1. Disinfection curves obtained from data on the action of phenol onBact. coliat 35° C. under conditions such that unfavourable circumstances, other than the presence of the germicide, were as far as possible eliminated, have been used for the calculation of the concentration exponent for phenol, i.e.nin the formulaCnxt=K.The death-rate was not constant throughout the germicidal process but showed initially a phase of slow but increasing death-rate which merged gradually into a phase which was treated (for reasons given) as one of constant rate. This was also the maximum rate for any given phenol concentration.2. The virtual sterilization times (v.s.t.'s), i.e. the times in min. required for the mortality to reach 99·999999 % as determined by slight extrapolation of the log survivors-time curves, the 99·9 % mortality times and the 99 % mortality times could all be used for the calculation of values ofnfor phenol as they all gave satisfactory linear relations between log concentration and log time.3. The 50 % mortality times did not show a satisfactory linear relation between log phenol concentration and log time over the full concentration range, and at this mortality level the concentration exponent appeared to increase for concentrations above 4·62 g. phenol per 1.4. The value ofnvaried according to the mortality level chosen. It was 5·8421 ± 0·1876, 6·6062 ± 0·2034 and 6·9638 ± 0·2164 when thev.s.t.'s,99·9 % mortality times or 99 % mortality times were used. The differences between the first and second and first and third values are significant, but that between the second and third values is not. The value calculated from thev.s.t.'sis regarded as being the most important.5. Evidence was obtained that, as expected on theoretical grounds,nincreases for very low concentrations of phenol. If the aberrant value obtained at the lowest phenol concentration be omitted from the calculations, the value ofncalculated from thev.s.t.'s becomes 5·6588 ± 0·1422, but the decrease is not significant.6. The maximum death-rate was related to the phenol concentration according to the expression km= 9·1743 × 10−6C5·0752, wherekmis the maximum (logarithmic) death-rate per min. andCthe concentration of phenol in g. per 1.

the results of experiments on the death of cultures of Bact. coli in the presence of phenol, using a special apparatus and standardized technique, were presented, and the reasons for adopting the particular methods were given. Those experiments, at various phenol concentrations, were all conducted at the same temperature, 350C., but a complete analysis ofthe reaction between phenol and Bact. coli requires that the temperature should also be varied. Such experiments have now been carried -out and the results repoiCted below have proved extremely fruitful. They throw considerable light on a number of aspects of the disinfection process, namely, the true shape of the logarithm of survivors-time curve, the distribution of resistance amongst the cells of the baQterial population, and the effect of both temperature and phenol concentration on the time taken to produce various degrees of mortality. It is impossible to deal adequately with the data from all these points of view in a single 'paper, and it is proposed, therefore, to discuss the various aspects in separate papers. Obviously, the true shape of the logarithm of survivors-time curve is of prime importance, since from such curves the times reqiiired to reach different degrees of mortality are obtained. Accordingly, the present paper is devoted to that aspect of the problem. METHODS AND RESULTSThe apparatus and methods used were the same as those previously described (Jordan & Jacobs, 1944a) except for a slight modification in technique necessitated by the employment of temperatures other than 350C. As before, the cultures were grown at that temperature to obtain the standard bacterial population, but immediately before the addition of the phenol solution the temperature of the thermostatically controlled water-bath was rapidly adjusted to that at which the experiment was to be carried out. Water was siphoned from the bath, hot or cold water added as required and the thermostat readjusted. The temperature of the culture within the flask rapidly attained the desired value, but in order to ensure complete equilibrium at the new temperature a period of 1 hr. was allowed before further operations were carried out. The required amount of phenol solution was then added and samples removed at intervals and plated out as previously described.Experiments were performed at several temperatures at each of five phenol concentrations which were chosen so that the data previously obtained at 350C. could be included in a study of the effect of varying temperature with phenol concentration constant. The results are given in Table 1 and the graphs of logarithms of survivors against time appear in Figs. 1-5. It is evident that the death-rate varied widely during the course of each experiment. At first the rate was low but increasing, and after rising sharply to a peak value it usually appeared finally to decline. Further, in many cases there were signs of an initial rush in the disinfection process, a feature which is apparent in Table 1 but not in the graphs. A table of the di...

1. Standard Bact. coli cultures have been heated at 51° C. in phosphate buffered media having pH values ranging from 2·8 to 8·8, and survivor curves obtained.2. Except at pH 3·9, the first continuous decline in viable numbers was followed by the establishment of a small fluctuating residual population which was apparently permanent.3. The colony counts showed a moderate excess of unduly high values of x2, the bulk of which were encountered when the mortality was between 95 and 99·999%.4. Prolonged incubation of the plates led to increased colony counts, but the rise was confined to the active disinfection phase. It occurred chiefly in the 95–99·999% mortality range and was moderate in degree. The x2 values were little affected.5. The log survivors-time graphs of the disinfection phase were convex in type but approached linearity in the faster reactions. The logarithmic death-rate became maximal at about 90 % mortality and thereafter remained constant, though under the more acid conditions a somewhat lower rate was maintained. At pH 7·0 and 6·4 the curves were exceptionally straight.6. Standardization of all the curves revealed a continuous change in shape with pH but, nevertheless, they could be placed in three closely knit overlapping groups which were best revealed by the variation in maximum death-rate.7. The 50–99·99% mortality times were obtained either from the calculated regressions of log survivors on time over the linear portions of the curves or, outside that range, from the freehand curves. Maximum survival at all mortality levels occurred at about pH 6·5–6·6, i.e. close to the neutral point of pure water at 51° C. On both sides of this point sensitivity increased regularly but was abnormally high at pH 7·0 and 6·4.8. Within these zones of abnormally high sensitivity the assessment of the effect of a given change in pH varied in amount and direction according to the mortality level chosen.9. The enhanced resistance of a culture regenerated from the survivors of a disinfection at 51° C. and pH 7.0, as previously reported, could possibly have been largely due to a small increase in acidity but not to a slight shift towards alkalinity.

