by the exhaustion of essential nutrients, or by accumulation limitation placed on it by the experimental conditions to increase

Transcription

1 THE INTERMITTENT GROWTH OF BACTERIAL CULTURES Re-search Laboratories, Bureau of Dairy Industry, United States Department of Agriculture Received for publication, August 3, 1929 In ordinary laboratory experiments bacterial growth is circumscribed by the exhaustion of essential nutrients, or by accumulation of the products of growth. The population is forced by the limitation placed on it by the experimental conditions to increase to the maximum for these conditions and then decline. This does not necessarily show what would take place if the unfavorable factors created by the growth of the bacteria were continuously removed. We can conceive such a condition to exist in the animal body, in which fresh food is constantly supplied, and by-products removed by the blood and other body fluids. Growth in a long tube of fluid medium like that used by Bibb (1925) does not exactly duplicate conditions in the body but permits a fluid colony to extend itself continuously into regions, at most only slightly affected by the previous growth. It is conceivable that nutrients may be drawn by the colony from an area slightly in adyance of the growth and that products of growth may diffuse along the tube faster than growth would progress. However, it is not probable that this interchange takes place rapidly enough to affect growth seriously and our experiments indicate that there is no material change in the medium beyond the limits of the colony. The apparatus used in this work consisted of a pyrex tube 7 mm. in diameter and about 15 meters long, wound in a flat spiral with a flask sealed to the inner end of the tube. The flask was of a capacity to hold sufficient medium to fill the tube completely with a slight excess for inoculation. In autoclaving, the broth 181 JOURNAL OF BA(?URIOLOGT, VOL. xix, NO. 3

2 182 was forced out of the tube but was drawn back as it cooled. All air bubbles were worked out of the tube by careful tilting. A broth of the following composition was used: Infusion broth, 1000 cc.; pepton, 5 grams; lactose, 0.5 gram. The small amount of sugar was sufficient to promote a prompt and vigorous growth of Es. coli or Str. lactis but not enough to produce a change in reaction beyond the normal limits of these organisms. Bromthymol blue was used as an indicator. The cultures used were fairly active laboratory cultures of Str. lactis and Es. coli. No essential difference in the growth of these cultures was note.d, except that, as would be expected, Es. coli grew much more rapidly than Str. lactis. Inoculation was made into the flask, the coil held at 300 and the progress of the growth through the tube marked from time to time by the change in color. Turbidity and decolorization were nearly identical but the demarkation between the decolorized and the unaffected indicator was not always sharp and some irregularity in the results was introduced. In order to obtain more complete records a motion picture camera was arranged to make a photograph of the coil automatically at fixed intervals.' The small room in which the apparatus was installed was held at 300. With the culture of Es. coli at this temperature the growth covered the entire coil in about eight days. The film obtained in this way was projected to the normal size of the coil and the rate of growth determined. It was sometimes difficult to determine on this reproduction the exact extent of the decolorization but with the Es. coli inoculations the reaction of the broth was raised to ph 7.4 and the amount of indicator increased to give a more pronounced change. This reduced the difficulty and while the indefiniteness of the end point produced some raggedness in the curves the results obtained approach very closely the actual conditions. At first glance there would seem to be no reason why the growth should not proceed at a uniform rate through the entire length 1We are indebted to Mr. Harry Greene of the Motion Picture Laboratory for the use of this apparatus.

3 INTERMITTENT GROWTH OF BACTERIAL CULTURES of the tube. Bibb (1925) has reported that this is what occurs. However, we have been unable to confirm his results. The uneven progress of the growth was evident, even on casual observation. Periods of rapid advance of growth were followed by intervals of very slow progress or complete cessation of change. Figure 1, which is representative of a number of experiments, shows that there is a considerable degree of periodicity to the alternation of rapid and slow growth. In considering this graph it- should be kept in mind that the curve represents not extent of growth, but rate of advance of growth for definite time periods. The number of bacteria in the different periods could not be determined. The objection that variations in rate of growth may have been caused by uneven temperatures, electric currents, or other physical factors connected with the control and the lighting, can be met with observations made on a smaller vertical coil held in an ordinary incubator. Observations could not be made often enough, or for sufficient time, to establish periodic variations, but the marked variation in the rate of growth was very evident. The growth in the tube is obviously a colony in a liquid medium, pushing its margin out into new territory. The physical conditions under which a colony grows on solid media are so different from those existing in a fluid medium that it would not be safe to reason from one to the other. However, it can be readily demonstrated that the growth of a spreading colony on the surface of an agar plate does not proceed uniformly. Motion pictures with exposures at five-minute intervals were made of spreading colonies and the film projected so that measurements could be made on an enlargement equivalent to 41 times the diameter of the colony. Since growth was evidently not taking place uniformly at all parts of the colony, measurements were made along one radius only. These measurements showed marked variations in the rate of growth. There were frequent intervals of from ten to twenty minutes in which no progress could be detected. One colony which grew 8.4 mm. in one direction in five hours produced practically all of this growth in eighty-five minutes. The growing periods were 183

