The Behaviour of Bacteria in Fluid Cultures as Indicated by Daily Estimates of the Numbers of Living Organisms

Extract

1. In dilute neutral meat extract cultures (without salt or peptone) inoculated with relatively small numbers of S. aureus, taken from agar cultures grown for 18 hours at 37° C. and incubated at 37° C., multiplication proceeds rapidly during the first day and more slowly on the second, when the maximum number, about 10,000,000 per standard loop (0·01 c.c.), is reached. Later the number of living organisms decreases at first rapidly, but later more slowly, until a low level is reached, which remains fairly constant or falls very slowly for a long period. During the period of relative constancy small oscillations are observed. The curve produced on plotting out the daily counts may be regarded as a “standard.”

2. The frequency with which the culture used for inoculation has been transplanted on agar slopes influences the growth on neutral meat extract. Several transplantations in rapid succession result in very rapid growth, a high maximum and a very rapid fall in the numbers. Less frequent transplantation over a long period seems to cause the maximum to be reached later than in the standard and the period of decline to be postponed.

3. In one series of experiments (Section III) the proportion of meat extract was varied in the different tubes employed. These experiments show that the greater the proportion of meat extract the greater is the multiplication, and the longer the period which elapses before the curve reaches its highest point, in fact the extent of multiplication appears to be closely related to the amount of meat extract present in the culture. The length of the period of rapid decline is also related to the amount of meat extract present.

4. The form of the curve of growth is influenced by the number of cocciinoculated. With a small inoculation into dilute neutral meat extract the maximum number of cocci present in the medium at any period does not usually exceed 10 to 12 millions per standard loop. If the initial dose greatly exceeds this figure multiplication proceeds relatively slowly for two days and subsequently there is a very rapid fall in the numbers. With an initial dose close to this figure a somewhat similar curve is produced, though the rate of fall is not so rapid. Much smaller doses produce “standard” types of curves.

5. Provided the numbers inoculated are small (50,000–50 per drop) the results after 24 hours' incubation in different experiments of the same kind are not materially affected.

6. If after the numbers have reached a low level small drops of concentrated meat extract, insufficient to cause appreciable dilution, are added to the culture further multiplication occurs, to some extent proportional to the amount of food material added. The fall in numbers, which follows the initial rise, is not due therefore to the accumulation of products, but seems to be caused mainly by the using up of food material.

7. By small regular additions of food material (concentrated meat extract) a definite concentration of Staphylococci can be maintained in a meat extract medium for a long period of time, and probably by suitable additions any desired concentration could be maintained. Accumulation of the products may gradually inhibit growth, but on this point the experiment gives little evidence.

8. Moderate dilution with distilled water at any stage of incubation has little effect. Events occur in the usual sequence, but the number of organisms in each standard drop is proportional to the dilution.

9. The incubation temperature has a great influence on the course of events in meat extract cultures of S. aureus. At 37° C. multiplication during the first 24 hours is very rapid, the maximum is attained on the second or third day, and the numbers fall very rapidly. At 27° C. the maximum is attained on the fifth or sixth day, and is considerably greater than that attained at 37° C. The fall is rapid. At 17° C. multiplication is very slow during the first 48 hours, but is subsequently rapid, and the maximum, which is higher than that attained at 27° C., is reached on the eighth day. The decline in numbers is slow.

At 8 to 10° C. very slight multiplication, if any, occurs during the first 24 hours and subsequently the numbers steadily decline for at least 60 days.

At lower temperatures the numbers fall rapidly and the cultures die. At — 1° C. the organisms were dead by the 19th day, at — 6° C. by the 13th day, and at — 10° C. by the 9th day.

10. If organisms such as S. aureus, B. coli or B. pyocyaneus are allowed to grow in meat extract medium at 37° C. until the numbers have reached a low level, and the tubes are then inoculated with the species originally present little or no multiplication takes place, but if one of the other organisms is inoculated multiplication of the added organisms occurs. If the cultures are sterilised by boiling before inoculation with fresh organisms the original strain or the others, when added, multiply. Boiling, therefore, appears to liberate some food for added organisms belonging to the strain which was originally present.

The growth of any of these organisms in the medium seems to remove most of the food for that species as well as a portion of the food substance used by other species, since in no case was the growth of the added species nearly so considerable in extent as in its primary cultures.

