STAINING METHODS iV46-15 



According to Kinyoun, smears are 6xed with heat, allowed to cool and stained 2-7 

 minutes. 



In the source of the original formula above cited, alisolute alcohol is specified; Kin- 

 youn calls for 9.5% alcohol. On theoretical grounds, indeed, absolute alcohol is not 

 indicated and the 95% strength may well be substituted even in the original formifla. 

 Although the Committee has had no personal experience with either formula, informa- 

 tion is at hand indicating the superiority of the Kinyoun modification. 



Flagella Staining — Recommended Procedures 



Flagella staining is a difRcult technic and there have been numerous 

 methods proposed for the purpose. It has k)ng be^n reahzed that 

 flagella are actually below the visual limit in size; but of recent 

 years the electron microscope has given a definite idea how small 

 they really are — around 0.02 to 0.03 /x in diameter. Electron 

 micrographs, in fact, indicate that with many kinds of bacteria even 

 the best stained preparations give a very inadequate picture of the 

 actual number or length of the flagella attached to a cell. Were 

 the electron microscope more simple to use, it is possible that it might 

 supplant the light microscope entirely in the demonstration of flagella. 

 Since that is far from the case at present, one must do the best he can 

 with staining methods intended to make the flagella visible. This is 

 usually done by a preliminary mordanting which causes precipitation 

 on the flagella and increases their apparent size — a principle intro- 

 duced by Loeffler (1890). 



A second difficulty in staining flagella is the ease with which bac- 

 teria shed these delicate appendages unless the cultures are properly 

 handled. To prevent this one ordinarily employs specially cleaned 

 slides and specially prepared smears on the slides. 



Methods for 'preparing slides. Ordinary cleaning of glassware is 

 not sufficient for the purpose. Various methods have been proposed, 

 but the following directions seem to give as good results as any: 



Use new slides if possible preferably of "Pyrex" glass or similar 

 heat resistant properties. (This is because under the drastic method 

 of cleaning to remove grease, old slides have a greater tendency to 

 break.) Clean first in a dichromate cleaning fluid, wash in water 

 and rinse in 95 per cent alcohol ; then wipe with a clean piece of cheese 

 cloth. (Wiping is not always necessary but is advisable unless fresh 

 alcohol is used after every few slides.) Pass each slide back and 

 forth through a flame for some time, ordinarily until the appearance 

 of an orange color in the flame; some experience is necessary before 

 the proper amount of heating can be accurately judged. 



Unless heat-resistant slides are used, cool slides gradually in order 

 to minimize breakage. An ordinarily satisfactory method of doing 

 this is to place the flamed slides on a metal plate (flamed side up) 

 standing on a vessel of boiling water; and then to remove the flame 

 under the water so as to allow gradual cooling. (Too rapid cooling 

 may result in breakage, sometimes as long as two weeks after the 

 heating.) 



Methods of handling cultures. Of various methods proposed, it 

 is not possible to recommend any one as unifc^rml}' the best. As any 

 laboratory worker becomes familiar with one particular method, 

 he soon finds he can get better results with that than with any other. 