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5 INTERMITTENT GROWTH OF BACTERIAL CULTURES distributed fairly uniformly over the entire time. The frequent exposure to an intense light may possibly have been a factor;,in this alternation of slow and rapid growth. The rate of growth of a colony on the surface of an agar plate may be influenced by physical conditions which tend to make observations on this type of colony of little value in studying the rate of growth of bacteria. It is a matter of common knowledge that the growth of spreaders is dependent, to some extent at least, on the moisture conditions on the surface of the agar. Bacteriologists who have had persistent trouble with this type of colony in making water and milk counts would be surprised to find how difficult it is to induce a colony to spread for useful purposes. To secure a spreading colony it is usually necessary to use measures which produce a moist surface, as for instance quick cooling of the melted agar. It is conceivable that differences in the amount of water on different parts of the surface of the agar may account for variations in the rate of growth. This, obviously, would not be comparable with the variations observed in the coil. When plates were made so that the surface was very moist and the growth of a colony of B. vulgatus followed under a microscope held at a constant temperature, movement of free cells into the water beyond the limits of the colony could sometimes be observed. When this happened the rapid multiplication of these free cells produced a sudden extension in the limits of the colony. Ordinarily, however, the boundary of the colony was sharply defined and its growth had the appearance of a flowing due to an accumulation of cells in the interior. Growth usually proceeded in the form of streamers with the space between filling in more slowly. Colonies of Es. coli grew continuously but at a rate which varied at times over 100 per cent. The relation between colony growth and growth in the closed tube seems so doubtful that these data are not included. Some observations of a different nature may possibly help to explain the variations in the rate of growth in the tube. If a culture of Str. lactis is grown in a collodion sac, suspended in a flask of broth containing a very small amount of sugar so that the 185

6 186 reaction will not become unfavorable, as the growth in the sac progresses the broth in the flask betomes more favorable to the initial growth of the same culture. However, when the growth in the sac has reached its maximum the bacterial population which the broth in the flask will maintain has been much reduced. This effect can also be shown by removing portions of a broth culture from time to time and freeing it from bacterial cells by filtration. The results of an experiment of this nature are shown in table 1. These results are typical of a number of similar experiments made by growing the culture in a collodion sac or by filtration. The broth had the same composition as that used inxthe coil except that no indicator was used. The results shown in table 1 were obtained by growing Str. lactis in a flask held at 300. At the TABLE I The accelerating and inhibiting effect obtained in a broth culture of BACTERIA AGE COUNT IN TUBE BACTERIA COUNT IN FILTERED BROTH Str. lactis FLASK 4 hours 7 hours 11 hour 27 houas 0 A 320,000 2,525,000 40,000, ,000, ,000 B 144,000 1,125,000 11,950, ,000, ,950,000 C 420,000 3,000,000 54,900, ,000, ,000,000 D 8,675,000 8,750,000 8,750,000 7,100,000 time of inoculation, and at four, eight, and twenty-four hours, portions of this culture were filtered into sterile tubes. In each case the flask was inoculated from a fresh milk culture. The bacterial content of the broth at these periods is given in the second column of the table. When the last sample (D) was taken, the normal population for this broth had been reached. The tubes of filtered culture were inoculated with the strain of Str. lactis grown in the flask and counts made at four, seven, eleven and twenty-seven hours. The difference in tubes A and B at four hours are probably not significant but there can be no question about the accelerating effect shown in tube D. This marked effect on the initial growth was always observed in the twenty-four-hour culture and some-

7 INTERMITTENT GROWTH OF BACTERIAL CULTURES 187 times at an earlier period. At twenty-seven hours, tubes A, B and C had each reached the normal population for this broth but D had not increased after the first four hours. This inhibiting effect exerted by a culture on itself and on other cultures is well known. The accelerating factor is apparently specific or at least Str. lactis does not accelerate Es. coli. This is shown in table 2. In this experiment duplicates of tubes A, ST. LACTIS AG AG N FLASK TABLE 2 The inhibiting effect of Str. lactis on Es. coli TUBE ES. COLI IN FILTERED BROTH 4 hours 6 hours 8 hours 10 hours 27 hours Aours 0 A 99,000, ,000, ,000,000 1,045,000,000 1,250,000, ,000 B 91,000, ,000,000 1,195,000, , 000, 000 1, 085, 000, ,950,000 C 2,045, ,600, ,000, , 000, 000 1, 325, 000, ,000,000 D 4,940,000 13,550,000 39, 600, ,250, ,000,000 TABLE 3 Growth of Es. coli in filtered broth AGED OF 11:8. COLI IN T1JBE ES. COLI IN FILTERED BROTH AGEA ES. COLI IN TUBE CULTURE FLASK 2 hours 4 hours 6 hours 24 hours 0 A 4,31,000 55,000, ,000,000 1,230,000, ,000 B 4,860,000 8,750, ,000,000 1,250,000, ,000 C 3,330,000 67,000, ,000,000 1,300,000, ,150,000 D 7,545,000 95,000, ,000,000 1, 195, 000, ,550,000 E 6,670, ,500, ,000,00 1,300,000, ,000,000 F 2,460,000 5,950,000 25,700, ,000, ,260,000,000 G 11,650,000 30,450,000 38,950, ,000,000 B, C, and D, as shown in table 1, were inoculated with Es. coli. A distinct inhibiting effect is evident from the beginning in tubes C and D. In tubes A, B, and C the inhibiting action was overcome and a normal population finally reached. In D, while an increase continued for twenty-seven hours, the population was still less than half of the normal. What has been said of the accelerating effect of Str. lactis on itself evidently does not apply to Es. coli. In table 3 are shown