SUMMARY : The effect of pH over the range 5-9 on the growth of Bacterium coli with a constant food supply was studied a t 20 and 30". Total and viable counts were made and growth curves constructed. To discover the effects of starvation, sampling was continued after stopping the food supply.The total count always substantially exceeded the viable. Each growth curve showed an ' initial phase ' of varying daily increment in cell numbers merging into a ' steady phase' of roughly constant increment, which continued as long as food was supplied. Low pH slightly shortened the initial phase, low temperature greatly prolonged it.In the early initial phase development was slow a t pH 5, but later became exceedingly rapid. Altogether, the conversion of the food supplied into (total) bacterial cells was best effected in conditions of low temperature and low pH, low temperature being the more important. These conditions also favoured high viable counts, and consequently smaller non-viability indices. During starvation the apparent total counts declined, except a t 30" and pH 5, when a steady increase occurred. Higher pH and lower temperature led to faster rates of decline. Viable counts remained approximately constant a t pH 5, but otherwise the numbers declined.Jordan & Jacobs (1944) showed that Bacterium coEi could be cultivated successfully in an apparatus which permitted accurate control of temperature, p H and aeration, and enabled food to be added a t a constant rate, so that the growth was controlled by the food supply. The apparatus was fully described and the results of preliminary experiments reported. After an initial phase, growth was shown to continue at an approximately steady rate so long as food was added. Subsequently, the apparatus was used to investigate the influence of temperature on the growth curves (Jordan 2% Jacobs, 1947), when it was shown that at pH 7 low temperatures were the most favourable to growth, in that they conduced to the formation of the largest amounts of bacterial cell substance from a given amount of food. Temperature also had a profound influence on the formation of viable cells. The proportion of viable cells formed was greatest at the lowest temperature used (15'), but a t 35", which is close to the temperature ordinarily regarded as the optimum for this organism, roughly half the cells formed were not viable, so that the number of viable cells remained virtually constant while the total cells steadily increased. At lower temperatures the viable count also increased. I n view of these interesting findings it was decided to extend the experiments to determine how the cultures would behave under other conditions of pH. The results of this work are reported below.

1. The action of phenol onBact. colihas been studied quantitatively in detail under such conditions that unfavourable circumstances other than the presence of the germicide were as far as possible eliminated. Briefly, this involved the addition of phenol solution directly to a large volume of culture developed under rigidly standardized conditions.2. When the mortality exceeded 95% the variation between the numbers of colonies on replicate plates, when determining the numbers of survivors, was often excessive.3. Under the conditions used the death-rate was not constant. First, there was a phase (1) of slow but increasing death-rate which merged gradually into a phase (2) of approximately constant rate which was also the maximum for a given phenol concentration. There are indications that the death-rate may decline towards the extreme end of the disinfection process.4. The virtual extinction time (the time taken for the survivors to fall to 1 per ml. as determined by extrapolation of the log survivors-time curve) increased as the phenol concentration decreased, as also did the duration of phase 1. The latter occupied about 60% of the virtual extinction time when the concentration was 3·48 g. phenol per 1., but with rising concentration its duration decreased at a greater rate than the virtual extinction time so that at about 7 g. per 1. phase 1 was very short though still detectable. The standard errors of the virtual extinction times varied between ± 1·5% and ± 5·43%.5. Using the probit method it has been shown that at low concentrations of phenol the distribution of resistance ofBact. coliis approximately normal when the resistance is measured in terms of the survival times.6. The distribution of resistance was found to be normal at all concentrations of phenol used, when that resistance was measured in units of the logarithms of the survival times, with the exception that at the lower concentrations there was a sudden change in the slope of the probit-log survival-time line at about 20% mortality. This change became less marked with rising concentration and was probably absent at 8·00 g. phenol per 1.7. After conversion into the dosage-mortality form, the data reveal that about 70% of the cells show an approximately normal distribution of resistance when the latter is expressed in terms of the dosage survived for a fixed time, provided that the exposure time is less than 50 min.8. The distribution tends to be normal for longer exposure times when the resistance is measured in terms of the logarithms of the dosages survived. On this basis a change of slope of the probit-log dosage line is observed as in the case when resistance is measured in terms of the logarithms of survival times.