206 LABORATORY EXERCISES IN BACTERIOLOGY. 



means of clinical diagnosis from the other bacteria apt to be met in persons suspected 

 of having tuberculosis which is practically reliable. A favorite method for determina- 

 tion of these germs in the sputum of consumptives may be detailed in illustration : 



1 . Spread sputum on a plate of glass over a black surface and with a forceps pick 

 out the small yellowish masses, which are likely to contain numbers of the bacteria. 

 (Do not mistake particles of food!) 



2. Place such material on a glass slide and spread by applying a second slide upon 

 the mass, drawing it along and across the slide evenly. (Warm if spreading is inter- 

 fered with by mucus.) 



3. Dry film in air, and fix by passing three times through the flame, smeared side 

 away from flame. (Do not burn!) 



4. Flood film with Ziehl's carbol-fuchsin (supra, a), and warm until vapor arises 

 (do not boil!) over flame for three or four minutes. 



5. Wash off excess of stain under tap, and apply as decolorizing agent and counter- 

 stain Gabbet's solution (6) for one-half to one minute. 



6. Wash in water until no more color is discharged. 



7. Dry film and examine directly in oil, or after covering with cover-slip. Mrco- 

 bacteria tuberculosis red ; all else in field blue. 



The examination of urine and vaginal discharge is similarly carried out, but 

 after discovery of acid-resisting bacteria one should apply a saturated alcoholic solution 

 of methylene-blue to the film for a minute. Should the bacteria be decolorized and 

 stained blue by this procedure they are not the organisms of tuberculosis, but the 

 smegma mycobacteria, non-pathogenic bacteria found in the cheesy secretion about 

 the foreskin or vulva. 



Staining Bacteria in Sections of Tissues. Tissues in which bacteria -are to 

 be stained should be at once fixed in absolute alcohol. After sections have been made 

 in one or other of the usual manners (freezing, paraffine, or celloidine methods for 

 technique of which, consult any work on histologic technology) a section should be 

 attached to a slide. This may be done by floating the section on a drop or two of a 

 solution of gelatine (best sheet gelatine, 0.5; chloral hydrate, 1 ; distilled water, 100) 

 for several minutes, after which the excess of the solution is drained off and the slide 

 set aside to dry (best until the following day under a bell jar; more quickly at 37 C. 

 in incubator). Thereafter it is plunged for five minutes in a five per cent, solution of 

 potassium bichromate to fix the gelatine glue , and render it insoluble in the staining 

 fluids to be used. (Specimens in paraffine should have paraffine removed prior to this 

 last step.) This done, the staining is carried out as if, instead of a section, the ordinary 

 film were on the slide. It is best, however, to use little or no heat and to allow the 

 stains a longer time for action before removal and application of the decolorizer. So, 

 too, in use of chemical decolorizers the best results usually follow the use of the less 

 concentrated solutions, and separate application of the decolorizer and counterstain. 

 The sections should be almost completely decolorized to gross inspection. In the 

 final dehydration before mounting, absolute alcohol should be used drop by drop on 

 the section, alternating with occasional absorption of all fluid possible by application 

 of a layer of folded unsized paper. Origanum oil is best suited as a clearing agent. 

 After clearing, xylol balsam and a thin cover are to be applied. Carbol-thionine (/) 

 is an excellent stain for general use with sections, alcohol being used as the decolorizing 

 reagent ; Gram's method and the Ziehl carbol-fuchsin (with Gabbet's solution, if desired) 

 are also entirely appropriate. As a counterstain in Gram's method borax-carmine 



