STAINING METHODS U 



the basic, dyes. Hence, the stains in common use by the bacteriologists 

 are rarely acid dyes. 



Preparation of Smears 



Pure cultures of bacteria can ordinarily be prepared for staining by the 

 simple process of making an aqueous suspension and drying a drop of it on 

 a slide or cover glass, without any fixation other than gentle heat. The 

 use of this simple procedure depends upon the fact that most bacteria, 

 because of their small size or their stiff walls, can be dried without great 

 distortion. For this reason it is not always necessary, as with higher 

 organisms, to coagulate the tissues before microscopic preparations can 

 be made, although for cytological studies and for accurate determinations 

 of size and shape of the cells, some fixation other than heat is needed. 



The best bacterial smears are usually made by removing a small amount 

 of surface growth from some solid medium and mixing it with distilled 

 water. It is often possible to use a drop of a culture growing in a liquid 

 medium, but such a smear is not always so satisfactory, since certain 

 constituents of the medium may prevent the bacteria from adhering to 

 the slide or may interfere with the staining. 



The suspension used should always be sufficiently dilute. Ordinarily, 

 only a faint turbidity should be visible to the naked eye, for it is always 

 best to avoid the occurrence on the slides of solid masses of bacteria, piled 

 one on top of the other. If a smear after staining does not show any por- 

 tions where the bacteria are well separated one from another, a new, more 

 dilute smear should be made. This is particularly important in the case 

 of the gram stain or flagella staining. 



The usual method of fixing the suspension to the slide or cover glass is 

 to pass it rapidly after drying through a bunsen flame two or three times. 

 Another very satisfactory method is to allow the drop of material to dry 

 on a slide lying on a flat, moderately hot surface, such as a plate of some 

 nonrusting metal resting on a boiling-water bath. With many bacteria 

 an aqueous suspension of the surface grow^th from agar can be dried in the 

 air at room temperature and stained without any fixing; this method is 

 not universally successful, however. 



For some cytological procedures special methods of making bacterial 

 preparations are necessary, sometimes calling for fixing solutions rather 

 than heat. It must be seen that the technic described for staining dried 

 smears is too crude for accurate measurements of cells or for studying 

 cytological details. One cytological method is given on page 30. 



It is also beyond the scope of this pubHcation to give staining methods 

 for other than pure culture work. 



In using any of the methods it must be remembered that blind adher- 

 ence to a staining technic is no guarantee that the result will be satis- 



