has a public health aspect since the unusual properties of the brine annually attract thousands of bathers to the resorts. Considering this aspect, Frederick (1924) records that the etiological agents of certain ocular, respiratory or gastrointestinal infections remain viable in lake water for extended periods and some of them actually multiply in lake water. However, she used massive inocula of stock cultures and other questionable techniques which may invalidate her conclusion that there is a real danger of infection from undiluted lake water. EXPERIMENTAL In order to determine to what extent bacteria from different habitats multiply in Great Salt Lake water, nutrient media were prepared having the following composition: Bacto-peptone grams Proteose-peptone grams Beef extract grams Bacto-agar grams Water ml. Either lake water, diluted lake water, distilled water or sea water from the Pacific Ocean was used. The reaction of all the media was adjusted to ph 7.5. Lake water must be heated higher than 1000 in an autoclave to dissolve the agar. At first considerable difficulty was experienced with the lake-water nutrient agar which congealed prematurely. Undiluted lake water containing 10 grams of Bacto-agar per liter congeals within a few minutes at 600 and even in lake water diluted one-half with distilled water, 10 grams of agar gives a medium which congeals before it can be cooled to 450 for plating. Five grams of agar per liter were found to be adequate to solidify lake water medium and it is necessary to cool it rapidly to 450 and pour it immediately or it will congeal in the tube. Six grams of agar per liter were adequate to solidify the 75 per cent lake water medium (750 ml. lake water plus 250 ml. distilled water); 7.5 grams were used in

3 GREAT SALT LAKE WATER the 50 per cent lake water, and 10 grams per liter in more dilute lake water. The different media were used to plate identical samples of raw lake water, sewage, sea water, soil and the pooled washings from the mouths of two individuals. Plates inoculated with the oral flora were incubated at 370 and the others at 250 until the counts became fairly constant. In general, the sewage bacteria developed most rapidly, there being little increase in the colony counts after four days. The bacteria from the lake developed most slowly, requiring two to three weeks for the colony counts to approach constancy; and even after prolonged incubation the majority of the lake water bacteria formed only minute colonies. TABLE 1 Number of bacteria per milliliter of Great Salt Lake water which developed on nutrient agar prepared with different concentrations of lake water (L.W.), distilled water or sea water SAMPLE 75 PER CENT 50 PER CENT 25 PER CENT 10 PER CENT DISTILLED SEA WATER NUMBER L.W. L.W. L.W. L.W. WATER The plate counts on six samples of lake water on the different media are given in table 1. The water was collected in glass bottles from the end of the Salt Company pier near Saltair resort in December 1935 at which time the water temperature was 1.20C. Although bacterial colonies appeared on the undiluted lake water medium, their small size and the crystallization of salt made it impossible to enumerate them. The highest counts were obtained on the 75 per cent lake water medium. Almost as many lake bacteria developed on the 50 per cent lake water medium, or that which was diluted one-half with distilled water. With further dilution the counts decreased sharply. There was an average of 3.8 bacteria from the lake water samples

4 256 C. E. ZOBELL, D. Q. ANDERSON AND W. W. SMITH which grew on distilled water medium for each 100 which grew on the 75 per cent lake water medium. Unlike the typical lake bacteria, most of the colonies which developed on the distilled water medium appeared within two or three days and were quite large. As a matter of fact, the designation "distilled water medium" in this experiment is misleading because when 15 ml. of nutrient medium prepared with distilled water is inoculated with 1.0 ml. of raw lake water having a salinity of 27.6 per cent, the resulting medium actually contains about 2 per cent salt. When some of the colonies which developed on the so-called distilled water medium were emulsified in sterile water and streaked on 75 per cent lake water medium, only a small percentage of TABLE 2 Relative numbers of bacteria from different sources which developed on nutrient agar prepared with various dilutions of Great Salt Lake water (L.W.), distilled water and sea water, expressed as ratios to the plate counts on the best medium expressed as PER 50 PER 25 PER 10 PER DIS-E SOURCE OF SAMPLE CENT CENT CENT CENT TILLED WATER L.W. L.W. L.W. L.W. WATERWAE Sewage Soil Oral cavity Pacific Ocean Great Salt Lake them grew although all of them grew when transplanted on freshwater medium. This is believed to indicate that at least some of the bacteria from the lake which develop on distilled water media are halotolerant freshwater varieties. Table 2 summarizes the relative numbers of bacteria from sewage, soil, oral cavity, the Pacific Ocean and Great Salt Lake which developed on the different media. The averages of two or more determinations are given expressed as ratios on a basis of 100 representing the number of colonies on the respective medium which proved to be best. The highest counts of sewage, soil and mouth bacteria were obtained on the medium prepared with distilled water. Therefore the microflora from these sources

