Some factors influencing lactose fermentation by Shigella Sonnei

Thesis (Ph.D.)--Boston UniversityThe fermentation of lactose by Shigella sonnei has been associated with the presence of mutants. We observed that lactose-fermenting (lactose) mutants of Sh. sonnei could be isolated from all lactose broth cultures which had become acid within about ten days of incubation. However, lactose mutants could not be isolated from cultures which had become acid after this time. It was thought that the absence of lactose-fermenting organisms in these cultures might be due to the death of the mutants because of increased acidity produced by lactose fermentation. The pH of all acid cultures was measured but no apparent difference was found between those from which lactose mutants were isolated and those in which lactose mutants could not be detected. The possibility existed that acid production might have been caused by lactose mutants present in numbers too few to be detected by loopful sampling. By the method described by Kacoyanis and Baker (Proc. Bact., 1935, p.103) entire bacterial cultures were screened for lactose mutants. However, lactose mutants could not be detected. The fact that Sh. sonnei did not produce acid in the same medium containing no lactose established that acid production was due to utilization of lactose. In the hope of explaining the apparent production of acid by normal Sh. sonnei, the effect of various cultural conditions on both the production of lactose mutants and the apparent production of acid by normal organisms was examined. Increased concentrations of lactose, meat extract or peptone in the medium; neutralized filtrates of acid cultures of lactose cells; and aging of ctutures in nutrient broth to which lactose was added did not increase the rate of lactose mutant production. Aeration of normal organisms in lactose broth favored the production of lactose mutants. Nearly all cultures became acidified within five days and then became alkaline again. Lactose mutants could be isolated for several days afterward. Eventually, the lactose mutants disappeared in the aerated cultures and only normal cells could be isolated. Lactose mutants could not be isolated from cultures of normal organisms when aerated in lactose-free nutrient broth. The stimulatory effect of aeration on the production of lactose mutants is apparent only when lactose is present. When lactose broth cultures of Sh. sonnei were grown under anaerobic conditions, all cultures became acid on the sixth day of incubation, but lactose mutants were not detected. The possibility that this phenomenon might be due to reduction of the indicator or to accumulation of carbon dioxide was examined and ruled out experimentally. Acid did not occur in cultures grown anaerobically in nutrient broth without lactose indicating that the presence of lactose was required for the production of acid in these cultures. When lactose cells were inoculated into lactose: broth and grown anaerobically, acid appeared within forty-eight hours. Under these conditions the lactose mutants produced considerably more acid than did normal organisms. Furthermore, lactose mutants could be isolated from these cultures. It is apparent that anaerobic conditions inhibit the production of lactose mutants. Furthermore, it seems probable that the production of acid by normal Sh. sonnei growth under anaerobic conditions occurs by some unknown mechanism different from that used by lactose-fermenting mutants. Relative growth rates of lactose mutants and normal organisms were studied. Lactose mutants grew at a better rate in lactose synthetic broth than normal organisms when subcultured from cultures grown in nutrient broth. However, the lactose mutants did not grow as well in lactose as in glucose synthetic broth, a lag occurring in the rate of growth. Lactose mutants subcultured from lactose broth medium into fresh lactose synthetic broth did not grow significantly better than lactose organisms which were subcultured from nutrient broth. Therefore, it appeared that enzymatic adaptation did not occur in this mutant. It seems that this lactose mutant may differ from mutants described by other investigators. Ultraviolet irradiation of normal Sh. sonnei neither increased nor decreased the rate of production of lactose mutants. In addition, chemical mutagenic agents (manganous chloride, formaldehyde, phenol and sodium desoxycholate) were used in an effort to induce an increase in the rate of production of lactose mutants. Under these experimental conditions, the physical and chemical agents used had a lethal but not a mutagenic action on Sh. sonnei. Preliminary work suggested that aeration of lactose mutants in nutrient broth induced the rise of lactose mutants. This was confirmed in detailed studies which also indicated that these lactose mutants are favored by aeration in the absence of lactose. The results seem to point to the selective action of lactose in the rise of lactose mutants. However, the possibility of a stimulatory effect of lactose cannot be eliminated