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Title: Some physico-chemical aspects of bacterial growth
Author: Jackson, Stanley
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1949
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Ability to accept changes in nutritional supply, and to become resistant to the action of drugs, are striking characteristics of bacteria. The mechanisms of these training processes have long been an issue of controversy. Alternative theories which have received some support have postulated, on the one hand, selection of mutant cells, and, on the other, direct adaptation of cells in response to their environment. One type of 'training' which has received some attention is exemplified in the behaviour of Bact. coli mutabile in lactose medium. In this medium, the species does not, on the first transfer, commence growth until after a long lag phase, but thereafter grows readily in the medium. Growth tests are usually made by inoculating a considerable number of cells into a medium containing the test substrate. The mutation theory suggests that a very small proportion of the cells in a test inoculum are, in virtue of chance mutation, able to grow on the new substrate, in this case lactose; the majority of the cells are not able to grow. Although the few mutant cells begin to grow very shortly after inoculation, the growth does not contribute to a detectable increase in total population until the number of mutant cells has become comparable with the total number of cells in the inoculum, so that there appears to be a long lag phase before growth begins. Once the culture has grown, the cells which are 'lactose-using mutants' are in a majority, and are subsequently able to grow readily in lactose medium. The alternative, adaptation theory postulates that all of the cells in the test inoculum are potentially capable of reorganisation, in response to the presence of lactose, until they are able to grow on this new substrate. The reorganisation process takes time – hence the long lag phase. The changed cells are afterwards able to grow readily in lactose medium. It is seen that the mutation theory requires that only a small proportion of the cells should be able to grow on lactose, fairly quickly, while the adaptation theory suggests that most of them are able to do so, but only after a long lag phase. In Part II, experiments are reported in which the behaviour of small numbers of cells was observed. Small inocula were tested in both liquid and solid lactose medium; in each case, parallel tests were made with a medium which is readily used by all the cells of the strain. With solid media, it was found that equal numbers of colonies appeared on lactose medium and on the control medium, in the former case after a long lag phase. Each colony springs from a single cell, or at most a very small number of cells. The result is in accord with the adaptation theory, but is incompatible with the mutation theory. With liquid media, it was found that inocula containing 100 cells or more grew in lactose medium after a long lag phase. Smaller inocula failed to grow, although inocula containing only one cell grew in the control medium. When a rich nutrient was added, to the tests which failed to grow in lactose medium, it was found that there was still no growth, and it was evident that the cells had died. The estimate most frequently put forward for the proportion of mutant cells in strains of bacteria is about one mutant per 106 normal cells, and, certainly, only such a low proportion could account for the length of the lag phase in this case. The growth of inocula containing only 100 cells is incompatible with the mutation theory, and it seems clear that the smallest inocula failed to grow because they were overtaken by death before adaptation was complete. Failure of small numbers of bacteria to grow in new substrates has frequently been reported, and has been interpreted as proof that only mutant cells were capable of growth in the new medium. Failure to grow is, however, inadequate evidence – only if it could be proved that cells which refused to grow after having had time to become adapted were viable would the mutant theory be vindicated. Similar experiments were carried out to investigate the long lag of Bact. lactis aerogenes in D-arabinose medium, and of a coli-aerogenes intermediate in sodium citrate medium. In liquid media, all inocula, even those containing only one cell, grey in the new substrate after the appropriate long lag phase. On solid media, equal numbers of colonies of Bact. lactis aerogenes grew on D-arabinose medium (after the long lag phase) and on the glucose control medium; the behaviour of the coli-aerogenes intermediate on solid media was erratic. It seems probable that, in both these examples, the bacteria train to the new substrate by an adaptation process. Experiments with Bact. coli, tested in ammonium sulphate medium, led to the conclusion that the cells of this strain only trained to use ammonium