5 GREAT SALT LAKE WATER 257 will be referred to as freshwater microflora. Virtually none of the freshwater bacteria grew on the more concentrated lake water media. Even 25 per cent lake water inhibited most of them. The number of bacteria from soil, sewage or the oral cavity which were capable of growing in 50 to 75 per cent lake water media was too small and variable to permit expression although occasionally such halophilic varieties are encountered. Soil bacteria were more euryhaline than those from the oral cavity or sewage. Only 0.23 per cent of the bacteria found in 11 different swimming pools in Salt Lake City and 1.84 per cent of those in the unchlorinated municipal water supply grew on 50 per cent lake water medium. It is of interest to note that the sea water medium was more bacteriostatic than the 10 per cent lake water medium for all the microflora except those from the sea although both kinds of media had approximately the same salinity and osmotic pressure. It has been shown by Lipman (1926), Korinek (1927) and others that the bacteria in the sea differ from freshwater varieties in their ability to live in sea water. According to ZoBell and Feltham (1933) who made similar observations on the specificity of marine bacteria, sea water possesses some factor besides its salt concentration which inhibits non-marine bacteria and which favors marine species. While most of the bacteria in the lake are obligate halophiles as indicated by the fact that few of them developed on media hypotonic to lake water, they do not require lake water media for their multiplication because many of them developed on isotonic sodium chloride media. When lake water lactose broth was inoculated with 1.0 ml. quantities of raw sewage there was rarely any evidence of fermentation and never 10 per cent gas production although freshwater lactose broth revealed the presence of thousands of gasifying Escherichia coli per milliliter of the sewage. Attempts to recover E. coli from the inoculated lake-water lactose broth after 48 hours by streaking on standard E.M.B. plates were all negative. This indicates that lake water is bactericidal as well as bacteriostatic for E. coli. Similarly pure cultures of recently isolated E. coli and Staphylococcus albus were inoculated into

6 258 C. E. ZOBELL, D. Q. ANDERSON AND W. W. SMITH Old stock cultures of E. coli were found to be much more lake water nutrient broth of different concentrations. In each case a loopful of the organisms from young agar slants was suspended in 10 ml. of sterile saline and then a loopful of the resulting suspension was used to inoculate tubes of lake water broth, care being taken not to touch the inside walls of the tube with the inoculating loop. These were incubated at 250C. since all of the stock cultures were able to grow at this temperature and since this approximates the temperature of the lake water in the summer time. After four days there was no evidence of growth in the 25 per cent or higher concentrations of lake water broth. S. albus clouded the 10 per cent lake water broth a little. Streaking loopfuls of the broth on nutrient agar revealed that the 50 per cent as well as the undiluted lake water broth was sterile but viable S. albus and E. coli were recovered from the 25 per cent lake water broth. The number of organisms recovered was only a small fraction of the number originally present, showing that even in the presence of peptone, 25 per cent lake water is bactericidal. salt tolerant than the recently isolated strains and even the latter were more resistant than those occurring naturally in sewage. Field observations reported by Smith (1936) also indicate that sewage bacteria cannot tolerate lake water. He inoculated 23 samples of lake water collected near places of sewage pollution into standard lactose broth none of which was fermented. Likewise all were negative for E. coli when streaked on E.M.B. plates. A search of the Utah State Board of Health laboratory records reveals that E. coli has not been found in undiluted lake water. However, it should be recognized that under certain conditions inflowing freshwater may actually flow over the surface of the denser lake water for considerable distances thereby forestalling the mixture of the salt water with the freshwater bacteria. Also the bactericidal potency of the lake water in nature will be greatly reduced by high concentrations of organic matter and by dilution with more than three volumes of freshwater. Quantitative data on the bactericidal effect of lake water were obtained by seeding it with appropriate dilutions of sewage or other sources of freshwater bacteria. After various periods of

7 GREAT SALT LAKtE WATER exposure, 1.0 ml. quantities were plated out on standard freshwater nutrient agar. Controls were run consisting of "formula C," a balanced salt solution which Butterfield (1932) has found to be superior to other dilution waters for the prolonged survival of water bacteria. The plates were incubated for four days at 250 and the colon es counted. Table 3 summarizes typical findings on the death rate of sewage bacteria in different concentrations of lake water. The results are expressed as ratios on a basis of the number of bacteria surviving in the "formula C" control being 100. TABLE 3 Relative numbers of sewage bacteria which survived exposure to Great Salt Lake water (L.W.) of different concentrations for various periods of time expressed as ratios to the number which survived in "formula C" expressed as 100 TIME OF "FORMULA LAKE 75 PER 50 Pt 25 PER 10 PER& 5 PEB DIS- EXPOSUZ C" WATER CENT 'CENT CENT CENT CENT TILLED L.W. L.W. L.W. L.W. L.W. WATER 1 minute minutes minutes hour hours hours hours Great Salt Lake water destroyed the viability of the majority of the sewage bacteria almost immediately. One minute's exposure to undiluted lake water killed over 95 per cent of the bacteria. It is recognized that part of the apparent bactericidal effect may be due to the bacteriostatic action of the lake water which is carried over with the inoculum, because when 15 ml. of nutrient agar is inoculated with 1.0 ml. of lake water, the medium contains about six per cent lake water or two per cent salt, and this is known to inhibit the growth of some sewage bacteria. However, bacteria do not multiply after being exposed to more dilute lake water which would not carry enough salt into the medium to inhibit growth. Therefore most of the decrease in the counts can be attributed directly to the bactericidal effect of

