Such solutions were never kept for more than one week.

lmmediately before the addition of

bacteria, the pH was brought to 7.1 by means of concentrated NaOH.

Inactivation curves of E. coli grown in complete broth when exposed to 1.M Hydroxylamine and 0.1
M Hydroxylamine. Salt concentration in both cases 1.35 M NaCL.

NaCI was added to the desired concentration and pH again adjusted to 7.5. At zero time the bacterial
suspension was added and incubation started in a 37 ° C waterbath.

Genetic methods: The inactivating effect of HA was analysed by determining the decrease in viable
cell count employing a plate-dilution method. In each determination the titre obtained represented
the mean of the number of bacteria on at least three plates. Mutagenic effects were determined by
an estimation of mutations from streptomycin sensitivity (str-s) to streptomycin resistance (str-r)
and from dependence to non-dependence of a particular growth-factor. Details concerning these
techniques have been described in the result section.

RESULTS (The inactivating affect of Hydroxylamine)

In phage the inactivating effect of HA is greatest at low concentrations of the compound. From the
experiment presented in Fig. 1 it seen that the same is also true for cultures of E. coli in the
stationary growth pase. When the cells are exposed to 0.1 M HA the inactivating effect is
significantly greater tan when exposed to 1 M of the compound.

In the two curves presented the salt concentration is the same (1.35 M NaCl). This is taken to
indicate that the concentration of HA is the main factor determining the rate of killing. When the
salt concentration was decreased to 0.2 M NaCl, inactivation curves were obtained which were
identical to those presented in Fig. 1. Thus, in contrast to the situation in phage the salt
concentration seems to be of little or no importance in determining the rate of killing in E. coli.

Successive series of experiments showed that concentrations of HA below 0.03 M were without
significant inactivating effect in the technique used. On the other hand, concentrations above 3 M
gave non specific killing, presumably due to the high osmotic pressure of the reaction mixture.

In the experiments reported in Fig. 1, the cells were grown in complete media (HIB). Growth in
minimal media regularly resulted in cells with greater resi tance towards HA. With such cells the
inactivation curves from separate experiments did not correspond so well. The highest
concentration of HA, however, always gave the slowest inactivation rate.

THE MUTAGENIC EFFECT OF HYDROXYLAMINE

The mutagenic effect of HA on cells of E. coli was first studied in a system which employed the
mutation from streptomycin sensitivity to streptomycin resistance. ln the experiments presented in
Table 1, cells were incubated in a reaction mixture which contained 1.2 M HA and 0.8 M NaCI. The
zero time control consisted of cells and salt solution (1.5 M NaCI) only. After an exposure time such
as indicated the cells were diluted 1:10 in slopping mixture, centrifuged and resuspended in saline
to the original volume before plating on complete agar plates.
After various hours of incubation, the agar was transferred to another agar plate of the same
composition and volume which in addition contained 200 µg streptomycin per mL. After an other
48 hours of incubation resistant colonies were counted.