IV46-4 MANUAL OF METHODS FOR PURE CULTURE STUDY 



usually necessary, as with higher organisms, to coagulate the tissues 

 before microscopic preparations can be made; although it has been 

 well demonstrated that for accurate determinations of size and shape 

 of the cells, some form of 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 satis- 

 factory, 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. Ordi- 

 narily, 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 portions 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 non-rusting metal resting on a boiling water 

 bath. With many bacteria an aqueous suspension of the surface 

 growth from agar can be dried in the air at room temperature and 

 stained without any fixing; this method is not universally successful, 

 however. 



For special staining procedures special methods of making bac- 

 terial preparations are necessary, sometimes calling for fixing solu- 

 tions rather than heat. It is beyond the scope of this leaflet, however, 

 to discuss them here, but it must be recognized that the technic 

 described above for staining dried smears is too crude for accurate 

 measurements of cells or for studying their cytological details. 



It is also beyond the scope of this publication to give staining 

 methods for other than pure culture work, although a few (e.g., 

 blood stains) have been given in previous editions.* 



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

 adherence to a staining technic is no guarantee that the result will 

 be satisfactory. Even experienced workers sometimes discover to 

 their dismay that they took too much for granted as to the purity of 

 their reagents, cleanliness of slides and covers, or proper compound- 

 ing of the staining solutions. A technic should, therefore, be checked 

 upon known organisms as controls. It is, furthermore, important to 

 know that the solutions and water used for dilution are reasonably 

 free from bacteria and their spores. 



*Those interested in other stains for microorganisms and for blood are referred to 

 the following leaflets of Staining Procedures (Conn and Darrow, 19-13-5): 



I D. Miscellaneous methods (blood, bone, marrow, fat). 



Ill A. Stains for microorganisms in smears. 



Ill B. Stains for microorganisms in sections. 



These leaflets can be purchased separately and are punched so as to 6t the cover to 

 this Manual. 



