50 THE PERMANENT FORMS OR SPORES 



be fatal to the vegetative forms. As in the following sections especially that 

 dealing with sterilisation occasion will often arise for a closer investigation of 

 this faculty, so important for the maintenance of the individual species, an 

 exhaustive and tedious list of individual cases need not be given here ; it will 

 therefore suffice if we cite one example, viz., Bacillus subtilis. According to the 

 researches of BREFELD (I.), which were confirmed by M. GRUBRR (I.), a con- 

 tinuous exposure of twenty minutes to the action of boiling water suffices to 

 destroy the sporeless rods of this microbe ; whereas to kill the spores requires 

 three hours' boiling at 100 C., or a quarter of an hour's exposure at 105 C., or, 

 finally, the action of a temperature of 110 C. during five minutes. The 

 assertion made by Koch, that the continuous action of steam at 100 C. for 

 fifteen minutes will destroy the spores of any of the bacteria, was subsequently 

 negatived by his pupil E. VON ESMARCH (II.). According to SWAN ([.), spores 

 of Bacillus megatherium, dried on a cover-glass, retained their vitality and 

 germinating power for more than three years. 



Use may be made of these powers of resistance for separating the sporiferous 

 from associated non-sporiferous bacteria. By skilful handling, e.g. by the aid of 

 sufficiently high temperatures, the weaker species in a mixture of bacteria can 

 be killed off, leaving only the spore-producing individuals. A process based on 

 this mode of procedure, and known as the boiling method, was introduced into 

 bacteriology by ROBERTS (I.) for obtaining pure cultures of the hay bacillus, 

 and the same method was employed by Prazmowski for preparing cultures of 

 Clostridium butyricum. 



The seat of this high power of resistance has already formed the object of 

 numerous researches. One school looks for it in a peculiar modification of the 

 spore plasma for instance, in the presumably low water-content thereof, as 

 suggested by LEWITH (I.). Others, again, attribute to the spore membrane an 

 exceptionally low heat-conducting power, and a very slight degree of permeability 

 by noxious substances. This latter opinion seems the more probable one, 

 considered from a physical point of view, and is further supported by 



54. The Behaviour of the Endospores towards Dyes. 



As already observed in a previous chapter, the dead plasma of the bacterial 

 cell absorbs colouring matters greedily and copiously. The staining of the 

 endospores is, however, more difficult, and consequently they have to be exposed 

 to the dye a much longer time before they will absorb any of it. However, the 

 colour thus taken up is retained by them more firmly than by the vegetative 

 forms. 



This property has been utilised in microscopy to obtain a differential staining 

 of the spore-bearing bacterial cells, for which purpose the latter are treated 

 with a suitable (e.g. red) colour solution until the spores are thoroughly im- 

 pregnated therewith, the preparation being then steeped in a decolorising liquid 

 (generally slightly acidified alcohol), wherein it is left until the vegetative cells 

 are deprived of the colour. These latter are thereafter stained anew by a short 

 immersion in a second colour (e.g blue) solution, a two-colour preparation 

 (double-staining) being thus obtained, the spores in this case being red and the 

 rods blue. More detailed directions for double-staining will be found in Hueppe's 

 handbook, Eisenberg's treatise, and in Bernheim's " Taschenbuch " (Pocket-book). 

 Fig. 20 gives a black and white reproduction of a cover-glass preparation of 

 spore -bearing Bacillus subtilis stained only once, so that the spores are unchanged, 

 and appear colourless (white). 



The aforesaid behaviour of bacterial endospores towards colouring matters is 

 characteristic of all. From this fact it is not infrequently, though erroneously, 

 assumed by medical bacteriologists that any formation, in the interior of the 