11. The effect of adding increasing quantities of N/10 hydrochloric acid up to 0·3 c.c. to each 5 c.c. of the medium is to retard the growth of the cocci during the earlier stages of incubation, though subsequently rapid growth takes place, and a high maximum is reached. With small inoculations of cocci the addition of more than 0·3 c.c. N/10 hydrochloric acid results in the death of the organisms within a short time.

With additions of N/10 soda varying between 0·4 and 1·2 c.c. there seems to be a progressive decrease in the height reached by the maxima, the rate of growth in the early stages is retarded, and the rate of decrease in numbers seems to be retarded. With the addition of 1·6 or 1·8 c.c. N/10 soda the rate of growth in the early stages is markedly retarded. With the addition of 2·0 c.c. N/10 soda no growth occurs.

B. coli seems to be more sensitive than S. aureus, especially to the addition of alkali.

12. More precise experiments with N/10 hydrochloric acid show that with the addition of increasing amounts of the acid the type of curve gradually changes from a “standard” with one peak to a curve with two peaks, separated by an interval in which the numbers are small.

13. By continuous growth in acid, neutral and alkaline meat extract the capacity of S. aureus to multiply when transplanted into media of different reactions is altered. When transferred into an acid medium all strains show a small primary rise followed, after a fall in the numbers, by a great secondary rise. In the case of the acid acclimatised cocci the secondary rise reached its maximum on the 7th day, in the neutral acclimatised cocci on the 13th day, and in the alkali acclimatised cocci on the 15th day. In the neutral medium there is also a primary and a secondary rise, but the former is much greater than the latter. In the case of the acid acclimatised cocci the primary rise was least in height and duration, and in the case of the alkali acclimatised cocci greatest both in height and duration. In the alkaline medium a primary rise only occurs and subsequently the numbers fall to a very low level. The rise was least in the acid acclimatised cocci and greatest in the alkali acclimatised cocci.

In comparing these experiments with others previously quoted it should be remembered that the organisms have been acclimatised to growth not only in media of different reactions, but also to continuous growth in fluid meat extract medium.

14. S. aureus can multiply to a small extent in neutral gelatin solution (8 per cent.). On a medium consisting of gelatin and meat extract the greatest multiplication takes place, much higher figures being obtained than the maxima of growth on gelatin solution and meat extract respectively added together. In agar solution (0·8 per cent.) also no multiplication takes place, and the cocci quickly die. In a medium consisting of agar and meat extract the maximum reached is lower than in meat extract, but the decline in numbers is slower.

15. When certain quantities of various acids are added to warm meat extract agar precipitates are formed, though little or no precipitate may be produced by lesser or even slightly greater quantities. In some instances no growth occurred in plates poured from those tubes in which a precipitate had formed.

16. The addition of glucose to the extent of 1 per cent. to dilute meat extract results in most cases in S. aureus multiplying rapidly during the first day. Subsequently the numbers decline and the culture dies. With increasing quantities of glucose the maximum figure attained diminishes, and the rate of the subsequent fall, at least from the second to the fourth day, increases. Even with a very small quantity of glucose the numbers begin to fall after 24 hours's incubation, instead of rising as they do in cultures without glucose.

If to cultures containing 1 per cent. glucose daily additions of small quantities of concentrated meat extract or of concentrated meat extract with glucose are made oscillations in the numbers occur, but the cultures remain alive and with large additions multiplication may take place. The death of the organisms is not hastened by small daily additions of glucose.

17. The addition at different times of small numbers of the cocci to growing cultures of S. aureus has no appreciable influence, but the addition of large numbers exerts a considerable influence.

18. In meat extract cultures of S. aureus incubated at 37° C. about 15 per cent. of the living organisms sink to the bottom after each daily shaking. If the tubes are left undisturbed about 25 per cent. sink to the bottom.

19. Meat extract incubated with chloroform for 24 hours at 37° C. and sterilised by boiling seems to be a slightly better medium than fresh meat extract sterilised by boiling or autoclaving immediately after preparation. Pancreas extract is a better medium than meat extract. The multiplication of cocci is greatest in pancreas extract incubated with chloroform for 24 hours at 37° C. and sterilised by boiling.

20. Organisms accidentally contaminating cultures of S. aureus may cause, according to their species, a sudden decline or a rapid increase in the number of the cocci.