54 MORPHOLOGIC VARIATION 



ments of the Congo red preparation were practically identical with 

 those of the living cells, whereas the methylene blue preparation 

 gave dimensions about one-third less. With the Congo red method, 

 therefore, the image of the cell is larger than with other staining 

 methods, and the measurements are correspondingly more accurate. 



Other negative staining methods could be used, as India ink or 

 nigrosin; but India ink is more granular and tends to give a less 

 sharp image, and at least some samples of nigrosin tend to become pre- 

 cipitated by certain substances associated with the bacteria, and 

 similarly give a granular background with an unsharp image. The 

 negative staining method also appears to give a much sharper image 

 than is the case with slides stained in the ordinary way. 



This negative staining procedure with Congo red gives such clear 

 preparations for certain types of morphologic investigation that a 

 word or two further concerning it will not be out of place. A per- 

 fect image of the cell is only obtained when the film of stain comes 

 up to the very edge of the cell but does not extend over it. It is 

 therefore not useful with organisms which accumulate any appreciable 

 amount of slime about them, and only those parts of the film are to 

 be used where the layer of stain is not so thick as to extend over the 

 top of the cell. This latter point may be readily determined by ex- 

 amination of the preparation ; if one observes the cells in a thick por- 

 tion it will be found that they are relatively slender and not clear 

 white, but as one proceeds to the thinner parts it will be found that 

 they become larger, and that one soon arrives at a place where no 

 decrease in the intensity of the blue is accompanied by an increase 

 in the size and brilliancy of the image of the cells, indicating that 

 the film is now so thin that it has not extended over the top of the 

 cells. The Congo red is precipitated by peptone, and with other 

 liquid media artifacts are produced by the precipitation of substances 

 dissolved in the medium, so that the method is not satisfactory 

 with organisms from liquid cultures unless they have first been 

 centrifuged out and washed with water. For this reason cell 

 measurements for the broth cultures described in Chapter IV have 

 been made from preparations stained with methylene blue. 



While in young actively growing cultures none of the cells show 

 any portion stained with the Congo red, the entire cell appearing as 