8 260 c. E. ZOBELL, D. Q. ANDERSON AND W. W. SMITH lake water. The continued decrease in the counts with increasing time of exposure lends support to this contention. Tests were also made on the viability of other freshwater microflora in lake water. Table 4 shows that the mixed microflora from the oral cavity dies off rapidly in lake water although a few varieties survive six hours' exposure. In general, there was a larger proportion of halotolerant bacteria in the mouth than in sewage. About 60 per cent of the soil bacteria died within the first minute when placed in lake water although 9 per cent were still alive after six hours' exposure. A larger proportion of soil bacteria were halotolerant than of other freshwater bacteria. TABLE 4 Relative numbers of bacteria from the oral cavity which were viable after exposure to Great Salt Lake water (L.W.) of different dilutions for various periods of time expressed as ratios to the number recovered from "formula C" expressed as 100 "FORM}ULA LAKE 75 PER 50 PER 25 PER 10 PER SPER DIS- TIME C WATER CENT CENT CENT CENT CENT TILLED L.W. L.W. L.W. L.W. L.W. WATER 1 minute minutes minutes hour : hours hours The death rate of freshwater bacteria is virtually the same in 25 per cent lake water as in 8 per cent sodium chloride,-solutions which are nearly isotonic. Likewise their death rates were the same in more concentrated isotonic solutions of lake water and salt. However, the bacteria survive longer in dilution waters consisting of 10 per cent or less of lake water than in isotonic solutions of sodium chloride. This is attributed to the favorable effect of the mineral balance offered by the dilute lake water whereas in the more concentrated solutions the high osmotic pressure or a specific effect of ions is injurious regardless of the mineral balance. The literature on the favorable effect of balanced salt solutions on bacterial viability is reviewed by Zeug (1920) and Falk (1923).

9 GREAT SALT LAKE WATER After the first few minutes both 5 and 10 per cent lake water were less injurious to freshwater bacteria than pure distilled water. In fact, concentrations of lake water ranging from 0.1 to 1.0 per cent compared favorably with "formula C" and in some cases even excelled it for maintaining the viability of freshwater bacteria for extended periods of time. This is not so surprising when one considers that "formula C" is based upon the average composition of river water, and Great Salt Lake water is essentially concentrated river water. Samples of lake water collected in December 1935 had the following composition expressed as grams per liter: Total salts grams Chloride and other halides grams Sodium and potassium grams Sulfate grams Magnesium grams Calcium gram Carbonate and bicarbonate.0.06 gram Traces of iron, phosphate, nitrate and ammonium were also detected. The hydrogen-ion concentration was ph 8.0 to 8.3 as determined calorimetrically and corrected for salt error (Parsons and Douglas, 1926). The presence of 0.61 mgm. of total iodine per liter may be significant. CONCLUSIONS Further evidence is presented of a bacterial flora indigenous to the Great Salt Lake in northern Utah. From water having a salinity of 27.6 per cent an average of 167 bacteria per milliliter were cultivated on nutrient lake-water agar. Most of the lake bacteria are obligate halophiles whose growth requires at least 13 per cent salt as indicated by the fact that diluting lake water more than 50 per cent inhibits their multiplication. Conversely, few or no bacteria from sewage, soil or the oral cavity grow on lake water media and even 10 per cent lake water inhibits the multiplication of over three-fourths of the freshwater bacteria. Lake water is likewise bactericidal for freshwater bacteria including Escherichia coli, Staphylococcus albus and the mixed micro- 261

10 262 C. E. ZOBELL, D. Q. ANDERSON AND W. W. SMITH flora of sewage, soil and the human mouth. Marine bacteria which are generally regarded as halotolerant are killed by a few minutes' exposure to Great Salt Lake water. Besides their halophilic properties, lake bacteria differ from those from other habitats in the slowness with which they develop and the smallness of their colonies. It is believed that they are species which have become acclimatized to the increasing salt concentrations during the time the water of old Lake Bonneville has evaporated leaving its saline remnant, Great Salt Lake. REFERENCES BUTTERFIELD, C. T Jour. Bact., 28, DAINES, L. L Amer. Naturalist, 51, FALK, I. S Abst. Bact., 7, 33-50, and FREDERICK, ELFRIEDE 1924 On the bacterial flora of Great Salt Lake and the viability of other microorganisms in Great Salt Lake water. Master's thesis, University of Utah, 65 pp. HOF, T Recueil d. Trav. bot. neerl., 32, KORINEK, J Centralbl. f. Bakt., Abt. II,71, LIPMAN, C. B Jour. Bact., 12, PARSON, L. B., AND DOUGLAS, W. F Jour. Bact., 12, PEIRCE, G. J Jour. Calif. Bot. Soc., 1, SMITH, W. W Evidence of a bacterial flora indigenous to the Great Salt Lake. Master's thesis. University of Utah, 101 pp. SMITH, W. W., AND ZOBELL, C. E Direct microscopic evidence of an autochthonous bacterial flora in Great Salt Lake. Submitted for publication. ZEUG, M Arch. Hyg., 89, ZOBELL, C. E., AND FELTHAM, C. B Proc. Fifth Pacific Sci. Cong., 8,