MICROSCOPICAL TECHNIQUE 49 



crystals, may be mistaken for spores in preparations stained 

 with simple aqueous dyes. A special stain may be employed 

 as follows : Place the dried and fixed films in chloroform for 

 two minutes, and then in a 5 per cent solution of chromic acid 

 for two minutes ; wash in water, cover with carbol-fuchsin, 

 and heat in the steam of a water bath for five minutes. Wash 

 off* the stain ; decolorize carefully in 1 per cent sulphuric acid ; 

 wash in water; counterstain with methylene blue at room 

 temperature for ten seconds; wash and examine. 



The principle of the process rests on the peculiar property 

 of the spores towards stains. They stain with difficulty, but, 

 when once stained, retain the color. Thus the first stain can 

 be removed from the cell with a weak acid, and the cell then 

 counterstained with a differential dye. The position of the 

 spores in the cells should be noted, whether medium or ter- 

 minal ; their shape, whether oval or round ; their size in rela- 

 tion to the mother cell, whether greater in diameter than the 

 mother cell, or of less diameter ; the presence of free spores, 

 i.e. outside of the mother cell. 



The only method by which the spores may be identified 

 as such in case of doubt is by observing their germination. 

 This also is often of diagnostic value, and can be observed in 

 the hanging block suggested by Hill. 



Exercise. Make spore stains from cultures furnished you. 



Examination of living bacteria. The examination of the 

 bacteria in a living condition will give much information of 

 morphological value. The method of procedure is as follows : 

 Place a drop of liquid medium broth on a cover glass or 

 slide, and inoculate it with a particle of growth from a solid 

 culture, mix carefully, and transfer a very small drop to a 

 cover glass; do not spread. Invert over the drop a hollow 

 ground slide around the cavity of which has been placed a 



