76 PRINCIPLES OF BACTERIOLOGY 



should be exposed to the maceration of the chromic acid. Even under 

 the best conditions it is almost impossible to stain some spores. 



Staining Flagella. For the demonstration of flagella, which are 

 possessed by all motile bacteria, we are indebted to Loeffler. The 

 staining of flagella satisfactorily is one of the most difficult of bacterio- 

 logical procedures. Special stains devised by him, by Van Ermengem, 

 by Pitfield, and others are employed. 



Preparation of Films. In all methods young (twelve to eighteen-hour) 

 cultures on agar should be chosen. A little of the culture is carefully 

 removed and placed in a few drops of filtered tap water. A tiny drop 

 of this rather thin emulsion is allowed to spread with as little manipu- 

 lation as possible over the cover-glass. 



Bunge's modification of Loeffler's method is carried out as follows: 

 Cover-glasses which have been most carefully cleaned are covered by a 

 very thin smear. After drying in the air and passing three times through 

 the flame the smear is treated with a mordant solution, which is pre- 

 pared as follows: To 3 parts of saturated watery solution of tannin 

 add 1 part of a 25 per cent, solution of ferric chloride. This mordant 

 should be allowed to stand for several weeks before using. After pre- 

 paring the cover-slip with all precautions necessary to cleanliness the 

 filtered mordant is allowed to act cold for five minutes, after which it 

 is warmed and then in one minute washed off. After drying, the 

 smear is stained with the carbol-fuchsin solution or carbol-gentian 

 violet, and then washed off, dried, and mounted. 



Frequently the flagella appear well stained, but often the process has 

 to be repeated a number of times. Overheating of the film prevents the 

 staining of the flagella. 



Van Ermengem 's method gives good results. 



Examination of Bacteria in Tissues. Occasionally it is of importance to 

 examine the bacteria as they are in the tissues themselves. The tissues 

 should be obtained soon after death, so as to prevent as much as possible 

 post-mortem changes, with consequent increase or decrease in the 

 number of bacteria in them. Selected pieces of tissues can be frozen by 

 ether and sections cut, but the best results are obtained when the material 

 is embedded in celloidin or, better still, in paraffin. From the properly 

 selected spots small portions, not thicker than one-quarter of an inch, 

 are removed and placed in absolute alcohol for one or two days if less 

 than one-eighth inch thick, and longer if thicker. For the larger pieces 

 it is better to change the aclohol after twenty-four hours. The tissue 

 pieces should be kept from falling to the bottom as the higher layers 

 of alcohol remain nearer absolute. If along with the bacteria one wishes 

 to study the finer structure of the tissue we must employ formalin or 

 corrosive sublimate. The tissue is put in formalin 4 to 10 percent, solution 

 for three to twenty-four hours, and then in alcohol. Corrosive sublimate 

 as a saturated solution in 0.75 per cent, sodium chloride solution is also 

 an excellent fixative agent. Dissolve the sublimate in the salt solution 

 by heat and then allow to cool, when the separation of crystals will 

 show that saturation is complete. For pieces of tissue one-eighth inch 



