770 



SCIENCE 



[N. S. Vol. XLII. No. 1091 



site sexes. Since one of the sexes may pre- 

 dominate in sporangial growth, the writer has 

 found it a surer method to pick out with fine 

 needles yotmg zygospores free from sporangial 

 spores and to plant them in Petri dishes on 

 nutrient agar. One or both suspensors are 

 likely to grow into mycelia which can be tested 

 out as suggested above. 



Inoculation of many sporangial spores 

 causes a dense growth of small sporangia and 

 a reduction of the mycelial growth at the point 

 of inoculation. It is therefore advisable to 

 inoculate only a small number of spores when 

 desiring zygospore production or better yet, to 

 make transfers of the mycelia from fresh tubes 

 of the fungus before they have produced 

 sporangia. In either way the opposite sexes 

 may be sown together or slightly separated so 

 as to cause a somewhat indefinite mass of 

 zygospores where the opposing growths meet. 

 If the nutrient requirements are satisfied and 

 the atmosphere is kept saturated, zygospores 

 may be thus obtained in abimdance and nearly 

 free from sporangia. 



To teachers on my regular exchange list I 

 am planning to send out dried male and fe- 

 male spore material of Bhizopus for use with 

 their classes, together with reprints of the 

 present article. I should also be glad to supply 

 .any other teachers with this material who may 

 request it. Cultures should be started from 

 this dried material within a month's time. 

 The male and female cultures may be kept 

 running by transfers to fresh nutrient about 

 every three or four months. 



A. F. Blakeslee 



Oaenegie Station fob 

 Experimental Evolution, 



Cold Spring Harbor, L. I., N. Y. 



NOTES ON THE FACTORS INVOLVED IN THE GER- 

 MICIDAL EFFECT OF FREEZING AND LOW 

 TEMPERATURES 



Many interpretations and conclusions on 

 the germicidal activity of low temperatures 

 and freezing have been given by earlier in- 

 vestigators. Cold was formerly considered a 

 powerful disinfecting agent, but now there is 

 a tendency to emphasize other factors than 



cold itself as potent. In fact we know that 

 cold may act as a preserver of germ life, as 

 the high bacterial content of frozen food stuffs 

 after weeks and months of refrigeration indi- 

 cates. Ice, on the other hand, tends to purify 

 itself upon storage. 



There are numerous variables which may 

 have an important bearing upon the experi- 

 ments. A partial list of these includes: (1) 

 The species or strain of bacteria used, (2) the 

 history and cultural manipulation of the or- 

 ganism prior to freezing, (3) the physical and 

 chemical composition of the medium in which 

 the organism is frozen, (4) the temperature of 

 the frozen mixture, (5) the duration of the 

 freezing, (6) the abruptness of temperature 

 changes, (7) the cultivation of the organism 

 subsequent to freezing. This list includes 

 those factors which we took special pains to 

 control. 



The bacteria may be killed by the mere fact 

 of low temperature interfering with metabol- 

 ism; by freezing of the cell contents and rup- 

 ture of the membrane by internal pressure ; by 

 external pressure or grinding developed dur- 

 ing crystallization, or by expansion of the 

 frozen medium within the receptacle; or by 

 more or less prolonged suspension of metabolic 

 activities, leading to slow death from old age 

 or starvation. 



We shall not take the space to give more 

 than a summary of our preliminary results. 

 I. The comparative germicidal potency of 



freezing on different species and strains of 



bacteria. 



B. coli and B. subtilis (twenty-four hour old 

 cultures, the latter presumably practically 

 spore-free), showed the former species to be 

 much more susceptible to freezing. Ninety- 

 nine per cent, and over of the B. coli suc- 

 cumbed to freezing in tap water in three 

 hours, while with B. subtilis the reduction was 

 not at all uniform, but seldom exceeded eighty 

 per cent. Three strains of B. coli tested 

 showed no appreciable variability in relation 

 to the disinfecting influence of cold and freez- 

 ing.^ 



1 The remainder of onr experiments were per- 

 formed with S. coli. 



