16 BACTERIOLOGY OF THE EYE 



especially in many members of the Coli group. I had a strain from 

 a blennorrhoea neonatorum, in which Bietti was only able to show move- 

 ment in agar or bouillon cultures less than ten to twelve hours old. 

 In twenty-four-hour-old cultures only cast-off cilia could be found. 

 Another strain of Coli from a case of keratomalacia, which had been 

 sent to me for control as non-motile, showed typical cilia and move- 

 ment in a twelve-hour-old culture. In deciding a diagnosis this must 

 be carefully watched, and it must be remembered that longer cultiva- 

 tion can influence motility. 



Staining Spores. 



Holler's Method. Combination of tubercle-staining with a previous mordanting 

 with chromic acid. 



(a) The cover-glass film (fixed in a flame or absolute alcohol) is dipped for two 

 minutes in chloroform, and washed with water. 



(b) Chromic acid 5 per cent, for one-half to two minutes. 



(c) Wash with water. 



(d) Carbol fuchsin heated to steaming for one minute. 



(e) Differentiate with 5 per cent, sulphuric acid till the red colour is almost gone. 

 (/) Thoroughly wash with water. 



(g) Aqueous methylene blue, or malachite green, one-half to one minute ; wash, 

 dry, etc. 



(The organisms of the Subtilis group are the only spore-forming ones which we 

 frequently meet with in ophthalmology. Tetanus is rarely seen; other spore- 

 formers very rarely.) 



Staining Methods for the Tubercle Bacillus. 



A. In Smear Preparations. 



Ziehl-Neelsen's Method : 



(a) Stain with carbol fuchsin (ac. carbol. cryst., 5 ; alcohol, 10 ; fuchsin, 1 ; aq. 

 dist., 100), warming till steaming for two minutes. 



(6) Decolorize in 20 per cent, nitric acid, or 25 per cent, sulphuric acid, for five 

 seconds. 



(c) Decolorize in 60 per cent, alcohol till film is quite colourless. 



(d) Contrast stain with aqueous methylene blue ; wash with water, and mount. 

 Friinkel-Gabbet combines the decolorizing and the contrast stain. From the 



carbol fuchsin the film is put into the following mixture : Alcohol, 50 ; sulphuric 

 acid, 25 ; aq. dist., 100 ; and sufficient solid methylene blue to produce a deep 

 blue colour ; stained, then washed, etc. 



To distinguish between the tubercle and smegma bacillus, Bunge and Trantenroth 

 recommend that the preparation be placed for fifteen minutes in absolute alcohol to 

 remove all fat. The smegma bacillus will then have lost its acid-fast property ; the 

 tubercle bacillus will not. Or (Weichselbaum) the stained preparation is put into a 

 saturated solution of methylene blue in absolute alcohol for five to ten minutes, 

 when the smegma bacillus will lose its red colour ; the tubercle bacillus will retain it 



B. Tubercle Bacilli in Sections (which must be very thin). 



(a) Place the sections hi Ziehl-Neelsen's carbol fuchsin for from twenty minutes to 

 several hours in the oven (celloidin sections longer) ; then in 25j per cent, nitric 



