APPENDIX 463 



(l>) Treat with alcohol for two minutes, and then with chloroform for two minutes ; 

 wash in water. 



(c) Treat with chromic acid, 5 per cent, aqueous solution, for from one to two 

 minutes ; wash and dry. 



(d) Pour on freshly filtered carbol-fuchsin and warm gently till it steams ; allow 

 it to act for ten minutes and wash off with water. 



(e) Decolorise with sulphuric acid (5 per cent. ) and water alternately, to remove 

 the carbol-fuchsin from the bacilli but not the spores. 



(/) Dry and counter-stain with Loffler's blue until the film is of a faint bluish 

 tint. Wash off stain, dry and examine. The spores will be stained red and the 

 bacilli blue. 



(2) Ziehl-Neelsen Method. 



(a) Stain the film as for tubercle bacilli. 



(/>) Decolorise with 1 per cent, aqueous solution of sulphuric acid, or alcohol 2 

 parts, acetic acid 1 per cent., 1 part. 



(c) Counter-stain with Loffler's blue. 



(d) Wash, dry, and examine. 



(3) Abbott's Method. 



Prepare films in usual way, and stain with Loffler's alkaline methylene-blue, 

 heating gently till steam rises (5 minutes). Then wash in water and decolorise with 

 nitric acid, 2 per cent, alcoholic (80 per cent.) solution, washing again in water. 

 Counter-stain with eosin, 1 per cent, aqueous solution. Wash, dry, and mount. 

 The spores are blue and the bacilli red. 



Bacteriological Diagnosis. The following points must be ascer- 

 tained in order to identify any particular micro-organism : 



(1) Its morphology : shape, size, etc. (bacillus, coccus, spirillum, etc.) ; 

 the presence or absence of involution forms ; motility, by the unstained 

 cover-glass preparation ("hanging drop"); note presence of flagella ; 

 presence of spores, their appearance and position. Staining reaction ; 

 whether or not the organism stains by Gram's method. 



(2) Cultural Characters. The character of the growth upon various 

 media (gelatine, agar, milk, potato, blood serum, broth, and special 

 media) ; the presence or absence of liquefaction in the gelatine culture ; 

 its power of producing pigment, acid, gas, indol, ferments, phenol, etc. 



(3) Biology : whether it is aerobic or anaerobic ; its powers of resist- 

 ance to external agencies ; agglutination reaction, etc. ; pathogenesis, its 

 effect upon animal tissues and the course of the disease produced ; its 

 toxins, etc. 



BACTERIOLOGICAL EXAMINATION OF WATER 



Collection of Samples, Water from streams or wells should be 

 collected in glass bottles or flasks closed with glass stoppers previously 

 sterilised (at 150 C. for three hours), or washed out with pure sulphuric 

 acid. When the latter method is adopted, the bottle should be well rinsed 

 with the water which is to be examined before the sample is taken. In 

 taking the sample, the bottle should be held below the surface before the 

 stopper is removed, in order to obtain a sample of the main body of water 

 and not the surface water only. If it is an ordinary water supply 

 through pipes or from a cistern, the tap should be turned on and the 

 water allowed to run for a few minutes before taking the sample : and 



