Determination of Bacilli. 571 



Milk must always be drawn under aseptic precautions (p. 558) and allowed 

 to settle m a conical vessel (protected from exposure) for 24 hours, or centrifuged 

 and the sediment examined. 



Methods of Staining. The acid-fast tubercle bacilli may be demonstrated 

 by various means which ai-e all based on staining coverglass preparations, fixed 

 in the usual manner, with anilin dyes, the staining properties of which have been 

 intensified by the addition of a mordant and then treating with a mineral acid 

 or with alcohol. The methods in common use are as follows: 



1.- Method of Ziehl & Nelsen. Staining solution: 1.0 gm. fuchsin, 10.0 gm. 

 absolute alcohol,. 90.0 gm. 5% carbolic acid solution; stain a few minutes under 

 the action of heat; decolorize a few seconds in 5% sulphuric acid, wash in alcohol 

 and re-stain in aqueous methylene blue solution. (The prepared staining solutions 

 keep well.) 



2. Gabbet's Method. Stain in hot carbol-fuchsin as above then place for 

 a few seconds in mixture of 1 to 2 parts of methylene blue and 100 parts of 5% 

 sulphuric acid to decolorize and double stain; rinse thoroughly in water. After 

 staining and drying the stained coverglasses are mounted in Canada balsam and 

 examined under a power of 500 to 600 diameters, preferably with an oil immersion 

 objective. 



Material supposed to contain only small numbers of bacteria may be treated 

 with antiformin before staining, as follows: Mix 20 to 30 parts of the material 

 to be examined with 15 parts antiformin and 55 to 65 parts of water, shake and 

 let stand for 1 to 2 hours. The antiformin will dissolve tissue cells and all non- 

 acid-fast bacteria but will not affect tubercle bacilli or other acid-fast bacteria 

 which accumulate in the sediment of the vessel (Uhlenhut and Kersten). 



Much's non-aeid-fast granular rods and granules may be stained by Gram's 

 method, but this requires 24 to 48 hours at body temperature. The Herman-Caan 

 method is more rapid, viz. Staining fluid: 3 parts of a 1% ammonium carbonate 

 solution in distilled water and one part of a 3% crystal violet solution in 96% 

 alcohol; stain with the aid of heat; decolorize with 10% nitric acid, then with 

 96% alcohol. 



Spongier and Betegh recommend special staining methods for the examination 

 of sputa -by means of which, it is claimed, human and bovine types may be 

 differentiated. These methods undoubtedly produce beautiful pictures but whether 

 they are of value for the purpose indicated, or in determining the spore characteristic 

 of the granules which they bring to view, is as yet a question. 



Acid-fast Paratubercle Bacilli. The property of the tubercle 

 bacillus which enables it to retain stains with which its tissues have 

 once become impregnated, even after treatment with mineral acids, is 

 not an exclusive characteristic of this bacillus. Soon after the discovery 

 of the tubercle bacillus it was found that, aside from the human lepra- 

 bacillus, the smegma of the genital organs and the cerumen of the 

 external auditory canal contained saprophitic bacilli that had the prop- 

 erty of retaining stains much like the tubercle bacillus, though not 

 as tenaciously. Spina, as early as 1883, demonstrated acid-fast bacilli 

 in decomposilig blood, in sewage and in the sputum of healthy human 

 beings. This property of the tubercle bacillus, however, was until very 

 recently regarded as a distinct characteristic in the examination of 

 pathological secretions until Petri (1896) and later Rabinowitsch found 

 bacilli in butter and milk that resembled true tubercle bacilli. There 

 is no doubt that these forms had previously frequently been mistaken 

 for true tubercle bacilli. Since that time bacteria with similar charac- 

 teristics have been discovered in raw animal products, food stuffs and 

 in human and animal secretions and excrements. The differentiation 

 of these various forms from true tubercle bacilli becomes the more 

 difficult since some of them produce lesions in experiment animals that 

 are very similar to those of tuberculosis. 



The bacteria of this class are without exception saprophytes that 

 may occur in soil, water and dust, decomposing substances, food stuffs, 

 manure, etc., occasionally gaining access to raw animal products, par- 

 ticularly milk, butter and oleomargarine. Occasionally they are also 

 found in the bronchial secretions, intestinal evacuations and urine of 