sulphate, after a long lag phase, when traces of broth, carried over from the previous medium, were also present. Part II is concerned with an attack on the problem from a different angle. The length of the lag phase of Bact. lactis aerogenes in D-arabinose medium was shown to be independent of the source of carbon previously used. If an attempt is made to account for this in terms of the mutant selection theory, it must be supposed that the strain contains an almost constant proportion of mutant cells which are capable of growth in L-arabinose medium. This condition would be satisfied if the rate of mutation were very small, and the small proportion of mutants had been established over a considerable period of time. However, cultures grown directly from single colonies showed the normal behaviour, proving that this was not true. If the phenomenon does depend on mutation, it is clear that a constant proportion of mutants can. only be maintained by balance with back-mutation, in a state of dynamic equilibrium. The implications of this theory were developed in mathematical form; it emerged that the minimum rate of back-mutation necessary to account for the observations would lead to steady 'de-training' if cells grown in D-arabinose medium were afterwards sub-cultured in glucose medium. The theory predicted that, as the 'trained' cells were grown in glucose medium, their lag, when tested in D-arabinose medium, should steadily increase. Experiments were carried out to test this prediction. It was found that the actual behaviour was quite different. Initially, there was a fairly rapid, but not very great, increase in the lag in D-arabinose medium, but thereafter there was no further increase during the course of the experiments, which were continued until the predicted lag was very much greater than the actual lag. By this criterion also, the mutation theory was incompatible with experimental observation. Although the behaviour of Bact. coli mutabile with regard to lactose was not completely investigated in the same way, the characteristics appeared to be similar to those just described for Bact. lactis aerogenes and D-arabinose. Adaptation may be supposed to occur by the synthesis of an enzyme pattern which plays an important part in metabolism of the new substrate. The pattern is probably present in a rudimentary form and in slight extent in the untrained cells. It appears that the adaptation process may result in a quite stable change in the structure of the cell, even if the process has not been completed. If cells of Bact. lactis aerogenes are exposed to D-arabinose medium for a period less than the lag phase, and are then allowed to grow in glucose medium, they afterwards require a correspondingly shorter lag phase when tested in D-arabinose medium. As already mentioned, cells of Bact. lactis aerogenes which have actually been grown in D-arabinose medium are subsequently able to grow in the medium after only a short lag phase. Prolonged culture in glucose medium has little effect on this property. In Part III, this stability is shown to be in sharp contrast with the changes observed in D-arabinose dehydrogenase activity. Glucose dehydrogenase activity, under the conditions used, was almost constant, at a high level, whatever the cultural history of the cells. Untrained cells possessed only very slight D-arabinose dehydrogenase activity. Cells which had grown in D-arabinose medium were equally active with either D-arabinose or glucose, but the D-arabinose activity was rapidly reduced by subsequent growth in glucose medium. The D-arabinose activity of very thoroughly trained cells was reduced less rapidly than that of cells less thoroughly trained. 'Training' by the criterion of the length of the lag phase in D-arabinose medium, is very stable during subsequent culture in glucose medium, but, on the other hand, the dehydrogenase activity in contact with D-arabinose is rapidly reduced by this treatment. The length of the lag phase does not directly depend on the dehydrogenase activity – it appears that 'training' primes the cell for synthesis of D-arabinose dehydrogenase enzyme, when this is required. These observations may be accounted for if it be supposed that, during the long lag phase, a fragment of protein in the untrained cells, an approximation to the ideal pattern for D-arabinose metabolism, is expanded, to form a suitable enzyme, and that the greater stability of D-arabinose activity in very thoroughly trained cells is due to perfecting and stabilising of the enzyme configuration during prolonged training. Further, if it be supposed that 'trained' cells, cultured in glucose medium, retain a small fragment of enzyme, which is more suitable for reaction with D-arabinose than the imperfect fragment of untrained cells, and can be readily expanded and activated to permit D-arabinose metabolism, the stability of the short lag phase of 'trained' cells in D-arabinose medium may be accounted for.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available