42 GENERAL PHYSIOLOGY OF BACTERIA 
development by cold in the preservation of food by refrigeration or by 
cold storage, and also for the preservation of laboratory cultures of 
many non-spore-forming bacteria by placing them in the ice-box at 
5 to 10° C. So resistant are bacteria to low temperatures that they may 
be actually frozen solid and kept in this state for days and even weeks 
without killing all the individuals of the culture. Alternate freezing 
and thawing is much more disastrous to them than simple freezing. 
Thus, typhoid bacilli may be suspended in water and exposed to a 
freezing mixture of ice and salt at —18° C. for several weeks without 
killing all the organisms, although the majority of them are killed 
within a few hours. At the end of a week fully 90 per cent are dead; 
over 95 per cent succumb by the end of four weeks' continual freezing; 
but from four to six months' continuous freezing is required to kill all 
of the typhoid bacilli. The survivors appear to be no more resistant 
to subsequent freezing than similar organisms which have not been 
frozen. It is a noteworthy fact that bacteria suspended in colloidal 
substances, as egg albumen, are much more resistant to freezing than 
similar organisms frozen in water. Alternate freezing and thawing in 
colloids is much less disastrous to bacteria, in other words, than the 
same freezing in aqueous solution. It is probable that the mechanical 
factor of crystallization which takes place when water is frozen actually 
crushes many of the bacteria, thus accounting, in part at least, for the 
greater death-rate in aqueous solutions than that observed in colloids. 
When bacteria are once frozen, further lowering of the temperature has 
surprisingly little influence upon the death-rate. Typhoid and colon 
bacilli will survive freezing, in moderate numbers at least, in liquid air 
( — 176° C.) or even liquid hydrogen ( — 252° C.) for several hours, and 
develop vigorously w^hen they are again placed in a suitable environ- 
ment at the optimum temperature. Shattuck and Dudgeon^ have 
kept certain sporeless types of bacteria in a vacuum surrounded by 
liquid air for one hundred and sixteen days without destroying the 
viability of these cultures. 
3. Heat. — Bacteria are distinctly injured by exposure to even slight 
increases of temperature above that optimum for their growth, although 
there are considerable differences met with among different kinds of 
organisms in this respect. Generally speaking, the saprophytic 
bacteria exhibit greater latitude than the pathogenic bacteria. If the 
maximum temperature of growth be exceeded by even a very few 
degrees, the death of the organisms follows rather promptly. The 
greater the degree of heat, the shorter the time required to kill them. 
Therefore, the thermal death-point of bacteria, that temperature at 
which specific organisms die, is dependent not only upon the actual 
temperature to which they are exposed, but also to the length of time 
of exposure. A standard exposure of ten minutes has been proposed, 
so that the termal death-point of the bacterium may be defined as the 
I Proc. Roy. Soc, Series B, 1912, 85, 127. 
