

APPENDIX 461 



Flag-ella Staining 1 



Successful staining of flagella is a matter of practice, and of careful 

 and exact technique. Whatever the method of staining adopted, the 

 preparation of the film is the same, and too much care cannot be 

 exercised at this stage. The slides should in no case have been 

 previously used, and they should be most carefully cleaned in the manner 

 described on p. 487. When taken out of the alcohol, the slide should be 

 carefully dried and wiped with a clean piece of old cambric, without 

 handling with the bare fingers. It should then be passed several times 

 through the flame, and set aside to cool. 



Preparation of the Film. 1. The cultures for examination should be 

 upon agar, and should not be less than six, or more than twelve hours 

 old, if incubation has taken place at 37 C. If incubated at 20, slightly 

 older cultures may be employed (twelve to twenty hours). 



2. Transfer from the young culture a small loopful by means of the 

 platinum needle, to a test-tube containing from 30 to 40 c.c. of sterile 

 water at room temperature. Or the emulsion may be made in a capsule 

 with a few c.c. of distilled water. Hold the loop in the water for a 

 few moments without shaking, until the water shows a slight turbidity. 

 Do not shake or handle the tube roughly. Incubate the emulsion for 

 five hours at 37 C. or for twelve to twenty-four hours at 20 C. 



3. With a finely looped pipette, take up a small quantity of the 

 surface water from the inoculated tube, and distribute it in small droplets, 

 upon the slide. 



4. Place aside to dry, carefully covered from chance of dust. When 

 dry, the staining can be proceeded with, according to the method 

 adopted. Do not fix the films in the flame ; the flagella are apt to be 

 injured thereby, and it will be found that the subsequent manipulations 

 will cause the organisms to adhere sufficiently to the slide. 



Staining the Film 



The three ordinary methods practised in this country are : 



(1) PitfieWs Method (Muir's modification). 

 The following solutions are required : 



A. The Mordant. 



Tannic acid, 10 per cent, aqueous solution . . 10 c.c. 



Corrosive sublimate, saturated aqueous solution . . 5 c.c. 



Alum, saturated aqueous solution . . . . 5 c.c. 



Carbol-fuchsin (Ziehl) . . . . 5 c.c. 



The above must be thoroughly mixed and the precipitate which forms must be 

 allowed to deposit. The clear supernatant fluid is then drawn off with a pipette and 

 placed in a clean dropping bottle. The mordant will remain good for one or two 

 weeks, but not longer. It should be centrifugalised before use. 



B. The Stain. 



Alum, saturated aqueous solution . . . 25 c.c. 



Gentian- violet, saturated alcoholic solution . . 5 c.c. 



Filter twice. The stain must be freshly prepared. 



The film is prepared as described above. The mordant is then dropped on to 