8 188 results obtained by growing Es. coli in tubes of broth removed from a culture by filtration at successive stages of growth. The limiting effect after the growth in the flask had reached some hund-reds of millions is evident, but there is no indication of an accelerating effect. The variations in the counts at two and four hours are not consistent and are probably only the differences which could be expected in a set of duplicate flasks. This conclusion was confirmed by results obtained in other experiments of a similar nature. Table 4 indicates that not only does Es. coli have no accelerating effect on Str. lactis but that the inhibiting effect is not marked. It is not surprising that such a difference between these cultures should be found. The lactics, while growing rapidly under TALBE 4 The effect of Es. coli on the subsequent growth of Str. lactis AGEC OF ZCO$ TEGROWTH OF 0T. LACTIS IN MAGEKO ES. COLI IN T_B_ FILTERED BROTH CULTURE FLASK 3 hours 7 hours 11 hours 24 hours hours 0 A 1,485,000 11,450o ,000, ,000, ,000 B 1,010,000-8,350, ,500, ,000, ,500,000 C 610,000 10,400, ,000, ,000, ,000,000 D 1,225,000 11,300,000 14,700, , 000, 000 favorable conditions, are sensitive to deficiencies in the nutrient medium. Es coli, on the other hand, grows luxuriently under conditions which would not support the growth of many bacteria. It does well in a very simple medium. It is possible to explain the accelerating and limiting effects observed in these cultures on nutritional grounds. It is conceivable that through a digesting effect a medium would become more favorable to initial growth, while at the same time some essential food constituent would become partially exhausted so that the normal population could not be attained. It is surprising, however, that through this action cultures of the lactic type should have such a decided effect on a vigorous species like Es. coli. while this organism has so little effect of Str. lactis.

9 INTERMITTENT GROWTH OF BACTERIAL CULTURES In the experiments in which a culture in collodion sac was suspended in a flask of broth, the broth in the flask showed the same effect of stimulating the initial growth and restricting the final growth. If this limitation of the final population was due to the partial exhaustion of one or more essential constituents of this broth this could only have taken place through their diffusion into the sac. If this had happened we would expect an increased population in the sac. This did not occur. The combined stimulating and inhibiting effect observed in the lactic cultures has some resemblance to the phenomena demonstrated by Carrel and Eberling (1923) who showed that the serum of a young animal contains a principle precipitated by CO2 which increases the proliferative activity of homologous fibroblasts while the serum of old animals has an inhibiting effect only. The application of the results obtained with the filtered cultures to the growth in the tube is not clear. It is possible that the growth of the lactic culture may have been influenced by the alternating stimulating and inhibiting effect. However, the coli culture showed. even more marked variation in rate of growth than the lactic, but if it possessed any self-stimulating action it was so slight that it was not detected. If we assume that there is an alternation of generations in bacteria a plausible explanation is available. The periods of rapid growth could be looked upon as active vegetative multiplication while the slow periods correspond to a time of sexual regeneration. It may also be possible to explain these variations by assuming periods of active motility alternating with periods in which there was little or no activity. Es. coli which is an active motile organism traveled more rapidly through the tube and showed greater variations in rate of growth. Str. lactis is generally considered to be immotile but we have had cultures in the laboratory which were actively motile. Ellis (1920) was able to demonstrate motility with various cocci, including streptococci, and believed that all cocci have cilia. In cultures which were ordinarily immotile, motility could be induced by rapid transfer 189

10 190 on a favorable medium. This condition would be stimulated in the growth through the tube. SUMMARY Cultures of Es. coli and Str. lactis inoculated into broth in a tube about 15 meters long grow through the tube in alternating periods of slow and rapid growth. Filtered cultures of Str. lactis stimulate the initial growth of Str. lactis but limit the final population. Filtered cultures of Str. lactis do not stimulate the initial growth of Es. coli but have a marked limiting effect on the final population. Es. coli does not stimulate itself or Str. lactis, and has onlya comparatively small effect on the final population of Str. lactis. REFERENCES BIBB, L. B Jour. Bacteriol., 10, 561. CARREL AND EBERLING 1923 Jour. Exp. Med., 38, 419. ELLIs, D Centr. f Bakt. Abth. II, 9, 546. Downloaded from on July 8, 2018 by guest