INTERMITTENT STERILISATION 83 



leaving the samples to stand at room temperature. At the end of twenty-four 

 hours the first treatment in the steamer is repeated, whereby the vegetative 

 forms that have in the meantime developed from the spores are killed. It 

 being, however, possible that, owing to the known irregularity of germination, 

 some of the spores have not developed, the samples are again left at rest for a 

 day, and thereafter steamed a third time to kill the residual cells proceeding 

 from these tardy spores. The medium, liquid, &c., will in this manner be 

 entirely freed from living germs without having been subjected to injury from 

 over-prolonged or excessive heating. This method is known as the intermittent 

 process of sterilisation, and is the only one in use for the preparation of nutrient 

 meat -juice gelatin. The success of this method of killing germs depends on the 

 whole of the spores being caused to germinate. Now we know, from statements 

 already made, that there are certain species of bacteria which will only develop 

 under high temperatures, and for whose germination the temperature of the air 

 of a laboratory is therefore insufficiently high. On this account it will be 

 evident that, under certain circumstances, the samples will have to be left to 

 stand at high temperatures. Since, however, these temperatures will, on the 

 other hand, retard the development of the spores of such species as thrive only 

 at low temperatures, it is therefore impossible to neglect either consideration. 

 The samples must, consequently, be kept for a certain time at room temperature, 

 and for another interval at higher temperatures. In no case, however, must 

 this be relied on without further examination, but it must be laid down as a 

 fundamental rule of conduct, that any nutrient medium apparently rendered 

 sterile by fractional sterilisation, may only be considered as actually sterile, and 

 used as such, when it is found that after a short storage, following the above 

 treatment, no spontaneous development has taken place. This regulation, 

 urgently necessitated by reason of the insecurity of the sterilising process in 

 question, must not be neglected. Nevertheless, if the work is cleanly done, it 

 will seldom be found necessary to reject samples on account of insufficient 

 sterilisation, since the highly resistant spores, now in question, are generally 

 absent in the majority of the substances employed in the preparation of artificial 

 nutrient solutions, and only creep in when the manipulations are performed 

 without due care. Cultivated soil is rich in such organisms, so that if such soil 

 is, by any means, introduced into these media, an unsuccessful result may readily 

 ensue, as was, for instance, observed by L. HEIM (II.). The occurrence of such 

 spores in meat-extract is no rarity, and the remarks just made should therefore 

 be recalled when such material is employed. 



It may happen that a nutrient medium, which cannot be exposed to a 

 temperature of 100 C. without decomposing, will have to be sterilised. An 

 instance of this is afforded by the solution employed in the study of uric 

 fermentation, which, in addition to the nutrient substances, contains also an 

 admixture of urea. This body, as is well known, is gradually converted at 

 1 00 0. (in aqueous solutions) into ammonium carbonate. In preparing a 

 medium containing this amide the directions of LEUBE (I.) should be fol- 

 lowed, the solution of the other nutrient substances (e.g. a bouillon) being 

 first treated by itself in the steamer, and the urea sterilised separately by 

 heating it in the dry state at 106 C. for half an hour. By this treatment 

 it is maintained unaltered, and may, when cooled, be added to the cold sterile 

 bouillon. 



Under particularly favourable circumstances, exposure to a temperature much 

 below 1 00 C. can bring about either the complete mortification of the germs 

 present in a solution, or else render them so debilitated that their further 

 development is prevented, so that the liquid will remain for a long time (months 

 and years) without alteration. This result is attainable when the influence of 



